Protein Export and Membrane Biogenesis, Volume 4 (Advances in Cellular and Molecular Biology of Membranes and Organelles)

ADVANCES IN CELL AND MOLECULAR BIOLOGY OF MEMBRANES AND ORGANELLES Volume 4 9 1995 PROTEIN EXPORT AND MEMBRANE BIOGEN...

Author: R.E. Dalbey

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ADVANCES IN CELL AND MOLECULAR BIOLOGY OF MEMBRANES AND ORGANELLES Volume 4

9 1995

PROTEIN EXPORT AND MEMBRANE BIOGENESIS

This Page Intentionally Left Blank

ADVANCES IN CELL AND MOLECULAR BIOLOGY OF MEMBRANES AND ORGANELLES PROTEIN EXPORT AND MEMBRANE BIOGENESIS

Series Editor: ALAN M. TARTAKOFF Institute of Pathology Case Western Reserve University Editor: ROSS E. DALBEY Department of Chemistry The Ohio State University

VOLUME 4

91995

@ Greenwich, Connecticut

JAI PRESS INC.

London, England

Copyright 91995 by JAI PRESSINC. 55 Old Post Road, No. 2 Greenwich, Connecticut 06836 JAI PRESSLTD. The Courtyard 28 High Street Hampton Hill, Middlesex TWI 2 1PD England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher. ISBN: 1-55938-924-9

Transferred to Digital Printing 2006

T LIST OF CONTRIBUTORS

VII

INTRODUCTION TO THE SERIES

Alan M. Tartakoff

ix

PREFACE

Ross E. Dalbey

xi

MEMBRANE PROTEIN ASSEMBLY

Paul Whitley and Gunnar von Heijne

MEMBRANE INSERTION OF SMALL PROTEINS: EVOLUTIONARY AND FUNCTIONAL ASPECTS

Dorothee Kiefer and Andreas Kuhn

PROTEIN TRANSLOCATION GENETICS

Koreaki Ito

BIOCHEMICAL ANALYSES OF COMPONENTS COMPRISING THE PROTEIN TRANSLOCATION MACHINERY OF ESCHERICHIA COLI Shin-ichi Matsuyama and Shoji Mizushima

17

35

61

PIGMENT-PROTEIN COMPLEX ASSEMBLY IN RHODOBACTER SPHAEROIDESAND

RHODOBACTER CAPSULATUS Amy R. Varga and Samuel Kaplan

85

IDENTIFICATION AND RECONSTITUTION OF ANION EXCHANGE MECHANISMS IN BACTERIA

Atul Varadhachary and Peter C. Maloney

105

CONTENTS HELIX PACKING IN THE C-TERMINAL HALF OF LACTOSE PERMEASE H. Ronald Kaback, Kirsten]ung, Heinrich ]ung, ]ianhua Wu, Gilbert G. PrivY, and Kevin Zen

129

EXPORT AND ASSEMBLY OF OUTER MEMBRANE PROTEINS IN E. COL/ Jan Tommassenand Hans de Cock

145

STRUCTURE-FUNCTION RELATIONSHIPS IN THE MEMBRANE CHANNEL PORIN Georg E. Schulz

175

ROLE OF PHOSPHOLIPIDS IN ESCHERICHIACOL/ CELL FUNCTION William Dowhan

189

MECHANISM OF TRANSMEMBRANE SIGNALING IN OSMOREGULATION Arfaan A. Rampersaud

219

INDEX

263

LIST OF CONTRIBUTORS Hans de Cock

Department of Molecular Cell Biology Institute of Biomembranes Utrecht University Utrecht, The Netherlands

William Dowhan

Department of Biochemistry and, Molecular Biology The University of Texas-Houston Medical School Houston, Texas

Koreaki Ito

Department of Cell Biology Institute for Virus Research Kyoto University Kyoto, Japan

Heinrich Jung

Departments of Physiology and Microbiology and Molecular Genetics University of California Los Angeles, California

Kirsten Jung


H. Ronald Kaback


Samuel Kaplan

Department of Microbiology and Molecular Genetics University of Texas Health Science Center at Houston Houston, Texas oo

VII

viii

LIST OF CONTRIBUTORS

Dorothee Kiefer

Angewandte Mikrobiologie Universit~t Karlsruhe (TH) Karlsruhe, Germany

Andreas Kuhn

Angewandte Mikrobiologie Universit~t Karlsruhe (TH) Karlsruhe, Germany

Peter C. Maloney

Department of Physiology Johns Hopkins University School of Medicine Baltimore, Maryland

Shin-ichi Matsuyama

Institute of Molecular and Cellular Biosciences University of Tokyo Tokyo, Japan

Shoji Mizushima

Tokyo College of Pharmacy Tokyo, Japan

Gilbert G. Priv~


Affaan A. Rampersaud

Department of Pathology The Ohio State University Columbus, Ohio

Geor8 E. Schulz

InstitOt for Organische Chemie und Biochmie A Ibert-Ludwigs-Universit~it Freiburg, Germany

Jan Tommassen

Department of Molecular Cell Biology Institute of Biomembranes Utrecht University Utrecht, The Netherlands

Atul Varadhachary

Department of Biological Chemistry Johns Hopkins University School of Medicine Baltimore, Maryland

Lbt of Contributors Amy R. Varga

Chemical and Agricultural Products Division Abbott Laboratories North Chicago, Illinois

Gunnar von Heijne

Department of Biochemistry Arrhenius Laboratory Stockholm University Stockholm, Sweden

Paul Whitley

Department of Biochemistry Arrhenius Laboratory Stockholm University Stockholm, Sweden

Jianhua Wu


Kevin Zen



INTRODUCTION TO THE SERIES The remarkable vigor and central importance of cell biology result from the realization that emphasis on structure/function relations at the cellular and subcellular levels is essential for a rigorous and satisfactorily complete understanding. Unlike many subdivisions of biomedical science, cell biology is not linked to any one methodology. It often emphasizes topological or topographic questions, and it is concerned with the structure, biogenesis, and turnover of macromolecular structures; however, there is no limit to the techniques and conceptual approaches which it brings to bear on these issues. Indeed, this has even been true since the term cell biology was first used. Certain scientists trace its origins back to ultrastructure and histology; others consider E.B. Wilson's The Cell in Development and Heredity to epitomize the foundations of cell biology; while others consider cell biology as an outgrowth of somatic cell genetics or the extension of biophysics to objects of increasingly large size. This varied and often changing identity makes it easier to specify what cell biology is not, rather than to describe it in a positive sense. It also suggests the rich intellectual mix which underlies today's successes in cell biology research. This series aims to match the continuing evolution of cell biology---with particular emphasis on cell membranes--by treating coherent areas in multi authored volumes This approach allows a multifaceted coverage of topics which benefits from the unity of vision of the volume editor(s), but does not rely on one individual to synthesize an entire subject. Hopefully, the result will be more readable and intrinsically richer than lengthy review chapters which attempt to be all encompassing. Alan M. Tartakoff Series Editor xi


PREFACE The incentive for putting together Volume 4 of this series was to review the wealth of new information that has become available in prokaryotic organisms in protein export and membrane biogenesis. Just in the last several years, protein translocation has now been efficiently reconstituted using defined components and the mechanism by which proteins are moved across membrane bilayers is now being examined at a higher resolution. In addition, because of a new technical breakthrough using osmolytes, it is now possible to reconstitute a number of channel proteins, ATPase, receptors, and transporters. In many cases, it is possible to successfully predict the membrane topology of these types of proteins using both "hydrophobicity analysis" and the "positive inside" rule. In this volume, two chapters focus on protein translocation across membranes

(Biochemical Analyses of Components Comprising the Protein Translocation Machinery orE. coil; Protein Translocation Genetics), while several others on how proteins assemble into the inner membrane of E. coil (Membrane Protein Assembly, Membrane Insertion of Small Proteins: Evolutionary and Functional Aspects; Pigment-Protein Complex Assembly in Rhodobacter sphaeroides and Rhodobac. ter capsulatus). Other sections review recent progress on transporters (Identification and Reconstitution of Anion Exchange Mechanisms in Bacteria; Helix Packing in the C-Terminal Half of Lactose Permease) and signal transduction (Mechanism of Transmembrane Signaling in Osmoregulation) as well as the assembly of porins into the outer membrane (Export and Assembly of Outer Membrane Proteins in E. coil) and their structures (Structure-Function Relationships in the Membrane Channel Porin). Although the emphasis of the book is on proteins, the role of xiii

xiv

PREFACE

phospholipids in controlling various cell surface processes is reviewed (Role of Phospholipids in Escherichia coil Cell Function). I should point out the reason for the rapid progress in bacteria research is because of the possibility to apply biochemistry and genetics to this organism. I would like to thank the contributors to this volume who gave their time and expertise and who made this task enjoyable. Ross E. Dalbey

Editor

MEMBRANE PROTEIN ASSEMBLY

Paul Whitley and Gunnar yon Heijne

I. II. III.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane Protein Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane Protein Topology . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Experimental Determination of Topology . . . . . . . . . . . . . . . . . . B. Prediction and Experimental Manipulation of Topology . . . . . . . . . . . IV. Helix Packing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Mechanism of Membrane Insertion . . . . . . . . . . . . . . . . . . . . . . . . A. Translocation of Periplasmic Loops . . . . . . . . . . . . . . . . . . . . . . B. Insertion of Transmembrane Segments . . . . . . . . . . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 3 3 4 5 6 6 10 11

I. I N T R O D U C T I O N Cells are made up of multip!e membrane-bound compartments, each containing a unique set of proteins. The sorting and insertion of proteins across and into the many membrane systems of eukaryotes and prokaryotes has been the subject of a vast amount of research. In this chapter we will deal with only one area of membrane

Advances in Cell and Molecular Biology of Membranes and Organelles Volume 4, pages 1-16. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-924-9

PAUL WHITLEY and GUNNAR VON HEIJNE

protein biogenesis, namely, the assembly of proteins into the cytoplasmic (inner) membrane of prokaryotes, notably Escherichia coll. We believe that many of the basic aspects of membrane protein assembly in E. coli are relevant to the assembly of proteins into other membrane systems such as the endoplasmic reticular (ER) membrane of eukaryotes, the thylakoid membrane of chloroplasts, and the inner mitochondrial membrane. Knowledge from E. coil can often illuminate the analogous processes in these other systems. As a background to the discussion on the membrane assembly processes per se, we will first give a brief introduction to membrane protein structure. This is followed by sections on membrane protein topology and helix packing in a lipid environment. Finally, the mechanisms of membrane insertion in E. coil are reviewed.

II. MEMBRANE PROTEIN STRUCTURE Although membrane proteins have diverse functional roles in the cell such as ion channels, transporters, and receptors, they appear to be limited in the types of structure they can adopt, due to the constraints imposed by the hydrophobic environment of their membrane-spanning regions. The hydrophobic interior of the lipid bilayer has for a long time been considered as energetically unfavorable for peptide bonds unless the polar groups are hydrogen bonded (32). Furthermore, amino acids with polar and potentially charged side chains are not favored energetically in a hydrophobic environment. Thus a-helices built mainly from amino acids with nonpolar side chains are structures likely to be found in membrane-spanning regions. Spectroscopic studies on a number of membrane proteins indeed have provided evidence that they are comprised largely of a-helical structure (34, 97). However, there is an embarrassing lack of high-resolution structural information for membrane proteins to confirm existing theories. The X-ray structure of the photosynthetic reaction center from Rhodopseudomonas viridis (25) and the structure of bacteriorhodopsin from Halobacterium halobium derived from electron microscopy studies (43), are among the few exceptions that confirm the predicted a-helical nature of membrane-spanning segments. a-Helices are not the only structures that can span the lipid bilayer. The crystal structures of bacterial outer membrane porins have recently been solved and shown to consist of large 16-stranded 13-barrelstructures in the membrane-spanning region (21, 98). Such closed ~-sheets fulfill the hydrogen-bonding criterion but do not possess the long hydrophobic stretches of amino acids characteristic of the helical bundle membrane proteins. Very little is known about the mechanism of insertion of the ~-barrel t y ~ membrane proteins. We therefore limit the following discussion to the assembly of proteins of the a-helical variety.

Membrane Protein Assembly Due to the difficulties in obtaining high-resolution structural information for membrane proteins by classical structural techniques, such as X-ray crystallography and NMR, it is necessary to turn to other low resolution techniques for looking at their structures. In the following sections we will discuss some of the theoretical and experimental methods that have been used to map or predict the topology of membrane proteins in the cytoplasmic membrane of bacteria.

III. MEMBRANE PROTEIN TOPOLOGY A. ExperimentalDetermination of Topology In order to establish accurately the topology of a membrane protein, we would ultimately like to know for each amino acid whether or not it is buried in the lipid bilayer, and towards which side of the lipid bilayer it is situated. One way to determine this is by so-called vectorial-labeling methods (46). Vectorial labeling requires a sealed membrane preparation of known orientation containing the protein of interest and a labeling reagent that only has access to one side of the membrane. Only those reactive amino acid residues that are accessible to the labeling reagent will be modified, and by analyzing the pattern of modification, their membrane disposition can be determined. Modifications can range from the covalent attachment of small probes, such as fluorescent, radioactive, and spin-labeled molecules, to binding of antibodies or proteolytic cleavages. The most frequently used methods to determine membrane protein topology in E. coli, however, rely on gene-fusion technology to produce hybrid proteins. Fusions to the reporter proteins PhoA (alkaline phosphatase), LacZ ([3-galactosidase), and 13-1actamase are the most commonly used. All of these fusions are useful in determining the topology of membrane proteins due to different activities of these proteins when located in the cytoplasm or periplasm. For example, PhoA is only active when it is periplasmic (68), and PhoA fusions to a portion of a membrane protein that is normally periplasmic likewise have a high activity. If fused to a portion of the protein that is normally cytoplasmic, the PhoA activity will be low (65). Another feature of PhoA that makes it useful in topological studies is the high protease resistance when situated in the periplasm, but not when located in the cytoplasm (79). The case is similar for 13-1actamase fusions that confer ampicillin resistance and give high [3-1actamase activity only when the [3-1actamase moiety is present in the periplasm (16). LacZ fusions, in contrast, are only active when fused to cytoplasmic domains (36, 38). Results from fusion protein analysis should, however, be treated with some caution. PhoA fusions may in certain situations show substantial activity, even when fused to normally cytoplasmic regions of the polypeptide chain. This phenomenon was observed when PhoA was fused to the beginning of proposed cytoplasmic domains of MalF (36), presumably because these fusions were translocated, albeit


slowly, across the cytoplasmic membrane (86). Fusions towards the C-terminal ends of the cytoplasmic and periplasmic regions are thus to be preferred (15). LacZ fusions sometimes exhibit low activity for reasons other than being attached to an export signal; for example, if the fusion is out of frame, there is often a low but detectable ~-galactosidase activity (41). Nevertheless, a combination of LacZ with PhoA and/or I~-lactamase fusions at different places within a polytopic membrane protein has been used successfully to determine the topology of many inner membrane proteins in E. coil [see (95) for a list of examples].

B. Prediction and Experimental Manipulation of Topology Although the powerful fusion protein techniques make topological mappings relatively straightforward, much can be deemed from the primary amino acid sequence. Apolar membrane-spanning helices are often easy to predict using hydrophobicity analysis. Essentially, a window of 15-20 residues is moved along the sequence, and the average hydrophobicity of. the residues inside the window is calculated for each position. Peaks on the hydrophobicity plot above a certain cutoff level correspond to candidate membrane-spanning segments and allow a tentative topological model that can serve as a basis for planning further fusion protein studies. However, although many transmembrane segments have a very high average hydrophobicity and are easy to spot, others have a more moderate hydrophobicity and can be difficult to distinguish from nonmembrane segments. In such cases, a considerably improved prediction can be obtained if the so-called positive-inside rule is taken into account. This rule is based on the observation that nontranslocated segments of inner membrane proteins have a 3- to 4-fold higher content of positively charged lysines and arginines than translocated segments (91) and that any predicted topology should thus be expected to show a similar bias. Ifa particular stretch is of moderate hydrophobicity, and thus difficult to assign unambiguously as a transmembrane segment, two tentative topologies may be constructed: one with and one without the doubtful transmembrane segment. The one with the highest Lys+Arg bias is chosen as the best prediction (95). In practice, this simple recipe gives a substantial improvement in prediction accuracy: For bacterial inner membrane proteins, one can expect to predict the correct topology about 9 times out of 10. Even more importantly, experimental studies have shown that the topology of an inner membrane protein can be manipulated by simply redistributing positively charged residues from one end of a transmembrane stretch to the other. Such topology-engineering was first demonstrated for the leader peptidase enzyme (93), where, in certain constructs, as few as one lysine residue can make the difference between Nin and Noet orientations (70). How could the positively charged residues influence topology? One possibility is that the electrical potential across the membrane (positive periplasmic) works

Membrane Protein Assembly

against the translocation of positively charged residues. Other possibilities are that negatively charged phospholipid head groups on the cytoplasmic face of the inner membrane may interact with the positively charged amino acid residues or that proteins of the secretory machinery such as SecA may bind preferentially to the positively charged end of a transmembrane segment (1).

IV. HELIX PACKING The actual membrane insertion process appears to follow a two-stage model (75), where independently stable a-helices are first inserted across the lipid bilayer to determine the topology of the protein. In the next stage, the membrane-spanning ct-helices pack together to form functional transmembrane structures (helix bundies). From the available data, it appears that interactions between transmembrane ct-helices can contribute substantially towards structural stabilization, and in some cases, the oligomerization of membrane proteins (20, 55, 66). Evidence for the importance of interactions between pre-inserted membranespanning helices that supports the two-stage model of protein folding comes from the observations that helix-containing fragments of membrane proteins can reassemble into native-like structures. B acteriorhodopsin can be cleaved by chymotrypsin to give two fragments containing 5 and 2 transmembrane helices, respectively. These two fragments, when reconstituted separately into lipid vesicles, were shown by circular dichroism to be ct-helical. When the two populations of vesicles were fused, the resulting protein complex acquired the ability to bind retinal and regained the spectroscopic properties of native bacteriorhodopsin (74). In fact, the same trick can be performed using two fragments of one transmembrane segment each, plus a third fragment containing the remaining five (48, 49). Further evidence comes from in vivo experiments where co-expression of fragments of lactose permease were shown to restore lactose transport in E. coli, whereas the fragments were rapidly degraded when expressed alone (9, 104). The types of interactions that take place between membrane o~-helices have been under investigation recently. Structures of membrane proteins known to sufficiently high resolution have been analyzed; it was found that residues that are buried in the interior of these proteins, on average, tend to be more hydrophilic than those in contact with the lipid and that the helix-helix interfaces are generally close-packed (78). There is no direct evidence for salt bridges in the known structures. However, there is indirect evidence for salt bridges between residues in different helices of lactose permease (26, 50, 59, 81). Interactions between potentially charged amino acids in transmembrane regions of eukaryotic proteins have also been proposed to be involved in oligomerization (20, 66), organellar retention (13), and degradation

(51). Not all helix-helix interactions appear to be driven by polar interactions. Giycophorin A from human erythrocytes has a single transmembrane domain and forms


very stable homodimers. Dimerization is driven by interactions between the transmembrane helices (14), and saturation mutagenesis of the transmembrane regions has shown the dimerization interface to be largely apolar (60). A general approach for analyzing helical packing that may be applicable to a wide variety of membrane proteins is to look for the formation of disulfide bridges between pairs of cysteines engineered into membrane-spanning helices. This approach was first used to look at the dimerization interface in transmembrane regions of the E. coil aspartate sensory receptor (Tar receptor) (63). The analysis was extended to include intrachain interactions between the two transmembrane helices in Tar receptor monomers (72). Recently, the interface between the two transmembrane helices of E. coil leader peptidase has been mapped by a similar approach (100). One problem with the disulfide approach is that it is not possible to say how the native structure of the membrane protein is perturbed by the formation of disulfide bonds between pairs of genetically engineered cysteines, and a fairly large number of mutants have to be analyzed to get a consistent picture. A couple of computational methods have been put forward to predict how helices may pack against one another (27, 28, 87, 88). However, without access to a sufficiently large number of 3-D structures, it is difficult to validate and refine predictive methods. It should be noted that some oligomerization of membrane proteins almost certainly takes place because of interactions between polar portions of the protein exposed outside the bilayer (89, 106). Interactions between polar domains within a membrane protein may also influence the packing of helices and will, especially in the case of short loops, impose constraints as to which helices can pack against each other. V. M E C H A N I S M O F M E M B R A N E I N S E R T I O N The assembly of a protein into the inner membrane of E. coli requires some portions of the polypeptide chain to pass completely through the lipid bilayer, some to partition into the lipid bilayer, and some to remain cytoplasmic. In previous sections we have dealt with features of the polypeptide chains of membrane proteins that contribute toward these topological decisions, but we have not discussed the mechanisms by which they are achieved. In what follows, we will focus on how periplasmic loops are translocated across, and how transmembrane segments are inserted into, the inner membrane.

A. Translocationof Periplasmic Loops One of the key questions in the study of membrane protein biogenesis is: How do hydrophilic segments of the polypeptide chain pass through the hydrophobic core of the lipid bilayer? This problem also arises when secretory proteins are to


be completely translocated across the lipid bilayer into the periplasmic space. The great majority of secreted proteins in E. coil (and also in eukaryotes) are synthesized with amino terminal extensions known as signal (or leader) sequences that are generally around 20 amino acid residues in length and are removed from the mature polypeptide on the periplasmic side of the cytoplasmic membrane by the leader peptidase enzyme. Despite the fact that signal sequences of different proteins do not appear to have any primary sequence homology, they do share common features (90): an amino terminal positively charged region (n-region, 1-5 residues long), a central hydrophobic part (h-region, 7-15 residues long) thought to be the key region in signal peptide function (37), and a more polar carboxy terminal domain (c-region, 3-7 residues) that contains the cleavage recognition site for leader peptidase (92). It is well established that the presence of a functional signal sequence is essential for efficient secretion, and mutations that impede export of a protein and result in the accumulation of its precursor in the cytoplasm nearly always map to the hydrophobic core of the signal sequence of the protein (30, 33). In general, signal sequences can be exchanged between proteins and still correctly direct export (8), [although this is not always the case (57)], which suggests the importance of secondary structure rather than specific sequence for correct function. Proposals as to how signal peptides function to direct the transfer of polypeptide chains across the lipid bilayer are diverse and are based on data from experimental techniques ranging from classical genetics to biophysics (see (37, 94)). It has thus been proposed that signal peptides interact directly with iipids and direct translocation through the lipid bilayer; in support of this idea, experiments with synthetic peptides corresponding to signal sequences have shown them to have substantial affinity for phospholipid mono- and bilayers and to have an o~-helical conformation in the lipid environment (7, 39). On the other hand, it has also been suggested that hydrophilic stretches of polypeptide must pass through the membrane in a polar proteinaceous channel (12, 84). This is supported by the observation that in electrophysiological experiments signal peptides can open aqueous channels when added to the cytoplasmic side of E. coil planar lipid bilayers (83). Also, translocating polypeptides can be crosslinked to protein components in the E.coli inner membrane (47) and the ER membrane (4, 40, 52). In vivo, it appears that interactions of signal peptides with both proteins (I0) and lipids (24) are important for efficient secretion. Components of the E. coil secretion machinery have been identified by both genetic and biochemical means (see chapters by Ito and by Matsuyama & Mizushima). At least six proteins are known to be involved; namely, SecA, SecB, SecD, SecE, SecE and SecY. In addition, a homologue of the eukaryotic SRP 54-kDa protein, called Ffh, has recently been implicated in the process (62, 73). A recent model as to how the secretion machinery functions in E. coli has been proposed by Wickner (102). Unlike translocation of proteins across the endoplasmic reticulum in eukaryotes (77), translocation across the cytoplasmic membrane


of E. coil is not obligatorily co-translational (76). After synthesis, some preproteins are thought to associate with secB, which acts as a molecular chaperone (2, 19) and keeps preproteins in an export competent state. SecB is not an essential protein; other molecular chaperones, such as GroEL and GroES, can substitute for secB function (3) and are, in fact, necessary for the efficient secretion of some proteins (56). SecA is a peripheral membrane protein (22) that probably binds with high affinity to SecB/preprotein complexes by virtue of its affinities for SecB (42) and both the signal (23) and mature (61) domains of the precursor proteins. This affinity for the secretory preprotein complex is greatly enhanced if SecA is in association with membranes containing acidic phospholipids (44) and the integral SecY/E protein complex (42). Thus SecA provides a pivotal link between the presecretory protein and the membrane components of the secretory machinery. SecA has an ATPase activity (the translocation ATPase activity) that is induced when it is in contact with the elements mentioned above (61). The binding of ATP to SecA without hydrolysis driveg limited translocation of the polypeptide chain (82). Hydrolysis of ATP then probably releases the precursor protein from SecA and proton motive force (AI~H+) dependent translocation ensues, although exactly how AI~H+ drives translocation is not known. Subsequent rounds of SecA binding to the preprotein, ATP binding to SecA, hydrolysis of ATP, release of the preprotein, and ApH + driven translocation drive the preprotein through the membrane until it is fully translocated. Since the problem of transferring hydrophilic stretches of polypeptide chain across the lipid bilayer is the same for membrane proteins as it is for secreted proteins, it would seem reasonable to assume that membrane proteins may also use the sec machinery. Additionally, signal sequences broadly resemble membranespanning sequences in their overall properties. In fact, it has been shown that a signal sequence can be converted into a membrane anchor (signal anchor) simply by increasing the length of the hydrophobic core (17) or by removing the consensus site for cleavage by leader peptidase (58). The converse is also true: A signal-anchor sequence can be converted to a signal sequence simply by inserting a recognition site for leader peptidase in the appropriate position (71). At least one membrane protein, leader peptidase, is known to require the function of the SecA and SecY proteins for its correct membrane assembly (103). Other proteins such as MI3 coat protein (101), fl coat protein (80), and lactose permease (105) do not appear to require the sec gene products. A possible distinction between the sec-independent and sec.dependent modes of insertion was first suggested by statistical analysis of inner membrane proteins (91, 96) and concerns the length of the translocated segment. Short periplasmic proteins or short periplasmic loops or tails in membrane proteins were postulated not to need the sec machinery--and indeed all of the sec-independent proteins mentioned above have only short translocated stretches of amino acids--whereas, longer pieces of chain, such as the large periplasmic domain of leader peptidase, were proposed to depend on the sec machinery for translocation.

Membrane Protein Assembly The first experimental evidence for this proposed correlation between length and

sec-dependence was the observation that when the periplasmic domain of M 13 procoat protein, which is sec-independent for assembly, had its length increased from 20 to 118 residues, it became sec-dependent (.54). Recently, a more systematic approach has shown that there is a linear relationship between the length of a translocated loop situated between two membrane-spanning segments and its degree of dependence on functional SecA and SecY proteins for lengths up to -60 residues (5). This observation suggests that there is no abrupt transition from a see-independent to a sec-dependent mode of translocation, but rather that there is a gradual increase with length in the need for functional SecA and SecY proteins. It should be noted, however, that results seemingly in conflict with this simple relationship between length and sec-dependence have been reported for certain MalF-PhoA fusions (67). It is not entirely clear what the see-independent mode of insertion is like in mechanistic terms, and proposals range from a simple direct insertion into the lipid bilayer (.53) to a partial utilization of the sec machinery (.5).

B. Insertion of Transmembrane Segments It has been proposed that polytopic membrane proteins insert into the membrane using an alternating series of uncleaved signal sequences (signal anchors) initiating translocation followed by stop transfer sequences that prevent the translocation of downstream amino acids (11). Evidence to support this mechanism comes from observations that internal hydrophobic sequences in membrane proteins can act as uncleaved signal sequences (6, 35). Furthermore, signal sequences can sometimes act as stop transfer sequences when they are artificially introduced downstream of another signal sequence (18, 64). According to this model, the integration of the first membrane-spanning sequence of a multispanning membrane protein determines the topology of the whole molecule. Thus, if the first hydrophobic membrane segment inserts in an Nin orientation, the second must insert in the opposite Nout orientation, the third again in the Nin orientation, etc. Some evidence for this alternating insertion process comes from an analysis of artificial polytopic membrane protein constructs made from tandem repeats of the human asialoglycoprotein (ASGP) receptor, which normally contains only one hydrophobic signal anchor (99). Constructs with up to four repeats of ASGP receptor were shown to be integrated into dog pancreas microsomes as polytopic membrane proteins. The first copy of the signal anchor indeed acted as a signal anchor, the second integrated into the membrane in the opposite orientation to the first and thus acted as a stop transfer sequence, and the third again acted as a signal anchor, etc. It was concluded that the most N-terminal of the signal anchors inserted first and determined the topology of the whole molecule. Because translocation across the ER membrane of eukaryotes is coupled to protein synthesis, it seems likely that the most N-terminal hydrophobic stretch integrates first as more downstream ones may not yet be synthesized when assembly


10

into the membrane begins. In prokaryotes, however, translocation is not mechanisticaUy coupled to protein synthesis, and therefore, it is not obvious that the most N-terminal hydrophobic segment necessarily integrates first. Thus, the amino terminal transmembrane segment in the MalF protein can be deleted with no apparent effects on the membrane integration and topology of the remainder of the molecule (29). Furthermore, the fact that polytopic bacterial inner membrane proteins have high numbers of positively charged residues in all of their cytoplasmic loops, whether they are at the amino or caboxy terminal ends of the protein (95), suggests that each transmembrane segment (or pairs of segments) inserts into the membrane independently of the rest of the molecule. In eukaryotes, the high number of positive charges in cytoplasmic loops is more pronounced towards the N-terminus of the protein (85), which may reflect the fact that if the N-terminus is inserted correctly then the rest of the molecule will follow. As has been mentioned above, the most characteristic feature of the membranespanning portions of a polypeptide chain is their high hydrophobicity which, in principle, could allow them to spontaneously partition into the lipid bilayer (31, 45, 69). However, if a polypeptide chain is translocating through an aqueous channel (a translocon) as is now the preferred model, interactions between the amino acid side chains of a hydrophobic stop transfer sequence and the core of the lipid bilayer cannot account for termination of translocation. Rather, proteins in the translocon must somehow recognize the hydrophobic stretch of amino acids as a stop transfer signal, and the translocon must presumably disassemble, or in some other way facilitate release of the membrane-spanning sequence (e.g., by letting it slip between helices of the translocon), in order for it to become integrated into the lipid bilayer. How this is achieved is still completely unknown.

VI.

CONCLUSIONS

Over the past few years, there have been major advances in our understanding of membrane protein biogenesis in bacteria. Thus, the components of the secretory machinery are basically known, and their functional roles have at least been partly defined. The most important characteristics of the substrates for the secretory machinery--the nascent membrane proteins--have also been clarified with the discovery that positively charged amino acids act as the major topogenic determinants during membrane integration. As a measure of our understanding, we can now predict the topology of an inner membrane protein from its amino acid sequence and expect to be fight 9 times out of 10. Nevertheless, a number of issues need to be addressed in the future. The obvious ones are of course related to the mechanism of translocation: Which proteins make the channel (if there is one)? What are the properties of the channel? How are signal sequence and stop transfer sequences recognized?


11

Other questions concern the nascent polypeptide: Can charge-pairs in or between t r a n s m e m b r a n e segments play a role during m e m b r a n e insertion? H o w should we approach the study of helix-helix packing? Can we design new m e m b r a n e proteins o f p r e d e t e r m i n e d topology and structure? Problems such as these should keep us busy for some time to come.

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12


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13

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14


58. Laforet, G. A., & Kendall, D. A. (1991). Functional limits of conformation, hydrophobicity, and

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15

0. Rohrer, J., & Kuhn, A. (1990). The function of a leader peptide in translocating charged amino acyl residues across a membrane. Science 250, 1418-1421. 81. Sahin-T6th, M., Dunten, R. L., Gonzales, A., & Kaback, H. R. (1992). Functional interactions between putative intramembrane charged residues in the lactose permease of Escherichia coil Proc. Natl. Acad. Sci. USA 89, 10547-10551. 82. Schiebel, E., Driessen, A. J. M., Hartl, E-U., & Wickner, W. (199 l). ApH~ and ATP function at different steps of the catalytic cycle of preprotein translocase. Cell 64, 927-939. 83. Simon, S. M., & Blobel, G. (1992). Signal peptides open protein-conducting channels in E-coli. Cell 69, 677-684. 84. Singer, S., Maher, P., & Yaffe, M. (1987). On the translocation of proteins across membranes. Proc. Natl. Acad. Sci. USA 84, 1015-1019. 85. Sipos, L., & van Heijne, G. (1993). Predicting the topology of eukaryotic membrane proteins. Eur. J. Biochem. 213, 1333-1340. 86. Traxler, B., Lee, C., Boyd, D., & Beckwith, J. (1992). The dynamics of assembly of a cytoplasmic membrane protein in Escherichia-coti. 1. Biol. Chem. 267, 5339-5345.

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104. Wrubel, W., Stochaj, U., Sotmewald, U., Theres, C., & Ehring, R. (1990). Reconstitution of an active lactose carrier in vivo by simultaneous synthesis of two complementary protein fragments. J. Bacteriol. 172, 5374--5381. 105. Yamato, I. (1992). Membrane assembly of lactose permease of Escherichia-coli. J. Biochem. Tokyo 111,444-450. 106. Yu, X.-M., & Hall, Z. W. (199 I). Extracellular domains mediating e subunit interactions of muscle acetylcholinerecelXOr.Nature 352, 64--67.

MEMBRANE INSERTION OF SMALL PROTEINS: EVOLUTIONARY AND FUNCTIONAL ASPECTS

Dorothee Kiefer and Andreas Kuhn

I. II. III. IV. V. VI. VII.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aminoterminal versus Carboxylterminal Translocation . . . . . . . . . . . . . Determination of Membrane Protein Orientation . . . . . . . . . . . . . . . . Transl~ Translocation . . . . . . . . . . . . . . . . . . . . . Insertion of Small Proteins into the Encloplasmic Reticulum . . . . . . . . . . Requirement of Chaperones . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17 18 20 21 23 26 27 28 28

I. I N T R O D U C T I O N Each protein in a living s y s t e m - - p r o k a r y o t i c or eukaryotic---resides in a characteristic cellular c o m p a r t m e n t to fulfill its biological function. With the increasing

Advances in Cell and Molecular Biology of Membranes and Organelles Volume 4, pages 17-35. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-$$938-924-9 17

18

DOROTHEE KIEFERand ANDREASKUHN

complexity during evolution, enzymatic mechanisms have been developed that guarantee the specific locations of proteins within certain compartments that are usually separated from each other by biological membranes. Since many translocared proteins have a rather complex conformation, often protein domains cannot cross or insert into the membranes spontaneously to reach their final destination. Therefore, a cellular export machinery evolved that allows the transport of more complex protein domains across a bilayer and that also prevents premature folding of these domains prior to the translocation event. In prokaryotes, as in gram-negative bacteria, the only membraneous system is the cell wall consisting of the cytoplasmic membrane and the outer membrane. Proteins synthesized in the cytoplasm, therefore, do not have to select between different membrane systems. Eukaryotic cells, however, possess several defined compartments and organeUes, such as the nucleus, the endoplasmic reticulum and the Golgi apparatus, as well as mitochondria, chloroplasts, and peroxisomes. It is obvious that, due to this differentiated organization, eukaryotes had to evolve sophisticated targeting systems to determine a protein's final destination. Both in eukaryotes and in prokaryotes, proteins that are not resident in the cytoplasm face the problem of translocating through a highly hydrophobic environment. Translocation machineries composed of mainly proteinaceous components were developed during evolution to facilitate this process. However, there are some proteins known to translocate through biological membranes independently of the transport components. Most of these proteins are small with simple secondary structures, and the mechanism of their membrane insertion may reflect an ancient situation before a complex translocation apparatus was evolved. II.

A M I N O T E R M I N A L VERSUS CARBOXYLTERMINAL TRANSLOCATION

Since membrane proteins are synthesized in the cytoplasm, they insert their hydrophobic portion from the cis side into the membrane. In many cases, this initial process leads to the formation of a structure with both hydrophilic regions that flank the hydrophobic region on the cis side (Figure 1). To achieve a transmembrane orientation either the aminoterminal or the carboxylterminal hydrophilic portion of the protein has to translocate across the bilayer. In nature we find that the majority of single membrane-spanning proteins are translocating their C-terminal regions across the membrane. In addition, most proteins that have their N-terminal portion at the trans side of the membrane use cleavable signal or leader peptides and leave the N-terminus of the translocating precursor at the cis side. After membrane translocation the signal peptides are proteolytically processed resulting in a new aminoterminus at the trans side. It is therefore possible that a cleavable signal sequence has evolved to initiate the translocation of the N-terminal domain of a protein. Multispanning proteins are

Membrane Insertion of Small Proteins

19

periplasm ~

.......... . .... ' .......

.

"

9

.......

~..

/

f

,

I

C N-terminal translocation

C

I

L

_

~

N C-terminal trarmlocat.lon

Figure 1. Amino- versus carboxylterminal membrane insertion of a bitopic membrane protein.

thought to insert as pairs of hydrophobic stretches synergistically forming single loops as initial insertion structures. The most simple case for such a synergistic insertion mode is the M 13 procoat protein, which forms a loop structure during membrane insertion (30). There are a few single membrane-spanning proteins in bacteria known to translocare their N-terminal region directly to the trans side. The most simple case is the coat protein of the Pseudomonas aeruginosa phage Pf3. When this protein of 44 amino acid residues is expressed in E. coli, the first N-terminal 18 hydrophilic residues are facing the periplasm (34). The N-terminal translocation of the Pf3 coat protein is limited to only simple protein regions. When the N-terminal region was replaced by one that contains 3 additional charged residues, membrane insertion was impaired (62). The same charged region, however, was translocated with an additional signal peptide derived from the coat protein of the bacteriophage M 13. Although the two phage proteins are functionally related, as they are both coat proteins of filamentous phages assembling in the bacterial plasma membrane, only the M 13 coat protein is synthesized with a cleavable signal sequence. Little is known regarding the translocation of aminoterminal regions since no involvement of the Sec components has been observed. It has been suggested that the N-terminal region of the Pf3 protein has an amphiphilic character and buries information to support an extracellular location (63). However, there is no direct evidence to support export by amphiphilic sequences in bacteria. Remarkably, when the 18-amino-acid long N-terminal region of the Pf3 coat protein is fused to the aminoterminus of leader peptidase, the Pf3 portion of the hybrid protein is clearly translocated to the periplasm (46). Since the region flanking the hydrophobic stretch C-terminally is highly charged, translocation of the C-terminal region is prevented in favor of the N-terminal. Consequently, Lee et al. (46) deleted the cluster of 7 charged residues and observed both species, either with the amino or

20

DOROTHEE KIEFERand ANDREAS KUHN

with the carboxy terminus on the trans side. The most likely molecular mechanism is the partitioning of the hydrophobic region into the bilayer, which results in a horizontal orientation to the membrane surface (Figure 1). Either hydrophilic region is then passively translocated in favor of an or-helical conformation of the transmembrane region then parallel to the membrane normal.

III. DETERMINATION OF MEMBRANE PROTEIN ORIENTATION A question arises when proposing a single membrane-spanning protein: Which of the two hydrophilic regions will be translocated across the membrane and which region will remain at the cis side? As predicted from the "positive-inside rule" (79, 81), the region bearing more positively charged residues remains at the cis side of the membrane. This was clearly demonstrated with a number of mutants of the leader peptidase leading to an inverted orientation (51, 80). Moreover, it has been shown for a number of proteins that the introduction of positively charged residues close to the hydrophobic region inhibits the translocation process (4, 45, 47, 92). These results might reflect a sensitivity of the Sec-components towards positively charged residues. However, the studies by von Heijne with the leader peptidase are confused by the fact that the insertion mode is altered from a Sec-dependent (wild-type leader peptidase) (17) to a Sec-independent mechanism (inverted leader peptidase) (6, 51, 80). Another reason for the influence of positive charges might be an electrophoretic contribution in the translocation process itself (22). There are, however, a number of exceptions to the "positive-inside rule." The subunit H of the photosynthetic reaction center of Rhodobacter capsulatus and R. sphaeroides has a string of 5 negatively charged residues flanking the hydrophobic core at the cis side of the membrane (1, 93). The same is true for the two small [l-subunits of the antenna complexes of R. capsulatus (75). Moreover, in the M13 coat protein, the reversion of the negatively charged periplasmic region to a 4-fold positively charged one does not affect the orientation, but does affect the velocity of the translocation process (35). These results question simple models proposing, for example, that the electrochemical potential orients horizontally inserted proteins or that charge interactions between the membrane surface and hydrophilic protein regions are the only factors that determine the orientation of the helix. The question of orientation of an inserting polypeptide has also been extensively studied in the eukaryotic system. As mentioned above, single-spanning (bitopic) membrane proteins can achieve two orientations, type H proteins leaving the aminoterminus at the cytoplasmic side of the membrane, while type I and type HI proteins translocate the aminoterminus to the luminal side. Type I proteins achieve their topology indirectly by a cleavable signal sequence creating a new aminoterminus. Because in this case the orientation is mainly directed by the signal peptide, only membrane inserting proteins with a single hydrophobic region (signal anchor)


21

are considered here. From a number of studies on different bitopic membrane proteins, it became clear that the properties of the regions flanking the hydrophobic core of the signal-anchor sequence are crucial (24, 41). Most of the work was done with type II bitopic membrane proteins, and it was shown that positively charged amino acid residues preceding the signal-anchor were important for the orientation of the transmembrane segment. For the asialoglycoprotein receptor the replacement of these N-terminal positive charges by negative ones resulted in an inverted orientation (9). Similarly, the orientation of paramyxovirus hemagglutinin-neuraminidase (HN) was inverted 75% by the introduction of charged residues in the flanking sides (54). Lamb and coworkers also showed that the orientation of the type III membrane protein M2 of the influenza A virus is not altered when the hydrophobic membrane-spanning region is replaced by one of the SH proteins of simian virus 5, a type II membrane protein (55). This clearly suggests that the orientation is not determined by the membrane anchor, but by the regions flanking the anchor. However, in further studies on P450, Interleukin-2 and the Na-K-ATPase, it was demonstrated that the hydrophobicity and length of the transmembrane segment can influence membrane orientation (42, 58, 64, 65). Conclusively, these results show that membrane orientation and functionality of topogenic signals are determined by a balance between the length of the hydrophobic domain and the charge distribution of its flanking regions. It should be mentioned that there are also a few eukaryotic proteins that do not assume the predicted "positive-inside" orientation, such as some mutants of a preprolactin and IgM fusion protein (7) or the adenovirus E3 14.5-kDa protein (29). Similar to prokaryotes, a simple explanation (e.g., an electrostatic effect between the inserting protein and the membrane surface) seems, therefore, not to be satisfactory. It has been suggested that the positive charges at either side of the hydrophobic region may interfere with components of the translocation channel (70). Eukaryotic proteins might need additional features to determine their correct transmembrane orientation. The components involved in this process might include SRP, ribosomes, GTP, and membrane bound transport components since most membrane proteins require these factors as do secreted proteins (26).

IV. TRANSLOCASE-INDEPENDENT TRANSLOCATION The translocation of small and simple protein regions (N-terminal or C-terminal to the transmembrane domain) might occur spontaneously, presumably driven by conformational changes generated by the transition from a hydrophilic to a hydrophobic environment (84). In E. coil, several small proteins have been investigated that insert into the membrane in the absence of a functional transiocase. In the Pf3 coat protein, the N-terminal 18 residues, of which two are negatively charged, are localized in the periplasm (34, 62). The translocation of this Pf3 region occurs without the help of the Sec proteins and does not always require the membrane

22


potential (46, 62). This is also observed very clearly in a Pf'3 leader peptidase fusion protein, since the Sec- and potential dependent translocation of the leader peptidase moiety at the C-terminus serves as an intramolecular control. The Sec-independent and potential-independent translocation of the Pf3 region, representing the N-terminus of the fusion protein, was blocked by the addition of two charged residues at positions 17 and 18 (46), whereas an extension at the N-terminus by 20 uncharged residues was not inhibitory (13b). The possession of a signal sequence does not necessarily determine the involvement of Sec proteins in the membrane insertion process, although all current models of the translocase mechanism predict a central role for the signal sequence, particularly in the early stages. Membrane translocation of M 13 procoat protein does not depend on the presence of SecB (36), nor on functional SecA or SecY (90), even though it has a signal sequence. In addition, no basic differences were found between a signal sequence of a Sec-independent and a Sec-dependent protein because both were interchangeable (33). Rather, the complexity of that part of the protein that is transported across a membrane calls for a support by the Sec components. It remains unclear, however, how the Sec components recognize their substrates and which regions actually interact. Two proteins have been extensively studied to decipher the Sec-requiring elements, the M 13 procoat protein and the inverted leader peptidase. Extension of the periplasmic loop of M 13 procoat by 98 amino acid residues derived from the mature region of the OmpA protein causes a See-requirement for membrane insertion (31). Similarly, the extension of the periplasmic loop of the inverted leader peptidase from 25 amino acid residues to 54 residues made membrane insertion dependent on SecA and SecY (80). For both proteins a study of a series of loop regions of different lengths showed that intermediate lengths resulted in a loss of translocation efficiency (.5). Presumably, protein segments of about 40 residues are already too complex for spontaneous insertion and too small for efficient interaction with the Sec components. Both hydrophobic regions are required in a synergistic way for the Sec-independent translocation of M 13 procoat. Substitution of valine 30 for an arginine in the center of the mature hydrophobic region results in a membrane-associated, but not translocated, species (32). The same substitution did not inhibit translocation when introduced into the Sec-dependent M 13 derivative with an extended periplasmic loop (13a), suggesting that the synergistic insertion mode requires a close proximity to the two hydrophobic regions. There are only a few examples of other small bitopic membrane proteins in bacteria other than the Pf3 and M 13 coat. Recently, some work has been done with the light-harvesting pigment-binding polypeptides of phototrophic purple bacteria. The o~- and the 13-subunits of the light-harvesting complex I of Rhodobacter capsulatus are 59 and 48 amino acid residues in length, respectively (73, 74). Their central domains of about 20 hydrophobic amino acid residues are proposed to form an 0~-helix (12). The N-termini of both subunits are located in the cytoplasm, whereas the C-termini are exposed to the periplasmic space (75). In accordance


23

with the "positive-insideomle," (81) the N-terminus of the (x-subunit is positively charged as it is known for various photosynthetic bacteria (99). However, the I~-subunit of R. capsulatus has multiple negatively charged residues at its N-terminus, although located at the cis side of the membrane. It was suggested that the opposite charges of the N-termini of the two subunits may stabilize the formation of the oligomeric complex. Recent work by DOrge et al. (21) with simultaneously exchanged N-terminal charges of the a- and [3-subunits, however, resulted in a destabilization of the antenna complex. Interestingly, the formation of the complex was strongly impaired in mutants with charge exchanges very close to the hydrophobic core; mutations in more distal positions had little or no effect on complex assembly as revealed by the respective absorption spectra. In a mutant with an (x-subunit carrying a negatively charged N-terminus no assembly of the light-harvesting complex was observed, whereas the mutation of the [3-subunit to a positively charged N-terminus led to a functional antenna, although with a decreased stability (72). In a more detailed analysis, it was shown that the subunits do not insert into the membrane independently of one another. The qx-subunit is absent in mutant membranes lacking the I~-protein, whereas the lS-subunit inserts independently of the a-protein (60). Remarkably, in a heterologous in vitro system, the oc-subunit inserts into E. coil membranes or mutant R. capsulatus membranes efficiently only if it was cosynthesized with the [3-subunit (Kiefer, unpublished results). Some highly conserved amino acid residues such as Trp8 or Pro 13 in the hydrophobic region of the o~-subunit seem to be important for a correct membrane assembly of the complex (8, 61). From the work with the light-harvesting proteins, it is obvious that one has to account for the way that the different subunits of a heterooligomeric membrane complex may insert when compared with the single polypeptide chains. It may well be that subunits of membrane-located oligomeric protein complexes insert in a synergistic manner, as proposed for monomeric multispanning integral proteins. The involvement of a translocase in these processes is yet not well characterized. Almost nothing is known about a protein export machinery in Rhodobacter, but it was suggested that cytosolic as well as membrane-bound transport components exist (77, 89). Recently, a signal peptidase has been partially characterized in this organism with similar specificities as the E. coil enzyme (88), but there is no evidence for components functionally homologous to the Sec proteins in E. coil (89). For the heat shock proteins GroEL and GroES, however, which are known to be involved in the export of l~-lactamase in E. coil, homologs in Rhodobacter sphaeroides have been characterized (76).

V. INSERTION OF SMALL PROTEINS INTO THE

ENDOPLASMIC RETICULUM

Protein transport into the endoplasmic reticulum (ER) of eukaryotic cells is in many respects similar to the mechanisms of the prokaryotic process (Figure 2). There also


24

ER-lumen

periplasm

il

J

| o

T

4.

4,

4.

'~

,IF

1 2

3

4

5

6

prokaryotlc translocatlon pathways

SRP

eukaryotictransloeatlon pathways

Figure 2. Comparative models of prokaryotic and eukaryotic insertion pathways. The prokaryotic soluble components are (B) SecB and GroE, the membrane-bound components are (A) SecA, (G) SecG, (E) SecE, (Y) SecY, (D) Sec D and F (SecF). The postulated pathways describe insertion mechanisms of (1) SecB-dependent proteins e.g. proOmpA, preMBP or preLamB; (2) SecB-independent proteins (e.g., leader peptidase or preRBP); (3) prel3-1actamasewhich uses GroE instead of SecB as a cis acting chaperone; and (4) translocase- and Sec-independent proteins M13 procoat, Pf3 coat or inverted leader peptidase. The components of the eukaryotic translocation machinery are the (SRP)signal recognition particle, (DP) docking protein, (Sec61) the Sec61 oc~7complex, (TRAM) translocating chain-associating membrane protein, (nO NEM-sensitive factor, (a0 azido-ATP-sensitive factor, (RR) ribosome receptor, (HspT0) and (Hspg0) heat shock proteins, and (BiP) the trans-acting chaperone. The postulated pathways describe the insertion mechanisms of (5) SRP-independent proteins (e.g., preprocecropin A or M13 procoat) and (6) SRP-dependent proteins (e.g., preproodactor or preprolactin). The TRAM protein is only required for the translocation of a subset of proteins and its role is not fully understood (22a). The E. coli leader peptidases and the ER signal peptidase are not depicted because they do not participate in the actual insertion or translocation process. exists a proteinaceous machinery consisting of soluble cytoplasmic (39, 52), as well as membrane-located components (22a, 25, 28, 65a). As is the case for the exported proteins in bacteria, the vast majority of ER-proteins carries a cleavable signal sequence that is recognized cotranslationally by the SRP transported to the cis side of the membrane where the precursor-SRP complex is recognized by the above mentioned membrane-bound components. During translocation across the membrane, the signal sequence is cleaved off by the signal peptidase, which in eukaryotes is a multimeric enzyme complex (18, 49). Interestingly, some ERoproteins are translocated in the absence of the central components of the export machinery. Usually these SRP-independent proteins

Membrane Imertion of Small Proteins

25

have, similar to the situation in prokaryotes, simple secondary structures and are small in size. Most of them are only 50 to 70 amino acid residues in length, are synthesized with a cleavable signal sequence, and usually are secreted from the cell-like the frog skin peptide GLa (66) or some insect venoms, discussed in detail in the following section. To our knowledge there exists only one exception of a non-secreted protein, the rat liver cytochrome bs. It is a 16-kDa integral protein of the ER-membrane and is synthesized without a cleavable signal sequence. Its membrane insertion does not depend on the presence of SRP (3). The viral M2 protein with 97 amino acid residues is an integral membrane protein in infected cells and does not contain a cleavable signal sequence. In contrast to the cytochrome, however, its membrane insertion requires SRP. It was shown that deletion of two residues in the hydrophobic domain strongly impaired translocation, while deletion of six residues completely abolished membrane insertion. As M2 requires the signal recognition particle, it was suggested that the deletion mutants were not recognized by SRP (27). One of the best studied small ER-proteins is melittin, the main component of the honeybee venom. It is synthesized as a precursor molecule (prepromelittin) of 70 amino acids with a cleavable signal sequence of 21 residues. The cleaved form is secreted as the inactive promelittin, which is subsequently processed to the mature toxin consisting of the carboxyterminai 26 amino acids (78), of which the basic C-terminus plays an important role in membrane binding allowing lysis to occur (53). Translocation of prepromelittin into dog pancreas microsomes does not require SRP or the docking protein and can also occur posttranslationally (50). Accordingly, if expressed in a prokaryotic in vitro system, prepromelittin is translocated into E. coil inner membrane vesicles in a SecA/SecY-independent fashion, as has been observed for M13 procoat in vivo (15, 31). However, for the mammalian system, treatment of the microsomes with N-ethylmaleimide (NEM) and trypsin, showed that a membrane-located proteinaceous component other than the docking protein, or any known membrane protein, is involved in prepromelittin and preprocecropin A sequestration (95, 97). Preprocecropin A is a small antibacterial peptide from the moth Hyalophora cecropia with 64 amino acid residues including the cleavable signal peptide of 26 amino acid residues (10, 28). Klappa et al. (28) discovered a novel microsomal component dependent on ATP hydrolysis that is required for membrane transport of preprocecropin A. This was shown by photoaffinity inactivation of the microsomal component and, consequently, the inhibition of precursor processing by azido-ATP. Interestingly, this membrane-bound transport component is also required for the translocation of SRP-dependent proteins such as preprolactin or yeast preproafactor (ppa) (28, 98). A striking feature of some very small secreted proteins in eukaryotes is that they are synthesized and translocated into the ER as longer precursors containing several copies of the same polypeptide. The proteins achieve their functional size by proteolytic processing. The precursor proteins are either a multiplication of a small peptide, as in the case of pp~ factor in yeast (40), or contain spacer sequences connecting the mature

26


peptides, as in the case of the thyrotropin releasing hormone in Xenopus (59). It is conceivable that very small peptides are not able to translocate through a lipid bilayer spontaneously because electrostatic and hydrophobic interactions are too weak. On the other hand, the interaction of the translocation machinery probably requires a minimal length of the secreted protein.

Vl. REQUIREMENTOF CHAPERONES The involvement of cis-acting chaperones in membrane transport has been established in different prokaryotic and eukaryotic systems. In E. coli, the best characterized systems are the GroE and the SecB chaperonins. The GroE complex consists of a 14mer of a 57-kDa protein (GroEL) and a heptarner of a 10-kDa protein (GroES). The complex appears as two stacked rings where the bound substrate is located as a molten globule in a central cavity of GroEL (!1, 48). In addition to the binding of GroE to a number of cytoplasmic proteins (71, 9d), it has been shown that GroE is essential for the efficient export of the periplasmic protein ]3-1actamase by maintaining the precursor in a loosely folded, transport-competent conformation (38, 43). A transport-competent folding of certain exported proteins is also catalyzed by the interaction with the SecB protein, which in contrast to GroE is not a heat shock protein (2). It has been shown that SecB forms a tetramer consisting of four identical subunits of 16 kDa (83). The majority of exported proteins in E. coil needs SecB for efficient translocation, although it is not essential for viability (37). SecB binds to the mature moiety of precursor proteins, probably by hydrophilic as well as hydrophobic interactions (16, '37), but it is not known what features specify a protein SecB-dependency or dependency on other chaperones. There are a number of heat shock proteins with chaperone activity, such as GroE, DnaK, DnaJ, and GrpE, that might interact with the newly synthesized proteins in a complex, cascade-like manner (23, 44). An open question is how the different chaperone proteins recognize their substrates and how they are passed on in the pathway. Nothing is known about the interaction of chaperones with small, Sec-independent membrane proteins. It might be that the small proteins like M 13 procoat bypass the stabilization by chaperones and insert into the membrane spontaneously. Alternatively, other translocation factors might be used, such as Ffh, a component of a ribonucleoparticle in E. coll. Strikingly, this protein is required for the efficient transport of the SecB-independent pre[l-lactamase and preRBP (56) and it was recently suggested that the SRP-like Ffh-ribonucleoparticle uses the FtsY protein as a receptor, a homologue of the eukaryotic docking protein (48a). Members of the heat shock protein 70 group are also required for efficient protein translocation in the ER from mammalian as well as from yeast cells (14, 19, 20, 39, 86). Strikingly, the transport of small, SRP-independent precursor proteins into mammalian microsomes is dependent on the presence of cytosolic Hsc?0 and ATP, as well as at least one additional NEM-sensitive soluble factor (85, 96). Unlike


27

preprocecropin A, the membrane transport of M 13 procoat does not require the azido-ATP sensitive membrane component described by Zimmennann and coworkers (28). In a heterologous in vitro system involving bacterial cellular extracts and dog pancreas microsomes, M 13 procoat is only processed to its mature form when the mixture has been supplemented with rabbit reticulocyte lysate or purified Hsc70 protein (87). The presence of the Hsc70 protein seems to be irrevocable for the transport process, since it could not be substituted by other chaperone-like GroE, Hsp90, Hsp60, BiP or DnaK in Mammalia (87) or BiP in yeast (13). This is in contrast to results with a prokaryotic in vitro system involving E. coli membrane vesicles, where M 13 procoat is translocated independently of the Sec components. In this system Hsc70 has no stimulating effect on membrane insertion. Hsp90 and GroE can bind M 13 procoat as shown by competition experiments, but obviously are not capable in mediating export competence of the precursor. Similarly, SecB does not support membrane insertion of M 13 procoat in E. coil and is incapable of binding (Kuhn et al., unpublished results). Addressing the problem of defining the features of SRP-dependence and -independence, respectively, it is first of all obvious that SRP-independent proteins are usually small in size. As shown by Siegel and Walter (69), there exists a critical size of a polypeptide (approximately 140 amino acid residues) that is necessary to interact with the SRP. The SRP cannot bind smaller precursor proteins probably because they are buried in the ribosome until their synthesis is completed. Thus, these short proteins have to take an alternate (-posttranslational-) pathway for membrane insertion not yet well characterized. It was shown that prepromelittin translocates into E. coil inner membrane vesicles in a SecA and SecY independent manner, whereas a longer derivative fused to dihydrofolate reductase (DHFR) needed these two components for export (15). Schlenstedt et al. (68) could demonstrate that preprocecropin A derivatives of up to 85 residues retained their SRP-independence, but longer hybrid proteins between preprocecropin A and DHFR (252 amino acid residues) were translocated in a cotranslational, SRP-dependent manner into dog pancreas microsomes. However, this construct could be translocated in a posttranslationai, SRP-independent way in the presence of the folate analogue methotrexate (67). To explain this, the authors propose that the substrate analogue leads to a looser, export-competent conformation of the fusion protein as indicated by its protease sensitivity. One could hypothesize that in an alternative export pathway, the chaperoning effect of SRP via a translational delay of precursor proteins (82, 91) can be substituted by other cytosolic chaperone such as the above mentioned Hsc70.

VII. CONCLUSIONS Spontaneous insertion of proteins into membranes seems to be limited to simple and short proteins. It involves a short stretch of hydrophobic amino acids that is

flanked by two hydrophilic regions. These two flanking regions are involved in orienting the protein in the membrane. Translocase-independent insertion has been observed in E. coli for small proteins and leads to the translocation of either the aminoterminal or the carboxyltenninal flanking region. So far, translocase-independent protein insertion has not been described in eukaryotes, although striking homologies between the eukaryotic and prokaryotic translocas systems exit (20a, 25a).

During evolution a number of different systems were developed that enable more complex proteins to transloeate across the membrane. Translocation machineries support the carboxylterminal translocation process of long polypeptide chains. To date, however, it is unknown whether a transloease enzyme complex can also support aminoterminal transloeation. To allow a topology with an extracellular location of the aminoterminus, cleavable signal sequences were added in front of the mature protein sequence. In the cytoplasm sophisticated chaperone systems assure a loose folding of the polypeptide chain that keeps the protein in an insertion-competent condition. And finally, chaperones in the periplasm control the folding of the secreted proteins and, most likely, membrane proteins as well.

ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (Ku 749/1-1 and Ku749/1-2).

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PROTEIN TRANSLOCATION GENETICS

Koreaki Ito

I. ll. Ill.

IV.

V.

VI.

Vll.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of Escherichia Coil Genes Involved in Protein Export . . . . . . Cytoplasmic Factors, Precursor Targeting, and Anti-Folding . . . . . . . . . . A. SecB and Other Chaperones . . . . . . . . . . . . . . . . . . . . . . . . B. SRP Homologs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SecA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane-Embedded Components of the Translocator . . . . . . . . . . . . . A. SecY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. SecE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. SecD and SecF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recognition of Signal Peptide and Early Part of Mature Sequence by the Translocator . . . . . . . . . . . . . . . . . . . . . . A. The prlA Mutations in SecY . . . . . . . . . . . . . . . . . . . . . . . . B. prig and Suppressor-Directed Inactivation . . . . . . . . . . . . . . . . . Sec Factor Interaction and lntramembrane Translocation Pathway . . . . . . . A. Intramembrane Steps of Translocation . . . . . . . . . . . . . . . . . . . B. Bieker-Silhavy Model of Translocation Complexes . . . . . . . . . . . . C. Insights into the SecY-SecE Interaction . . . . . . . . . . . . . . . . . . .

Advances in Cell and Molecular Biology of Membranes and Organdies Volume 4, pages 35-60. Copyright 9 1995 by JM ~ Inc. All rights of reproduction in any form reserved. ISBN: I-$$938-924-9 35

36 36 38 38 39 40

41 41 43 44 44 44 45 47 47 48 49

KOREAKI ITO

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VIII. AdditionalFactors Possibly Involved in Translocation . . . . . . . . . . . . . . A. Band I andPl2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B. C. D. E.

PspA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Skp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ydr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Orfl2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E FtsH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Are There Translocation Factors in the Periplasm? . . . . . . . . . . . . . IX. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51 51 51 51 51 52 52 52 53 53 54

I. INTRODUCTION Biological membranes are restrictive to the passage of small and macromolecules and, hence, maintain the integrity of the cell as well as its intracellular compartments. At the same time, membranes are surprisingly dynamic, asymmetrically interfacing the communication between aqueous milieus on both sides. A class of proteins that must localize out of the cytoplasm (the compartment fed with the genetic message) experience, once in their lives, a critical process of translocation across the membrane. While extracytoplasmic proteins of prokaryotic cells and eukaryotic cells cross different membranes (the plasma [cytoplasmic] membrane vs the endoplasmic reticulum membrane), their mechanisms of translocation appear to be well conserved. The conservation includes the exchangeability of substrate secretory proteins and their signal sequences (1) as well as the occurrence of the SecY/SEC61p family of integral membrane "translocator" components (2). In addition, at least some elements of the signal recognition particle exist and seem to function in bacterial cells. Thus, elucidation of the translocation mechanisms in any system will positively contribute to the understanding of similar processes in other organisms. This review is intended to update the progress in studies of the protein translocation system in E. coli, with emphasis placed on genetic analyses of the translocation machinery. More general accounts of the secretory pathway in gramnegative bacteria have recently been reviewed (3).

II. IDENTIFICATION OF ESCHERICHIA COLI GENES INVOLVED IN PROTEIN EXPORT Because the selection and screening systems that have been successfully designed for obtaining mutants with altered activities of protein export have been reviewed extensively (4), only outlines of these approaches will be summarized. Fusion proteins between the N-terminal regions of exported proteins, such as MalE, LamB, and the cytoplasmic ~-galactosidase (LacZ), play central roles in the

Protein Translocation Genetics

37

genetics of protein export. Such fusion proteins can initiate translocation but cannot complete it because of the presence of the translocation-incompatible sequence or conformation within the LacZ part (5); they jam up the translocation machinery when induced by maltose, rendering the cell carrying them maltose-sensitive. ~l-galactosidase activity of the fusion proteins is low, presumably because the membrane localization does not effectively allow the formation of the active tetramer structure. The maltose-sensitive phenotype provided conditions for the initial isolation of the signal sequence mutations that conferred maltose-resistance (4). The Lac- phenotype allowed the isolation of not only the signal sequence mutations on the fusion gene, but also the secA and secB mutations that conferred lactose utilizing ability. Similar Lac+ selection using the phoA-lacZ fusion allowed the isolation of the secD and secF mutations (4). More recently, a Lac + selection using a malF-lacZ fusion, in which PhoA was attached to a periplasmic domain of the MaIF membrane protein, allowed the isolation of mutations in the dsbA and dsbB genes, whose products are involved in disulfide bond formation of proteins after export (6, 7). A clear and simple indication of an export defect is the accumulation of precursor proteins with unprocessed signal peptide. Direct screening for precursor-accumulating mutants, without involving the bias imposed by a particular selection, yielded mutations in the secY gene (8--10). Another "brute force" screening for plasmids overexpressing the leader (signal) peptidase activity allowed the identification of the lep gene (11); (note that this enzyme [12] will not be discussed further because its activity is not essential for the protein translocation reaction itself [13]). The most powerful method of isolating export-impaired mutants is to use the peculiar regulatory property of the secA gene whose level of expression elevates in response to an export defect (other than that caused by a defect in cytoplasmic factors such as SecB). Using cells carrying the secA-lacZ gene fusion, mutants with increased expression of the fusion were selected or screened for by virtue of their increased ~-galactosidase activity (14). Mutants of the secE, sec u secDlsecF, and secA genes were thus isolated. The wide spectrum of the sec mutations isolated by the secA-lacZ approach raised the possibility that the "sec" genes had been exhaustively identified in E. coil (4, 15). It should be noted, however, that the secA-lacZ method does not unravel defects in the cytoplasmic events, and the earlier Lac § method did not yield a secY mutant. It seems safe to say that each selection method contains inherent bias and that not all the genes for protein export have been discovered. Although secB is dispensable (at least in synthetic media), other sec gene functions appear essential for cell viability (as has conclusively been shown for secE; [16]), since their mutational alterations can give conditionally lethal phenotypes. Strikingly, most conditional mutations are cold-sensitive (14). Temperaturesensitive export mutations are only known for secA (17) and secY (9, I 0). Pogliano and Beckwith (18) proposed that the E. coil export pathway includes some intdn-

38

KOREAKIITO

sicaUy cold-sensitive steps, and lowered activity at a step following the cold-sensitive step invariably leads to the cold-sensitive export phenotypes. Another successful approach to identifying export components was the prl selection in which mutations that suppress export defects caused by signal sequence mutations were selected (19). The prlA, prlD, and prig mutations that were isolated are alleles of secY, secA and secE, respectively (19-21). A more specific genetic strategy for dissecting the translocation mechanisms and additional components that possibly participate in the system will be discussed in the following sections.

III. CYTOPLASMIC FACTORS, PRECURSOR TARGETING, AND ANTI-FOLDING A. SecBand Other Chaperones Protein export in E. coil occurs both posttranslationally and before polypeptide chain completion. The latter mode of translocation could be called cotranslational but it occurs after a substantial proportion (some 80%) has been synthesized; the polypeptide chain does not move through the membrane residue-by-residue as it elongates (22). The cellular number of the translocation machinery in the membrane (about 500 per cell) (23) appears to be too small to account for the strictly cotranslational biogenesis of cell envelope proteins. In other words, such a small number of translocators should translocate envelope proteins in periods shorter than those required for their translation (23). Tightly folded precursor or its segments cannot effectively enter or complete the translocation pathway (24, 25). Thus, E. coil cells possess cytoplasmic factors that prevent premature folding of secretory precursors (26). In the secB mutant cells, a subset of envelope proteins (OmpA, MalE, PhoE, LamB, and OmpF) are retarded in their export. The secB mutational effects are epistatic or additive to phenotypes of many other export mutations (27, 28), placing its action point early in the export pathway. Purified SecB protein binds to MalE and other precursor proteins, after their in vitro synthesis or denaturation, and keep them competent for subsequent translocation across inverted membrane vesicles (29). It also retards refolding of urea-denatured proteins (30). In vivo, mutations in the mature part of exported proteins that destabilize the protein conformation exhibit decreased dependence on secB (31). These observations established the notion that SecB is an anti-folding factor and important for the posttranslational mode oftranslocation. However, SecB is, in fact, cotranslationally associated with incomplete nascent chains of some exported proteins and found in the polysomal fraction (32). Biochemical studies indicate that SecB binds rather indiscriminately to unfolded proteins, which can be in kinetic competition between SecB-binding and folding (30). Closer examinations revealed that SecB, under low ionic strength, has some specificity to polypeptides enriched in positively charged residues, whereas hydro-


39

phobic interactions are probably more important at physiological ionic conditions (33). Since signal peptides retard the folding of the mature part (34), the apparent specificity of SecB for signal sequences (35) could be secondary. The "translocation-competent" states of precursors could have substantial tertiary structure (36) or could be more unfolded (30, 33), depending upon the precursors examined. Nascent chains bound to SecB in vivo are mainly secretory proteins such as MalE, LamB, OmpE and OmpA (32). Signal sequence exchange experiments between MalE (secB-dependent) and ribose-binding protein (secB-independent) showed that the mature portions are solely responsible for their SecB dependence/independence (37). However, such discrimination is not absolute because ribose-binding protein becomes partially secB-dependent when its export efficiency is lowered

(28). The fact that the secB nul mutation (secB::Tn5) does not confer the Lac § phenotype to MalE-LacZ fusion-bearing cells (38) suggests that some other factors might substitute for the simple loss of function of SecB. In other words, the original secB missense mutations as well as those isolated recently have some dominant characters (38). The latter mutations have been shown to increase SecB's affinity for unfolded proteins (38). SecB's ability to release secretory precursors should be important for targeting the membrane-associated components of the export system; SecB is believed to have a targeting role in the cell (26, 35). Cell-free experiments showed that SecB can bind to SecA (39), the peripheral component of the translocator, although genetic evidence for the SecB-SecA interaction is still to be provided. The observation that a mutation in the secA locus that creates a duplication of most of the SecA sequence partially compensating for the loss of SecB function (40) is consistent with the notion that SecAreceives precursors from SecB. Cytoplasmic accumulation of secretory precursors under secB-deficient conditions induces heat shock proteins (41). Further induction of heat shock proteins, DnaK and DnaJ in particular, compensates partially for the loss of SecB, whereas the secB nul and the dnaK/dnaJ mutations are synthetically lethal (42, 43). Thus, the DnaK/DnaJ chaperone can substitute for the SecB function to a certain extent. Export of PhoA may naturally be dependent on DnaK/DnaJ (42), while it also depends on SecB at low temperature (44). In contrast, ~-lactamase (Bla) is totally independent of secB in vivo and in vitro (45). The export of Bla is retarded in the groEL or groES Ts mutants (44), as well as under the conditions of GroE depletion (46). Taken together with the fact that Bla precursor is a preferred substrate of GroEL (47), it is indicated that Bla utilizes this chaperone as the cytoplasmic escort factor.

B. SRP Homologs Prokaryotic cells possess homologs of some components of the signal recognition particle that mediates cotranslational attachment of the translocation complex containing nascent secretory precursors to the endoplasmir reticulum membrane.

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KOREAKI ITO

The 4.5 S RNA of E. coli (thefts gene product) has a sequence homology to the 7 SL RNA (48) of SRP, and the./~ gene product is homologous to an SRP subunit, SRP54 (49). Also, the ftsY gene product is homologous to the SRP receptor r subunit (50). The C-terminal "methionine-rich" domain of SRP54 may interact with a signal peptide while its N-terminal "GTPase" domain engages in the SRP-SRP receptor interaction (2). While evidence indicates that the 4.5 S RNA is involved in general protein synthesis (51), it also associates with the Ffh protein in a ribonucleoprotein complex that interacts with the signal sequence part of nascent preprolactin in a heterologous cell free system (52). Depletion or overexpression offfs causes retardation of Bla export but not of other exported proteins (53, 54). Depletion of Ffh causes growth arrest and export retardation of not only Bla, which is most severely blocked, but also OmpA, MalE, and PhoA (55). The Ffh protein can substitute for SRP54 in a mixed reconstitution with the eukaryotic SRP components with respect to the particle formation and the translation arrest activities (56). However, the chimeric particle cannot interact with the eukaryotic SRP receptor and cannot facilitate translocation activity. These results suggest that the SRP homologs in E. coli have a role in preprotein export, possibly in the targeting process. It is interesting to note that, while the eukaryotic SRP is believed to act in the cotranslational pathway of translocation, Bla that is most clearly affected by the ffs.ffh mutations shows the slowest (posttranslational) export kinetics in E. coli (57). The relationship between the SRP pathway and the Sec pathway is not clear at present. A similar situation also holds for protein translocation in Saccharomyces cerevisiae. In these cases, different proteins show different dependence on the two systems (58). The SRP pathway may not be unique in its timing of action but may be unique in that it recognizes the signal peptide segment of the precursor protein for targeting to the membrane. Thus, it could be complementary to the chaperone pathway that recognizes the mature part.

IV. SECA SecA has recently been reviewed by Oliver (59) and detailed discussion on its biochemical properties has been provided. SecA does not contain any segment that is sufficiently hydrophobic to traverse the membrane, but a fraction of it is associated with the membrane. While its membrane association under certain conditions is strong enough to be regarded as "integral" (60-62), a major fraction of SecA is in the cytoplasm or only loosely bound to the membrane (60). SecA is essential for the in vitro translocation reaction (63). It possesses an ATPase activity (translocation ATPase) that is activated by anionic phospholipids as well as vesicles containing functional SecY/SecE proteins (64). SecA translocation ATPase is inhibited by azide. The identification of the azide-resistance gene with secA indicates that secA is the primary target of low azide concentration in E. coli (65).


41

SecA binds to precursor molecules probably by recognizing both the N-terminal basic region and the hydrophobic core of the signal peptide as well as the mature part (64, 66, 67). Genetically, the occurrence of prlD mutations is consistent with SecA's ability to recognize the signal sequence. Biochemical results and "intracistronic" complementation suggest that SecA acts as a dimer (60, 68). ATP- and preprotein-binding sites are located at the N-terminal region (69, 70). Model experiments in vitro show that binding of ATP to SecA stimulates the SecA-preprotein binding (71), allowing subsequent penetration of some 20 residues of the preprotein (72). ATP hydrolysis then allows release of the preprotein from SecA. Translocation of the preprotein can be continued by repeating the cycles of SecA-binding, translocation of the limited segments, and release. If proton motive force is imposed, more efficient translocation occurs upon the ATP hydrolysis-dependent discharge of precursor from SecA (72, 73). There is a likely possibility that SecA itself penetrates into the membrane along with the preprotein (60-62, 74), although in the model in vitro system ATP (presumably its hydrolysis) weakens the membrane binding of SecA (60, 62). Biochemical results suggest that SecA binding sites on the membrane include SecY or its vicinity (39). Genetic evidence, such as specific suppression, remains to be provided in support of the SecA interaction with SecY (75). SecA (of B. subtilis) with a mutation in the ATP-binding site sequence is inactive or somewhat inhibitory and binds strongly to the membrane (76, 77). The SecA52 mutant protein also partitions mostly to the membrane (60). The ATPase mutant is defective in release of bound preprotein and hence in the proton motive force-dependent transiocation (77). Normally, SecA should undergo dynamic transitions among different conformations, localizations, and degrees of membrane penetration. ATP-induced conformational changes should be central to this problem. Mutational "freezing" of SecA into one state is destructive. The existence of different isoforms of SecA provides the cell with a means to monitor its export states and regulate the synthesis of SecA itself. Thus, the cytoplasmic form of SecA can bind to the secA mRNA and presumably shut down its own synthesis (78).

V. MEMBRANE-EMBEDDED COMPONENTS OF THE

TRANSLOCATOR A. SecY

SecY (PrlA) is defined by the prlA suppressor mutations and export-defective, conditionally lethal secY mutations. It encodes the first identified integral membrane translocation factor among prokaryotes and eukaryotes (79, 80). SecY homologs are now identified in gram-positive bacteria (81), plastid (82), chloroplasts (83), yeast (the SEC61 gene product; 84), and mammalian cells (85). E. coil SecY contains 10 transmembrane segments, 6 cytoplasmic domains, and 5 perip-

Figure 1. Mutations in the secY gene. prl mutations suppress signal sequence mutations, while other mutations (except for s e c Y l 2 l and secY161) retard export; most of the latter are Cs, but secY24 and secYlOO are Ts. secY121 and secY161 are mutations that cannot support export of staphylokinase ( 103). Note that prlA4 and secYlOO cause more than one amino acid alteration.


43

lasmic regions (Figure 1; [86]). The estimated cellular concentration of SecY (and also of SecE) is about 300--500 molecules per cell (23). Mutations that impair SecY function include an amber mutation in rplO that lowers the SecY amount (8), and Ts and Cs missense mutations (9, 10, 14, 75, 87). Recently, we isolated 10 new export-defective secY alleles (Figure 1; [87]). Most of them are Cs and the one Ts mutant contained the same mutation in the cytoplasmic domain 4 (C4; see Figure 1) as the classical secY24 mutant (9). Many Cs mutations are in the cytoplasmic domains, C3, C5, and C6, but some can be within transmembrane segments, TM2 and TM9, as well as in the periplasmic domain 1 (P l). The CS-TM9 region is the preferred site of Cs export-defective mutations (Figure 1). The occurrence of export-defective mutations all over the SecY sequence contrasts to the secE case where defective mutations affect only the expression levels (16; see below). Because SecY probably functions in a complex with other components, its "dominant-negative" variants could be obtained. For instance, "active site" mutations can be dominant-negative if they do not abolish the SecY's ability to complex with other component(s); the mutant protein will sequester the interacting components in an inactive complex and hence will compete with wild-type SecY for the formation of the functional translocation complex. An internal deletion (AArg372Leu-Thr) of SecY (Figure 1; secY -a 1) is dominant negative when expressed from a multicopy plasmid (88). Of the secY Cs mutations, three mutations in C5 and one in C6 also dominantly interfere with export when cloned onto multicopy plasmids (87). Evidence suggests that these dominant effects are mainly due to sequestration of SecE (88, 89; see below). These results with the Cs and the dominant mutations suggest that the carboxy-terminal (C5, TM9 and C6) region will be important for the "catalytic activity" of SecY. As discussed in a later section, the central region of SecY is important for its interaction with other factors, such as SecE. B. SecE

The gene secE (priG) is defined by mutations that lower the expression level of the gene and retard protein export and growth at low temperature (15, 16), as well as by missense prig mutations (21). secE is essential for the viability of the E. coli cell (16). Reconstitution studies indicate that SecE and SecY are the principal membrane-embedded components for the in vitro translocation activity (90, 91). SecE contains three transmembrane segments, but the most C-terminal one (TM3) plus some surrounding sequences can function in vivo (16) and in vitro (92). Recently, a B. subtilis gene encoding a 59-amino acid peptide was shown to complement the E. coli secE mutant (93). The general feature of this peptide resembles that of TM3 and the preceding cytoplasmic region of SecE; the SecE counterparts in B. subtilis and B. licheniformis naturally contain only the equivalent of the C-terminal half of SecE (93).

44

KOREAKI ITO C. SecD and SecF

Also defined by Cs export.defective mutations (94), secD and secF constitute an operon and encode proteins containing both a large periplasmic domain and six transmembrane segments (95). No prl mutations are known for this pair of genes. The large periplasmic domains of these gene products are speculated to play a role in the late stage of translocation (95). Although purified SecD and SecF do not stimulate reconstitution of translocation activity as assayed by sequestration of precursor molecules into the proteoliposomes (23), experiments using semi-intact cells showed that antibodies against SecD inhibited precursor protein release to the aqueous milieu when added outside of the intact spheroplasts (96). Thus, complete translocation may require SecD (and SecF?). The role of SecD-F may be to facilitate either protein release from the membrane or post-translocational folding.

VI. RECOGNITION OF SIGNAL PEPTIDE AND EARLY PART OF MATURE SEQUENCE BY THE TRANSLOCATOR A. The prlA Mutations in SecY The existence of priG and prlA mutations suggests that the two integral membrane proteins, SecE and SecY, interact somehow with the signal peptide. Recent evidence indicates that the signal peptide and the following 30 or so residues of the mature part comprise a special domain called export initiation domain (97). The mature part of this domain was defined as the segment in which artificial placement of positively charged residues is not tolerated for efficient translocation. The prl mutations are not specific to the signal peptide since the translocation-incompatible existence of basic amino acids in the early mature part can be overcome by some prl mutations (98). Interestingly, one such mutation, prlA666, alters a residue of the P1 domain of SecY (Fig. 1). The same residue can be mutated to another prlA allele, prlA3. Other prlA mutations are located either in TM2, TM7, or TM 10 (Figure 1, 99, 100). The lack of clear allele specificity is sometimes interpreted to mean that the prl suppression does not reflect a change in specific protein-protein interaction (101). However, it must be remembered that the protein-protein interaction here is special in that it is an interaction between a protein factor and its "substrate." Such an interaction should be very dynamic. Signal sequences themselves are only conserved in their general features, such as hydrophobicity and charge balance flanking the hydrophobic core, but not in their specific sequences. Even an artificially designed sequence, MKQSTLIoA6, can function perfectly well as a signal sequence in vivo (102). If a common apparatus recognizes signal sequences of different exported proteins, the recognition cannot be sequence specific to the extent that the classical concept of allele specificity would predict. Flexible protein-protein inter-


45

actions could be a key feature in the action of protein translocation factors, which should deal with polypeptide segments of diverse sequences that are even moving. In reality, there is some specificity for the prlA suppression. Some prlA mutations preferentially suppress mutations of the hydrophobic core while others can suppress those reversing the charge balance (98). Of the Met to Arg changes at the 18th residue and the 19th residue of the MalE signal sequence, the prlAlO01 mutation at TM2 prefers the former, while many other prlA mutations prefer the latter (I 00). Some of the prlA mutations as well as other mutations in TM7 render the translocation system refractory to some heterologous proteins, such as staphylokinase (99, 103) and streptokinase (104). Overcoming mutations can then occur in the signal peptide region of these proteins (104, 105). Taken together, these observations suggest that P l, TM2, TM7, and TM 10 of SecY somehow recognize the signal peptide and the region immediately following the signal. Membrane insertion of this region should be a critical early phase event in translocation, in which the precursor polypeptide will assume a loop-like configuration (Figure 2). Such a configuration of the translocation initiation domain may explain the periplasmic localization of some prlA mutations. The Pl domain could be involved in acceptance/rejection of the early mature part (89; Figure 2). The existence of two cold-sensitive mutations (secY125 and secY124) and three prlA mutations (prlA3,prlA666,andprlAI OO1)in the P I-TM2 segment (Figure 1) points to the importance of this segment (100). It may be speculated that the P I-TM2, TM7, and TM l0 regions together recognize the early part of the precursor allowing its insertion. The TM3 region of SecE could also participate in this hypothetical channel (Figure 2). in addition, the CS-TM9 region of SecY, the importance of which has been suggested by the occurrence of a number of Cs and dominant-negative mutations, may additionally participate and confer mid- to late-phase translocation activities on the channel (Figure 2). The notion that theprlA mutations alter the SecY recognition of signal sequences has been questioned by the fact that some of the prlA mutations allow slow export of periplasmic proteins (PhoA and MalE) whose signal sequence had been completely eliminated. (106) However, these signalless proteins behaved anomalously in cell fractionation (106). The mature portion of these proteins appears to contain some elements that target them to the periphery of the prlA or the wild-type cells (106). It is possible that SecY recognizes such an element (in the mature portion of the translocation initiation domain?), and the prlA mutation enhances the acceptance of the signal-less precursor molecules for further translocation.

B. priG and Suppressor-Directed Inactivation Silhavy and his colleagues used a LamB-LacZ fusion protein with a mutation in the signal peptide of the LamB part to selectively inactivate the prl mutated component of translocation machinery (SDI or suppressor-directed inactivation; 107). They reported that the LamB-LacZ fusion protein with the LamB signal

KOREAKI ITO

46 K

i P1 ~~1"1 .:i M2-~ ~'M7 TM9

NH2 o4 I *

cs

Figure 2. A hypothetical pathway of preprotein entry and transit. On the basis of the existence and locations of the prl (stars) and export-defective (or interfering) sec (asterisks) mutations, transmembrane segments of SecE and SecY are supposed to provide a pathway for both an early phase of translocation in which the amino terminal part of the preprotein inserts, and a late phase in which the bulk of the mature part moves across the membrane. Not all the sec mutations (see Figure 1) are shown. Also, only the prlGl mutation is shown for SecE (16). The hatched region of the bold line represents the signal sequence part of the preprotein and the filled part represents the mature part. See the text for more detailed discussions. sequence mutation assumes a different location in the cell, depending upon whether it is present in a strain carrying theprlAd ortheprlG1 mutation. The signal sequence has been processed for the prlA-suppressed fusion protein, but not for the priG-suppressed one. Thus, it was proposed that the former (termed translocation complex) is in the transmembrane configuration, but the latter (termed pretranslocation complex) is only associated with the cytoplasmic surface of the membrane. This provided a basis for the proposal that SecE acts prior to SecY (or more exactly SecY-SecE complex; see Section VII.B). However, the location of theprlGl mutation close to the periplasmic side of TM3 of SecE (16) poses an apparent difficulty. As already pointed out, we consider the possibility that TM3 of SecE, along with some of the SecY TM segments (TM2, TM7 and TM I0), forms the entry pathway (Figure 2). Because the lack of signal sequence cleavage does not necessarily exclude a transmembrane (but leader


47

peptidase-inaccessible) configuration of the pr/G-suppressed fusion protein, establishment of its disposition by independent methods will be crucially important. Curiously, the fusion protein with wild-type signal peptide is subject to rapid cleavage of the signal (107). If the pretransloeation complex and the translocation complex indeed represent sequential events in translocation, why does not the "wild-type" fusion protein jam at the former step? Also, why does aprlA suppressor work without having a prig mutation (see 108 and 89, for further discussion)? The priG1 mutation actually exacerbates the processing of the LamB-LacZ fusion protein with mutated signal peptide (107). These peculiar properties of the prlG l mutation could have "mutation-specifically" contributed to the SDI properties. A more fundamental complication in the SDI analysis is that it relies on the prl-specific signal recognition on one hand and jamming by LacZ on the other. Simply stated, these are separate events only linked with some translocation process itself occurring between them. One cannot assume a priori that LacZ jams the very component that recognized the signal. Pogliano and Beckwith (18) proposed that the initial insertion of the signal peptide is intrinsically cold-sensitive and it subsequently interacts with the integral membrane components. The proposal that two kinds of molecules accumulate in different signal sequence mutants is interesting in their relationship to the pretranslocation and translocation complexes.

VII. SEC FACTOR INTERACTION AND INTRAMEMBRANE TRANSLOCATION PATHWAY A. Intramembrane Steps of Translocation In a model in vitro experiment, an engineered OmpA-dihydrofolate reductase (DHFR) fusion protein was forced to be stuck in the membrane because of the folded DHFR domain. This transmembrane intermediate could be photo-crosslinked with SecA and SecY, but not with phospholipids, via a modified cysteine residue introduced close to the DHFR moiety (67). This result is consistent with the notion that translocating polypeptides move through the proteinaceous translocator, but not directly through the hydrocarbon phase of the membrane, from the early through the late phases. If mutations in the translocation components cause conditional arrest in the intramembrane translocation reactions, similar transmembrane intermediates could be detected in vivo. Indeed, late phase intermediates of MalE can be detected in some secY and secA mutant cells (109) as well as under conditions of dissipated proton motive force across the membrane (110). They ("the processed immature forms") have already been processed for the signal peptide cleavage but remain associated with the membrane and unfolded. A similar intermediate of B la has been detected at low temperature (111).

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Pogliano and Beckwith (18) proposed that a number of Cs sec mutants are non-specifically defective in the translocation step and accumulate metabolically unstable transmembrane intermediates. However, taken together with the results of our screening of a number of Cs secF mutants (87), it should be stated that no mutation has been fully characterized that accumulates a transmembrane intermediate as a major product. The small number (about 500 per cell) of membraneembedded translocators (23) will limit the upper number of translocation intermediates that remain in direct contact with the translocator within the translocation pathway. Thus, one of the most interesting classes of mutations that primarily affects an intramembrane process might be difficult to distinguish from mutations affecting other processes; a translocation defect will rapidly back up the system and lead to the secondary accumulation of earlier step intermediates.

B. Bieker-SilhavyModel of Translocation Complexes Bieker and Silhavy (107) proposed that the pretranslocation complex and the translocation complex, as defined by the SDI analysis, have different compositions. The former contains SecA in addition to SecE (PriG), whereas the latter contains SecA, SecE, SecD, and SecF, in addition to SecY (PrlA). Thus, two different molecular assemblies sequentially participate in translocation, and SecE cycles between association with and dissociation from the other integral membrane components. This model is based on "Sec titration" experiments (107, 112). When the LamB-LacZ fusion protein with the signal sequence mutation is induced in the priG or prlA mutant cells, it inhibits general protein export and cell growth. Growth of partially diploid cells containing both the prl and the prF alleles is not affected. This result sounds reasonable, because the wild type (pr/+) gene product that cannot recognize the signal-mutated fusion protein should not be interfered with directly by the fusion protein. However, it is also inferred from the diploid analysis results that SDI at neither Prig (SecE) nor PrlA (SecY) functionally titrates out other Sex gene products that participate in the system. In other words, it seems either that the interaction is only transient (or nonexistent), or that the interacting components exist in excess. According to Bieker and Silhavy, the "Sec titration" experiments discriminate between the above possibilities. Thus, they combined a series of export-defective sec mutations with the prlGl/prlG + or the prlA4/prlA + partial diploid construction. Growth inhibition by the induced fusion protein was examined under "semi-permissive" conditions with lowered functional levels of the mutated Sec protein. The prIGl/prlG +diploid cell was not sensitized to the fusion protein by either secY, secD, or SecF mutations, indicating that SexY, SecD, and SecF are not stably contained in the "pretranslocation complex." In contrast, the prlA4/prlA § diploid cell became fusion protein-sensitive when it received the secE, secD, or secF mutation. Thus, the "translocation complex" does contain SecE, SecD, and SecF, but SecE, SeeD, and SecF might normally be present


49

in excess over SecY (112). Interactions between SecY and SecD, as well as between SecY and SecF, were also supported by our observations on the multicopy suppressing or exacerbating effects of SecD and SecF on the phenotypes of specific secY mutations (87). The revised experiments of Bicker and Silhavy showed that the secA mutant affected both of the SDI systems (112). Thus, they predicted that precursors first bind to the membrane via the SecA-SecE complex forming the pretranslocation complex to which SecY, SeeD, and SecF subsequently associate to form the translocation complex. Upon completion of translocation, the translocation complex is disassembled. Although the Bieker-Silhavy model provides a very important guide to studies of the intracellular pathway of protein translocation, it should not be accepted without critical examination. In addition to the points already raised about the use of the LacZ fusion protein as the secluding molecules and the disposition of the priG-suppressed fusion protein, the following questions about the See titration experiments are addressed. (i) Since the translocation capacity of the wild-type cell is not conclusively estimated, the apparent recessiveness of SDI does not necessarily indicate the excess presence of the interacting components over the one affected by SDI. (ii) Although the Sec titration experiment concerns the number, but not quality, of the Sec factor, only the secE501 mutation was defined as reducing the quantity of SecE (16). Can the results of other undefined sec mutations be compared meaningfully with each other or with secESOl? (iii) As far as SecY and SecE are concerned, mutational reduction of the SecE level destabilizes SecY and leads to a reduced accumulation of the latter (see below), whereas, SecE is stable by itself. Such asymmetric mutational effects can complicate the interpretation; the secE501 effects on the phenotypes of the SDI at PrlA (SecY) should have been affected by the instability of SecY.

C. Insights into the SecY-SecE Interaction When solubilized membrane components of the wild-type cells were fractionated and assayed for reconstitution of proteoliposomes with SecA-dependent translocation activity, a complex containing SecY, SecE, and Band 1 was obtained (90). Although SecY and SecE can be isolated separately from the overproducing cells and can then be reconstituted for active translocation (91), they are in close interaction in vivo. Although prlA and prig mutations generally do not affect cell growth, one particular combination, prlA4 and prlGl, gives a synthetically lethal phenotype, providing genetic evidence for the interaction between SecY and SecE (107). SecY can only be overproduced by overexpressing both the secY and the secE genes (113). This is due to rapid (half life about 2 min) degradation of excess (uncomplexed) SecY (114); simultaneous overproduction of SecE completely

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stabilizes the excessive SecY (113, 114). SecE is the primary determinant of the stability of SecY in the wild-type cell, because the secE501 mutational reduction (by about 50%) of the secE expression level results in destabilization of a fraction (again about 50%) of SecY (114); one of the roles played by SecE should be to stabilize SecY. In the secE501 mutant cells, about 50% of the newly synthesized SecY molecules are immediately committed to rapid degradation, while the remaining 50% remain completely stable (114). The existence of two distinct populations of SecY when availability of the partner SecE molecules is limited implies the following. First, newly synthesized SecY immediately associates with SecE in the wild-type cell. Second, the SecY-SecE complex, once formed, does not dissociate to the extent that dissociated SecY is attacked by the SecY-degradation system. This seems to invalidate a simple version of the Bieker-Silhavy model. The dominant negative secF mutation, secF'dl, provided a useful system for genetic analysis of SecY-SecE interaction (88). Our working hypothesis is that the mutation inactivates an essential SecY activity without abolishing its binding to SecE, and the mutant protein sequesters SecE. In accordance with this hypothesis, SecE overproduction effectively overcame the export interference. The dominant effects of a series of SecY-PhoA fusion proteins focuses the region necessary for the dominant export interference to the central part of SecY (P3 to TM7 interval; 88). To define more precisely the region of SecY important for the interaction with SecE, second site insertion/deletion mutations that suppressed secF-dl but did not knock out the synthesis of SecY itself were isolated using a derivative of the sec Fdl gene to which the lacZa sequence had been attached in frame to the 3' end (115). The intragenic suppressor mutations were found to reside mostly in the C4 domain. The secF24 mutation, a single amino acid substitution in this region, also suppressed the export interference by secF-dl. These suppressor mutations do not restore the translocation activity of the SecF-dl protein itself, but impairs its interaction with SecE. It was found that the SecY24 mutant protein, when it was overexpressed, was no longer stabilized by the overexpression of SecE, although stabilization by Ydr (see below) was unchanged. While SecY-SecE complexes in the wild-type cell survived the immunoprecipitation experiments under appropriate solubilization conditions, only uncomplexed SecY24 protein was found in the anti-SecY immunoprecipitates from secu mutant cells. In addition, the basal level SecY24 protein was destabilized at 42 C, the nonpermissive temperature for this Ts mutant. Thus, the Gly to Asp change in the C4 domain weakens the SecY's interaction with SecE (115). These results establish genetically that the SecY-SecE interaction is crucial for the translocator function and suggest that the C4 domain of SecY is important for this interaction. How the C4 domain facilitates actual binding between SecY and SecE, that could well involve TM-TM interaction (see the preceding sections), remains to be determined.

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VIII.

A D D I T I O N A L FACTORS POSSIBLY I N V O L V E D IN TRANSLOCATION A. Band 1 and P12

These factors have been identified by biochemical studies. They appear distinct and their in vivo roles remain to be established. Band I is found in the active fraction purified as membrane-embedded components of"translocase" (90, 116). Chromatographic and immunoprecipitation behaviors suggest that it is associated with SecY/E. The association is labile at ambient temperature. The translocase preparation reconstituted with Band 1 is as active as native membrane vesicles (117). PI2 is a membrane protein which confers markedly higher translocation activity when present during reconstitution of proteoliposomes from purified SecY and SecE (118).

B. PspA This protein is induced when abnormal secretory proteins, such as mutant forms of PhoE and MalE-LacZ fusion protein, enter the translocation pathway and are stuck in the membrane, as well as upon infection by filamentous phages (phage shock protein). ApspA mutant cell shows export retardation of several cell surface proteins including Male and B la (119).

C. Skp This protein stimulates protein translocation in vitro when added to membrane vesicles prepared from secA mutant cells. Although it was purified from a ribosomal fraction, its amino terminal sequence puzzlingly proved to be identical with the mature part of an outer membrane protein (120). D. Ydr Ydr has been identified as a multicopy suppressor of the dominant negative

secY-dl mutation (89, 121). It is a 181-residue hydrophilic protein with its C-terminal region potentially forming an amphiphilic helix. Ydr is loosely associated with the cytoplasmic membrane in a saturable fashion, possibly via SecY. Its overproduction can partially stabilize the oversynthesized SecY that is otherwise unstable. While its overproduction improves protein export in the presence of secy-dl on the one hand, it strikingly exacerbates export and viability of the secY24 mutant cell on the other hand. As already discussed, the sec}24 mutation weakens the SecY-SecE interaction. Taken together with the observation that ydr can be disrupted without appreciable phenotypes, one hypothetical role of Ydr is to control

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the formation of active translocator by negatively intervening in the SecY-SecE interaction.

E. Orfl2 This gene, located in the upstream of secD, constitutes an operon with secD and secF (95,122). Its overexpression, like that ofydr, suppresses secF'al and stabilizes oversynthesized SecY. Its disruption causes synthetic growth impairment with the sec F39 mutation, but is otherwise silent. The deduced amino acid sequence suggests that the Off12 product is a membrane protein. F. FIsH

In an attempt to identify genes involved in membrane protein anchoring and assembly, we devised a screening system that allowed the isolation of mutations that enhance translocation of a PhoA reporter attached to a cytoplasmic domain of a membrane protein (123). A mutation thus obtained, std101 (std for stop transfer defective), proved to be an allele offisH, that encodes a putative membrane-bound ATPase with significant sequence homology with the members of an eukaryotic ATPase family such as SEC 18p, NSF, and PAS I p (124, 125). A temperaturesensitive ftsH mutation (ftsH1), depletion of FtsH, and expression of either a C-terminally truncated form of FtsH or an ATP-binding sequence mutant of FtsH, lead to the stop transfer defective phenotype for the PhoA fusion protein and translocation-defective phenotypes for several exported proteins (123, 126, 127). Thus, impaired FtsH function leads to translocation enhancement of normally anchored proteins and translocation inhibition of normally translocated proteins. Among the exported proteins tested, Bla was most affected by the ftsH mutations (126), but OmpA export is also significantly retarded by the fish depletion (123) or by dominant mutations offish (127). FtsH could functionally interact with the translocation machinery or facilitate correct assembly of membrane proteins, including SecY, SecE, or other translocator components.

G. Are There Translocation Factors in the Periplasm? Molecular chaperones in the trans side of the membrane, such as the mitochondrial and the endoplasmic reticulum HSF70 homologs, can actively participate in the translocation reaction (128,129). They either drive translocation by binding the incoming polypeptide chain or facilitate assembly or recycling of translocator components. In E. coli, Skp may reside outside the cytoplasn,ic membrane, but its role has not been established. SecD (and SecF?), that contains a large periplasmic domain and possesses a release/folding facilitating role (96), can be regarded as a periplasmic translocation factor. A well-defined folding factor in the periplasm is DsbA (6, 130) that, in concert with the membrane-bound DsbB (7), facilitates the


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formation of disulfide bonds in a set of envelope proteins after membrane translocation (131). In the absence of DsbA, some proteins such as PhoA remain on the periplasmic surface of the membrane in the reduced and unfolded form (130). It remains to be established whether polypeptide-binding chaperones and/or folding enhancers (including the DsbA system) in the periplasm have roles in the late phase translocation reactions.

IX. CONCLUSION The significance of genetic analysis is at least two-fold. First, it is useful to identify actors that participate in the system. Second, it is a powerful tool in assigning their intracellular roles. Protein translocation genetics in E. coli has been successful in both of these aspects. In particular, elaborate genetic dissection, such as the SDI/Sec titration and the dominant negative analyses, now approaches the central question: How proteinaceous components in the membrane interact with one another in providing the pathway through which movement of substrate polypeptide is facilitated. Although, as discussed in detail, each approach has its own limitations or uncertainties, use of complementary biochemical experiments will be of great assistance in the search for a solution. For instance, mutational defects in the membrane traversing reaction might effectively be studied only in vitro. Recent and remarkable progress in reconstitution and energetics of the in vitro translocation reaction and its substeps (132) indicates that it is now feasible to blend biochemistry and genetics to study molecular events that occur in the intracellular pathway of protein topogenesis.

ACKNOWLEDGMENTS The author would like to thank the membersof his laboratory,especially Yoshinori Akiyama, Tohru Yoshihisa, Tetsuya Taura, Tadashi Baba, Takashi Shimoike, and Satoshi Kishigami for fruitful collaborations, and Kurt Cannon for reading the manuscript. The work from the author's laboratory has been supported by grants from the Ministry of Education, Science and Culture, Japan; the Naito Foundation, Takara Shuzo, Mitsubishi-Kasei Corporation, and Yamada Science Foundation.

NOTE ADDED IN PROOF Band I and P12 are identical and given the name of Sec G. Ydr is now called "Syd."

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85. Gorlich, D., Prehn, S., Harunann, E., Kalies, K.-U., & Rapoport, T. A. (1992). A mammalian

homolog of SEC61p and SecYp is associated with ribosomes and nascent polypeptides during translocation. Cell 71,489-503. 86. Akiyama, Y., & lto, K. (1987). Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli. EMBO J. 6, 3465--3470. 87. Taura, T., Akiyama, Y., & Ira, K. (1994). Genetic analysis of SecY." additional export-defective mutations and factors affecting their phenotypes. Mol. Gen. Genet. 243, 261-269. 88. Shimoike, T., Akiyama, Y., Baba, T., Taura, T., & Ira, K. (1992). SecY variants that interfere with Escherichia coli protein export in the presence of normal secY. Mol. Microbial. 6, 1205-1210. 89. Ira, K. (1992). SecY and integral membrane components of the Escherichia coil protein translocation system. Mol. Microbial. 6, 2423-2428. 0. Brundage, L., Hendrick, J. P., Schiebel, E., Driessen, A. J. M., & Wickner, W. (1990). The purified E. coil integral membrane protein SecY/SecE is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell 62, 649--657. 91. Akimaru, J., Matsuyama, S., Tokuda, H., & Mizushima, S. (1991). Reconstitution of a protein translocation system containing purified SecY, SecE, and SecA from Escherichia coll. Prec. Natl. Acad. Sci. USA 88, 6545--6549. 92. Nishiyama, K., Mizushima, S., & Tokuda, H. (1992). The carboxyl-terminal region of SecE interacts with SecY and is functional in the reconstitution of protein translocation activity in Escherichia coli. J. Biol. Chem. 267, 7170-7176. 93. Jeong, S. M., Yoshikawa, H., & Takahashi, H. (1993). Isolation and characterization of the secE homologue of Bacillus subtilis. Mol. Microbial., in press. 94. Gardel, C., Benson, S., Hunt, J., Michaelis, S., & Beckwith, J. (1987). secD, a new gene involved in protein export in Escherichia coll. J. Bacterial. 169, 1286-1290. 95. Gardel, C., Johnson, K., Jacq, A., & Beckwith, J. (1990). The secD locus of E. coli codes for two membrane proteins required for protein export. EMBO J. 9, 3209-3216. 96. Matsuyama, S., Fujita, Y., & Mizushima, S. (1993). SecD is involved in the release oftranslocated secretory proteins from the cytoplasmic membrane of Escl~richia coli. EMBO J. 12, 265-270. 97. Andersson, H., & van Heijne, G. (1991). A 30-residue-long "export initiation domain" adjacent to the signal sequence is critical for protein translocation across the inner membrane of Escherichia coli. Proc. Natl. Acad. Sci. USA 88, 9751-9754. 98. Puziss, J. W., Strobel, S. M., & Bassford, P. J. (1992). Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring pd suppressor mutations. J. Bacterial. 174, 92-101. 99. Sako, T., & lino, T. (1988). Distinct mutation sites in prlA suppressor mutant strains of Escherichia coli respond either to suppression of signal peptide mutations or to blockage of staphylokinase processing. J. Bacterial. 170, 5389-5391. 100. Francetic, O., Hanson, M. P., & Kumamoto C. A. (1993). prlA suppression of defective export of maltose-binding protein in secB mutants of Escherichia coll, J. Bacterial. 175, 4036--4044. 101. Randall, L. L., Hardy, S. J. S., & Thorn, J. R. (1987). Export of protein, a biochemical view.Annu. Rev. Microbial. 41,507-54 I. 102. Laforet, G. A., & Kendall, D. A. (1991). Functional limits of conformation, hydrophobicity, and steric constraints in prokaryotic signal peptide cleavage regions. J. Biol. Chem. 266, 1326-1334. 103. Sako, T. (1991). Novel prM alleles defective in supporting staphylokinase processing in Escherichia coll. J. Bacterial. 173, 2289-2296. 104. Mtiller, J., Reinert, H., & Malke, H. (1989). Streptokinase mutations relieving Escherichia coil K- 12 (prlA4) of detriments caused by the wild-type skc gene. J. Bacterial. 171, 2202-2208. 105. lino, T., & Sako, T. (1988). Inhibition and resumption of processing of the staphylokinase in some Escherichia coil prlA suppressor mutants. J. Biol. Chem. 263, 19077-19082. 106. Derman, A. I., Puziss, J. W., Bassford, P. J., & Beckwith, J. (1993). A signal sequence is not required for protein export in prlA mutants of Escherichia coll. EMBO J. 12, 879-888.


59

107. Bieker, K. L., & Silhavy, T. J. (1990). PrlA (SecY) and PriG (SecE) interact directly and function sequentially during protein translocation in E. coll. Cell 61,833-842. 108. Bieker, K. L., & Silhavy. T. J. (1990). The genetics of protein secretion in E. coli. Trends Genet. 6, 329-334. 109. Ueguchi, C., & Ira, K. (1990). Escherichia coli sec mutants accumulate a processed immature form of maltose-binding protein (MBP), a late-phase intermediate in MBP export. J. Bacterial. 172, 5643-5649. 110. Geiler, B. L. (1990). Electrochemical potential releases a membrane-bound secretion intermediate of maltose-binding protein in Escherichia coll. J. Bacterial. 172, 4870-4876. 111. Minsky, A., Summers, R. G., & Knowles, J. R. (1986). Secretion of ~lactamase into the periplasm of Escherichia coil: Evidence for a distinct release step associated with a conformational change. Proc. Natl. Acad. Sci. USA 83, 4180-4184. 112. Bieker-Brady, K., & Silhavy, T. J. (1992). Suppressor analysis suggests a multistep, cyclic mechanism for protein secretion in Escherichia coli. EMBO J. 11, 3165-3174. 113. Matsuyama, S., Akimaru, J., & Mizushima, S. (1990). SecE-depcndent overproduction of SecY in Escherichia coll. FEBS Len. 269, 96-100. 114. Taura, T., Baba, T., Akiyama, Y., & Ito, K. (1993). Determinants of the quantity of the stable SecY complex in the Escherichia coli cell. J. Bacterial. 175, 7771-7775. I15. Baba, T., Taura, T, Shimoike, T., Akiyarna, Y., Yoshihisa, T., & Ira, K. (1994). A cytoplasmic domain is important for the formation of a SecY-SecE translocator complex. Proc. Natl. Acad. Sci. USA 91, 4539-4543. 116. Brundage, L., Fimmei, C. J., Mizushima, S., & Wickner, W. (1992). SecY, SecE, and Band I form the membrane-embedded domain of Escherichia coil preprotein transiocase. J. Biol. Chem. 267, 4166-4170. 117. Bassilana, M., & Wickner, W. (1993). Purified Escherichia coil preprotein translocase catalyzes multiple cycles of precursor protein translocation. Biochemistry 32, 2626-2630. 118. Nishiyama, K., Mizushima, S., & Tokuda, H. (1993). A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli. EMBO J. 12, 3409-3415. 119. Kleerebezem, M., & Tomassen, J. (1993). Expression of the pspA gene stimulates efficient protein export in Escherichia coil. Mol. Microbial. 7, 947-956. 120. Thome, B. M., Hoffschulte, H. K., Schiltz, E., & Mtiller, M. (1990). A protein with sequence identity to Skp (FirA) supports protein translocation into plasma membrane vesicles of Escherichia coli. FEBS Lett. 269, 113-116. 121. Shimoike, T., Taura, T., Kihara, A., Yoshihisa, T., Akiyama, Y., Cannon, K., & Ira, K. (1995). Product of a new gene, s)~/, functionally interacts with SecY when overproduced in Escherichia coll. J. Biol. Chem., in press. 122. Reuter, K., Slany, R., Ullrich, E, & Kersten, H. (1991). Structure and organization of Escherichia coil genes involved in biosynthesis of the deazaguanine derivative queuine, a nutrient factor for eukaryotes. J. Bacterial. 173, 2256-2264. 123. Akiyama, Y., Ogura, T., & Ito, K. (1994). Involvement of FtsH in protein assembly into and through the membrane. I. Mutations that reduce retention efficiency of a cytoplasmic reporter. J. Biol. Chem. 269, 5218-5224. 124. Tomoyasu, T., Yuki, T., Morimura, S., Mori, H., Yamanaka, K., Niki, H., Hiraga, S. and Ogura, T. (1993). The Escherichia coli FtsH protein is a prokaryotic member of a protein family of putative ATPase involved in membrane function, cell cycle control, and gene expression. J. Bacterial. 175, 1344-1351. 125. Tomoyasu, T., Yamanaka, K., Murata, K., Suzaki, T., Bouloc, P., Kato, A., Niki, H., Hiraga, S., & Ogura, T. (1993). Topology and subcellular localization of FtsH protein in Escherichia coli. J. Bacterial. 175, 1352-1357.

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126. Tomoyasu, T., Yamanaka, K., Niki, H., Hiraga, S., & Ogura, T. Effects of a thermosensitiveftsH mutation on localization and assembly of cell envelope proteins in Escherichia coll. Personal communication. 127. Akiyama, Y., Shirai, Y., & lto, K. (1994). Involvement of FIsH in protein assembly into and through the membfme. II. Do~nant mutations affecting FtsH functions. J. Biol. Chem. 269, 5225-5229. 128. Kang, P. J., Ostenmn, J., Shilfing, J., Neupert, W., Craig, E. A., & Pfanner, N. (1990). Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature 348, 137-143. 129. Sanders, S. L., Whitfield, K. M., Voge, J. P., Rose, M., & Schelunan, R. W. (1992). Sec61p and BiP directly facilitate polypeptide Iranslocation into the ER. Cell 69, 353-365. 130. Kamitani, S., Akiyama, Y., & Ito, K. (1992). Identification and characterization of an Escherichia coli gene required for the fommtion of correctly folded alkaline phosphatase, a periplasmic enzyme. EMBO J. i 1, 57-62. 131. Akiyama, Y., Kamitani, S., Kusukawa, N., & Ito, K. (1992). In vitro catalysis of oxidative folding of disulfide-bonded proteins by Escherichia coli dsbA (ppfA) gene product. J. Biol. Chem. 267, 22440-22445. 132. Wickner, W. Driessen, A. J. M., & Hard, E-U. (1991). The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu. Rev. Biochem. 60, 101-124.

BIOCHEMICAL ANALYSES OF COMPONENTS COMPRISING THE PROTEIN TRANSLOCATION MACHINERY OF ESCHERICHIA COL/

Shin-ichi Matsuyama and Shoji Mizushima

I. II. III. IV. V.

Vl.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Components Involved in Protein Translocation . . . . . . . . . . . . . . . . . Overproduction and Purification of Sec Proteins . . . . . . . . . . . . . . . . Numbers of Molecules of Sec Proteins in One E. coli Cell . . . . . . . . . . . Functions of Sec and Related Proteins . . . . . . . . . . . . . . . . . . . . . . A. SecB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. SecA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. SecY and SecE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. SecD and SecF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. SecG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signal Peptidases and Signal Peptide Peptidases . . . . . . . . . . . . . . . . A. Signal Peptidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Signal Peptide Peptidases . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in CeU and Molecular Biology of Membranes and Organelles Volume 4, pages 61-84. Copyright 9 1995 by JAI Press Inc. All fights of reproduction in any form reserved. ISBN: 1-55938-924-9 61

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SHIN-ICHI MATSUYAMAand SHOJIMIZUSHIMA

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VII. Other Factors Involved in Protein Translocation . . . . . . . . . . . . . . . . . VIII. Model for Protein Translocation . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.

73 75 77 78

INTRODUCTION

Proteins synthesized on ribosomes in the cytosol have to be targeted to their final destinations for them to exhibit their intrinsic functions. Cells of gram-negative bacteria, such as Escherichia coil, comprise four compartments: the cytoplasm, cytoplasmic membrane, periplasm, and outer membrane. Proteins of the outer membrane and of the periplasm have to be translocated across the cytoplasmic membrane to reach their destinations as genuine secretory proteins do. Proteins are usually translocated across the cytoplasmic membrane in a precursor form (preprotein) that possesses an extra polypeptide, called the signal peptide, at the N-terminus. The translocation shares common features with that of the endoplasmic reticulum membrane of eukaryotes. The protein translocation in both cases requires ATP (25, 38, 44,103,128,132) and a signal peptide, which comprise the N-terminal positively charged region and a long hydrophobic stretch (54, 91). Some prokaryotic preproteins can be translocated across the endoplasmic reticulum membrane and some eukaryotic ones across the bacterial cytoplasmic inembrane (74, 117). Homologues of E. coli SecY, one of the membrane components of the translocation machinery, have been identified in the endoplasmic reticulum membranes of yeasts (114) and mammals (40). Homologues of the mammalian signal recognition panicle, which is involved in the targeting of preproteins to the endoplasmic reticulum membrane, have been found in E. coli (13, 95, 96, 100, 102) and yeasts (9, lO, 42, 43). These facts suggest that the mechanisms underlying the translocation of presecretory proteins are similar in all organisms. In this chapter, we summarize recent progress in biochemical studies on protein translocation across the cytoplasmic membrane of E. coil, focusing on our own biochemical studies, including reconstitution ones. We will, of course, mention relevant work done in other laboratories.

II. COMPONENTS INVOLVED IN PROTEIN TRANSLOCATION Protein translocation across membranes is believed to be catalyzed by a specific proteinaceous complex. Extensive genetic studies on E. coil have revealed that.at least six gene products (SecA, SecB, SecD, SecE, SecF, and SecY proteins) are involved in the general protein translocation across the cytoplasmic membrane (14). In addition to these gene products, several factors have been reported to participate in the translocation process. They are summarized in Table 1 together with Sec

Table 1. Components Participating in the Protein Translocation across the Cytoplasmic Membrane in Escherichia coli Component

Molecular Weight

CellularLocalization

SecB

18 K(subunit)

Cytoplasm

SecA

102 K (Subunit) 204 K (homodimer)

CytopCytoplasmic mtmbrane (WPM)

SecY

Cytoplasmic membrane (integral)

SecE

Cytoplasmic mcmbrane (Integral) Cytoplasmic membrane (with a large periplasmic domain)

SecD

SecF Sed.3 ( ~ 1 2 )

67 K

Cytoplasmic membrane (with a large paiplasmic domain) Cytoplasmic membrane (in@@)

Function

Translocation-specificchaperone Interact with SecA General molecular chaperone General molecular chaperone Essential component for translocation TranslocationATPase Interact with preprotein (signal peptide), SecB, SecY. and SecE Essmtial component for translocation Interact with preprotein (signal peptide), SecA. SecE SecD, SccF, p12, and Ydr Essential component for translocation Interact with signal peptide, SecA, and SecY ihcntial component for translocation Interact with SecY and SecF Involved in release of secretory proteins from the cytoplasmic membrane Component required for hanslocation Interact with SecY and SecD Interact with SecY

Table 1. (continued) Molecular Wig&

Co??pnent

Ydr

19 K

Signal pcptidasc I

36 K

Signal peptidasc 11

18 K

Signal pcptide peptidase

34 K

Cellulor Localization

Cytoplasmic membrane (@PM) Cytoplasmic membrane (integral) cytoplasmic manbnme

Ffh

g 4.5s RNA P~PA

cyt0pb

26 K

Cytoplasmic mmbnmc

Fvnction Intaact with SecY

Cleavage of signal peptides of pnprotcins except prolipoproteins Cleavage of signal peptides of prolipoproteins Rotease 1V:Hydrolysisof cleaved signal pcptih Homologue of 54 K subunit of mammalian SRP Form a complex with 4.5s RNA Homologue of 7s RNA of mammalian SRP Fonn a complex with Ffh Stimulate translocation InmmoIecular disufide bound formation in

Protein Translocation of E. Coli

65

proteins. In the following sections, the roles of these components will be discussed, focusing on Sec proteins from the biochemical point of view.

III. OVERPRODUCTION AND PURIFICATION OF SEC PROTEINS The cellular contents of Sec proteins in E. coil are not high. To obtain large quantities of purified Sec proteins for biochemical studies, including reconstitution ones, the overproduction of Sec proteins by means of gene manipulation was carried out. Since the overproduction of proteins, especially membrane proteins, often causes cell death, the sec genes encoding Sec proteins were placed under a controllable expression promoter on a plasmid. The tac and T7 promoters were judged to be suitable for this purpose. The construction of plasmids carrying the sec genes controlled by these promoters and overproduction of all Sec proteins were successfully achieved (23, 60, 77, 79, 129). SecA, SecB, SecE, and SecF could be overproduced alone, whereas the overproduction of SecD or SecY alone resulted in its rapid breakdown (4, I03a). The overproduction of SecD and SecY was achieved with the simultaneous overproduction of SecF and SecE. (77, I03a), suggesting the occurrence of interactions between SecD and SecF, and SecY and SecE. Interaction between SecY and SecE has also been suggested genetically (15) and biochemically (22). In addition, the SecF-dependent overproduction of SecY was observed, suggesting interaction between SecF and SecY (K. Sagara, S. Matsuyama, and S. Mizushima, unpublished data). All the overproduced Sec proteins have been purified (Figure 1). SecA was purified from the cytosol fraction, which contained most of this protein in the overproducing cells (2, 60). During the purification more than 50% of the SecA molecules lost the eight amino acid residues comprising the N-terminus (82).. Intact SecA was only obtained when the purification was performed in the presence of six protease inhibitors (82). SecD, SecE, SecF, and SecY were purified from the detergent-solubilized membrane fraction containing the overproduced amount of the individual Sec proteins (1, 79, 120). Sarcosyl was used for the purification of SecF and octylglucoside for the other Sec proteins.

IV. NUMBERS OF MOLECULES OF SEC PROTEINS IN ONE E. C O L I CELL The amounts of SecA, SecD, SecY, SecE, and SecF in overproducing cells were determined densitometrically after SDS-gel electrophoresis of the cellular fractions and staining with Coomassie Brilliant Blue. The overproduction (fold)of individual Sec proteins was then determined by means of immunoblot analysis of the cellular fractions of normal and overproducing cells. From these values, the numbers of individual Sec proteins in one normal E. coli cell were estimated (Table 2) (79).

66

SHIN-ICHI MATSUYAMA and SHOJI MIZUSHIMA

SecA SecE SecY SecD SecF p12

F~ure 1. SDS-Polyacrylamide gel profile of purified Sec proteins, pl 2 is now called

SecG (86).

SecE, SecY, SecD, and SecF are cytoplasmic membrane proteins, whereas only 10% of SecA is usually located in the cytoplasmic membrane of normal cells (79). Thus, numbers of molecules of Sec proteins, except SecF, in the cytoplasmic membrane of one E. coli cell were assumed to be approximately 500. The number of molecules of SecG (p 12), a recently found membrane protein involved in protein translocation (86), is similar. This figure may represent the upper limit for the amount of protein translocation machinery in one E. coli cell. It is unclear how many SecE, $ecY, SecD, and SecG molecules exist in one such unit of machinery. SecA was found to exist as a dimer (3). The smaller number of SecF molecules suggests that the role of this protein is different from those of the other Sec proteins.


67

Table 2. Number of Molecules of Sec Proteins in one E. coli cell Protein

SecA SecE SecY SecD SecF SecG ( p 1 2 )

Protein Content (%)

Overproduction(fold)

25.0/WC 3.4/M 5.3/M 6.9/M 16.5/M 15.7/M

50.-100 80-160 50-100 20-40 1500-3000 200-400

Number of Molecules per One Normal Cell 2500-5000 300-.6(g) 206..400 450-900 30-60 650-.1300

Note: WC: Wholecell;M: Membrane

V. FUNCTIONS OF SEC AND RELATED PROTEINS A. SecB

SecB, a cytosolic protein, is a homotetramer of a 16-kDa subunit (127) and is required for the translocation of a subset of secretory proteins, including maltosebinding protein (MBP) and LamB, in vivo (65, 66) and in vitro (127, 129). The conformation of preproteins is often crucial for their translocation across membranes (./2, 98). Preproteins folded into a tight conformation cannot be translocated. SecB binds directly to preproteins to inhibit their folding into a translocation-incompetent conformation (26, 64, 71, 126), and the signal peptide was assumed to play a role in this interaction (8, 126). However, this does not necessarily exclude the participation of the mature domain in the binding. In fact, SecB-dependent in vivo protein translocation was inhibited by the overproduction of mature MBP lacking the signal peptide (11, 26). The exchange of signal peptides between MBP and alkaline phosphatase, of which the translocation is SecB-dependent and SecBindependent, respectively, has no effect on their SecB-dependence (35). Finally, SecB forms complexes not only with preproteins but also with denatured mature proteins (45, 71). It is suggested, therefore, that SecB can bind to the mature domain of preproteins and that the signal peptide is not absolutely required for the recognition by SecB. In fact, genetic analyses have revealed that SecB-binding regions are located between amino acid residues 151 and 186 (26) and 72 and 200 (35) of the mature domain of MBR and between 320 and 380 of that of LamB (7). No characteristic features common to these regions have been found, however. Randall proposed that SecB recognizes a positively charged region of proteins but not a specific amino acid sequence (97). Her and another group proposed that signal peptides play a role in the retardation of the folding of the mature domain (45, 70,

99). A factor that exhibits antifolding activity, such as SecB, is called a molecular chaperone (39). Some other chaperones have also been reported to be involved in

68

SHIN-ICHI MATSUYAMAand SHOJl MIZUSHIMA

protein translocation: GroEL is essential for the secretion of ~-lactamase in vivo (69) and in vitro (18). Overproduced amounts of GroEL and DnaK improved the export of LamB-LacZ fusion proteins (92). It is probable that several molecular chaperones play similar roles in keeping the conformation of preproteins translocation-competent. The trigger factor was originally identified as a factor that interacts with proOmpA to facilitate in vitro translocation across the cytoplasmic membrane (27). In vivo genetic analyses revealed, however, that the translocation of proOmpA is normal in trigger-factor-deficient cells (41). B. SecA

SecA is a homodimer of a 102-kDa subunit (3, 108) and is located both in the cytoplasm and on the inner surface of the cytoplasmic membrane (28, 89). The direct involvement of SecA in protein translocation has been demonstrated both in

vitro (23, 60) and in vivo (88, 89). The interaction between SecA and preproteins was revealed by a chemical cmss-linldng study (2). The interaction was signal-peptide-dependent, and an increase in the N-terminal positive charges of the signal peptide resulted in an increase in the efficiency of the cross-linking of preproteins to SecA. This result, together with the fact that the cross-linking was inhibited under high salt conditions, indicates that an electrostatic interaction is important for the SecA-preprotein interaction. SecA is negatively charged as a whole. Very recently evidence for recognition by SecA of the signal hydrophobic region was also presented (H. Mori and S. Mizushima et al., unpublished data). SecA alone exhibits weak ATPase activity (72). The activity was enhanced in the presence of prepmteins and membrane vesicles that contain acidic phospholipids and SecY/SecE (29, 72, 73). The enhanced ATPase activity was termed translocation ATPase activity. Both the translocation ATPase activity and the SecA-dependent protein translocation were inhibited by sodium azide (90), and azide-resistant mutations have been mapped to the secA gene (34, 90), suggesting that SecA is the primary target of this inhibitor. The regions in the SecA molecule that interact with ATP and preproteins have been determined by us (62, 80). The ATP-binding region of SecA is located within the first 217 amino acid residues from the H-terminus. A study involving 8-azido ATP by Lill et al. (72) demonstrated three ATP-binding sites in SecA, although their locations were not determined. The ATP-binding region determined by us is presumably the high-affinity one of the three. The preprotein-binding region of SecA was determined to be located between amino acid residues 267 and 340 (62), and the N-terminal half of SecA was reported to be involved in the interaction with the membrane (24). All the secA(ts) mutations so far reported have been mapped within the first 200 amino acid residues (108). Thus, all these results support the importance of the H-terminal region for the SecA function.

Protein Translocation orE. Coil

69

The ATP-binding region is adjacent to the preprotein-binding region in the SecA molecule (62), suggesting that these two regions may be functionally related. Consistent with this view, (i) ADP/ATP on the SecA molecule was released upon the addition of preproteins (110); (ii) ATP enhanced the cross-linking of preproteins to SecA (2); and (iii) a protease-sensitivity test revealed that SecA takes on different conformations in the presence of ATP and preproteins and that the conformation taken on in the presence of ATP disappeared upon the addition of preprotein (110). Furthermore, evened membrane vesicles caused SecA to take on another conformation. These results further suggest that SecA takes on different conformations upon interaction with different components in the process of protein translocation. Since SecA is the only protein of the secretory machinery that is known to interact with ATP, it is likely that such changes in the SecA conformation constitute the driving force for the transmembrane movement of preproteins. Phosphatidylglycerol and carcliolipin, acidic phospholipids of E. coli, stimulated protein translocation and SecA translocation ATPase activity (30, 68, 73, 94). These phospholipids made SecA sensitive to V8 protease (110), suggesting that they also modulated the conformation of SecA. The insertion of SecA into a phospholipid monolayer was enhanced when the layer contained these acidic phospholipids (19). Several lines of evidence support the view that preproteins first interact with the soluble SecA to initiate the translocation reaction: (1) Preproteins can directly bind to soluble SecA in a manner that is dependent on the strength of the positive charge at the N-terminus of the signal peptide, as is the translocation reaction (2); (2) In vitro protein translocation is stimulated by externally added soluble SecA in an amount that is much more than that which can be retained by the membrane (131); and (3) Different preproteins require different concentrations of SecA for efficient in vitro translocation (48). If the membrane-bound SecA is the initial site of preprotein binding, such a difference in SecA requirement would not be observed. It is likely, therefore, that SecA molecules alternate between the two forms in the process of protein translocation. The following evidence suggests that ATP, and probably its hydrolysis, are involved in the SecA alternation: (1) Exchange between membrane bound B. subtilis SecA and soluble E. coli SecA was only observed in the presence of ATP (122); (2) SecA (Ts) proteins, which possess mutations within the ATP-binding region, were preferably localized in the cytoplasmic membrane (24); and (3) ATR but not nonhydrolyzable ATP analogues, inhibited the insertion of SecA into the phospholipid monolayer (19).

C. SecYand SecE SecY and SecE are integral membrane proteins that span the cytoplasmic membrane ten and three times, respectively (.5, 105). Mutations on the secu and secE genes confer general protein export defects (56, 101), suggesting the direct participation of these proteins in protein translocation. Re,constitution studies have shown this directly. Proteoliposomes reconstituted from purified SecY, SecE, and E. coli

70


phospholipids exhibited protein translocation activity that was dependent on SecA and ATP (1). Omission of either one of these Sec proteins resulted in the complete loss of the activity, suggesting that SecY, SecK and SecA are indispensable proteinaceous components of the protein translocation machinery. Reconstitution of translocation-active proteoliposomes from a complex composed of SexY, SecE, and another protein called band 1 was also performed (22). Recently band 1 was found to be identical with SecG (30a). The SecE molecule has three transmembrane segments. Genetic and reconstitution studies have revealed that a truncated fragment containing only the C-terminal segment was 50% as active as intact SecE (85,104). This fragment stabilized SecY in the membrane, as does intact SecE, suggesting that the third transmembrane segment directly interacts with Sexy (85). A gene in Bacillus subtilis corresponding to E. coil secE has been identified and found to complement the E. coil secE (cs) mutation (57). The deduced amino acid sequence suggests that 8. subtilis SecE has only one transmembrane segment (57). The homology between this segment and the E. coil C-terminal segment is quite low, however. What are the functions of SecY and SecE in the translocation reaction? Since the primary structure of the mature domains of preproteins that can pass through the cytoplasmic membrane is highly hydrophilic (81), it is unlikely that secretory proteins can be translocated directly through the lipid phase of the membrane. The interaction of SecA with SecY and SecY/SecE was demonstrated (15, 22, 33, 72, 73, 77, 106). High affinity binding of SecA to the membrane required SecY/SecE (46). These facts suggest that SecY/SecE functions as a receptor for SecA, enabling the transfer of preproteins from SecA to the SecY/SecE-containing translocation machinery in the membrane. It is assumed that protein translocation takes place through a specific channel. SecY possesses ten membrane-spanning segments, all of which are seemingly required for the translocation reaction (Nishiyama et al., unpublished data), whereas only one such segment is required for SecE to function (85, 104). It is likely that SecY constitutes the major portion of the channel. In this respect, it is interesting that translocation intermediates arrested in the membrane were directly crosslinked to SecY but only limited cross-linking to phospholipids (58). It should be noted, however, that SecY is highly hydrophobic. It is unclear how SecY can be responsible for the formation of a hydrophilic pore, if one exists. Simon and Blobel electrophysiologically observed translocation-dependent channel activity by fusing E. coil membrane vesicles to a planar lipid bilayer (112). The channel activity, observed for ion (K+) passage, was signal.peptide.dependent. Schiebel and Wickner (107) demonstrated that SecA- and AlP-dependent protein translocation increases the membrane permeability to halide anions. Kawasaki et al. (61), on the other hand, observed the countermovement of protons during the protein translocation into membrane vesicles that contain overproduce~ amounts of SecY and SecE. The proton effiux depended on a presecretory protein, ATP, SecA, and SecY. The addition of the mixture of signal peptide and mature protein


71

did not cause the proton efflux in this case. It is likely that the translocation reaction is coupled with the opening up of a channel, of which the major constituent is SecY. It is still unclear, however, whether the channel thus formed is responsible for the protein passage. D. SecD and SecF

SecD and SecF are integral membrane proteins that appear to span the cytoplasmic membrane six times (36). Genetic studies have suggested that both proteins are important for in vivo protein translocation (36). Extensive biochemical analyses including reconstitution studies, however, have failed to demonstrate the involvement of SecD and SecF in protein translocation (79). It has been suggested that SecD and SecF each possess a large periplasmic domain (36). Furthermore, the prl mutations, which suppress signal peptide defects, have not been mapped to the secD and SecF genes (36, 113). These facts suggest that SecD and SecF play a role in a late step of protein translocation, when the signal peptide is no longer required. If this is the case, the functions of SecD and SecF may not be detectable with the current in vitro assay method, in which translocation activity is measured as the resistance of preproteins to externally added protease, namely, the entry of preproreins into membrane vesicles. Recently, the role of SecD in a late step of protein translocation was demonstrated by employing E. coli spheroplasts (78). The spheroplasts secreted authentic periplasmic and outer membrane proteins into the medium. When the spheroplasts were pretreated with anti-SecD antibody, the secretion of MBE a periplasmic protein, and OmpA, an outer membrane protein, into the medium was inhibited, and accumulation of their precursors in the spheroplasts was observed. Interestingly, a time-course experiment revealed that the anti-SecD antibody treatment resulted in simultaneous accumulation of the mature form of MBR which has been processed for the signal peptide and localized on the outer surface of the spheroplasts. These results indicate that SecD is involved in a late step of protein translocation and probably plays a role in the release of translocated secretory proteins from the cytoplasmic membrane. It is unclear, however, whether SecD is directly involved in the release from the membrane or the final stage of the translocation reaction (i.e., export of the C-terminus of secretory proteins). The secD and secF genes constitute an operon (36). The membrane topologies of SecD and SecF are thought to be similar, and their primary sequences are partially homologous, suggesting that the two proteins perform related functions (36, 78). However, no biochemical studies, including ones involving anti-SecF antibody, have demonstrated the functioning of SecF in the translocation reaction. Very recently, Sagara et al. (103a) of our group showed in vivo that SecF stabilizes not only SecD but also SecY. It is probable, therefore, that SecF plays a role in the translocation event by interacting with both SecD and SecY.

72

SHIN-ICHI MATSUYAMAand SHOJI MIZUSHIMA

E. SecG All the components of the protein translocation machinery described so far were originally identified through genetic studies. Recently, a new proteinaceous factor for protein translocation was discovered using a reconstitution system and named p 12 (86) and later on SecG (86a). SecG is a 12-kDa cytoplasmic membrane protein. When purified SecG was added to the reconstitution mixture together with SecY and SecE, the translocation activity of the reconsfitmed proteoliposomes was enhanced more than 20-fold. The translocation thus enhanced was ATP- and SecA-dependent. Furthermore, anti-SecG antibody inhibited the translocation of proteins into everted membrane vesicles. The gene encoding SecG was identified and sequenced, and the amino acid sequence of SecG was deduced. This protein possesses two or three possible membrane-spanning domains. The cloning of the gene enabled the overproduction of SecG. The overproduction of SecG supported the simultaneous overproduction of SecY, as SecE does, suggesting that this protein also interacts with SecY in the membrane (K. Nishiyama, S. Mizushima, and H. Tokuda, unpublished data). A secG::kan null mutant was cold-sensitive for protein export and growth (86a). VI.

SIGNAL PEPTIDASES A N D SIGNAL PEPTIDE PEPTIDASES

A. Signal Peptidases E. coil has two types of signal peptidases that cleave the signal peptides of preproteins during protein translocation. Signal peptidase II (lipoprotein signal peptidase) cleaves the signal peptides ofglyceride-modified prolipoproteins. Signal peptidase I (leader peptidase) apparently cleaves the signal peptides of all preproteins except prolipoproteins. Signal peptidase H is an integral membrane protein that spans the cytoplasmic membrane four times (83). The amino acid sequence, Leu-(Ala Ser)-(Gly Ala)-Cys, surrounding the cleavage site, is conserved in all prolipoproteins, suggesting this domain to be the site of recognition by the enzyme (47). Glyceride modification of the cysteine residue immediately after the cleavage site is a prerequisite for the proteolytic processing (51). This processing is specifically inhibited by the antibiotic, globomycin (50). Signal peptidase I is a cytoplasmic membrane protein that possesses a large periplasmic region functioning as the catalytic domain (17). There are some common features around the cleavage site of signal peptides, the target domain of this enzyme: Small apolar amino acids (Ala, Ser and Gly) at- 1 and-3 are important for the processing by signal peptidase I (91, 109, 123). Pro and Thr at +1 strongly inhibit the cleavage of signal peptides (75,109). The {3-turn structure at the cleavage

Protein TranslocationorE. Coli

73

site for recognition by signal peptidase I was purported to be important (31, 91). No protease inhibitor has been found to inhibit signal peptidase I activity. Recently, the Ser90 residue was determined to be essential for the function of this peptidase (115), suggesting that signal peptidase I belongs to a novel class of serine proteases. The precise structure of the catalytic domain remains unclear, however. About 500 signal peptidase I molecules exist in one E. coil cell (130). This number is close to that estimated for See proteins comprising the translocation machinery (79), suggesting that signal peptidase may be part of the translocation machinery together with Sec proteins. It should be noted, however, that the cleavage of signal peptides is not essential for protein translocation in vivo (53) or in vitro (133), although the release of translocated proteins from the membrane requires cleavage. Purified signal peptidases actively cleave signal peptide, even in the absence of See proteins (130). Furthermore, the sec mutation has not been mapped to the signal-peptidase-encoding genes. It is likely, therefore, that the function of signal peptidases is not directly involved in the translocation event, although the cleavage of signal peptides is usually tightly coupled with protein translocation.

B. Signal Peptide Peptidases Signal peptides cleaved from preproteins by signal peptidases are rapidly degraded in the membrane. Protease IV, a cytoplasmic membrane protease named signal peptide peptidase, was found to specifically hydrolyze cleaved signal peptides (52). Signal peptides attached to the mature domain were not attacked by this enzyme. The disruption of the protease IV-encoding gene, spp, on the chromosome did not result in accumulation of signal peptides (116), indicating that other peptidases could also be involved in the signal peptide hydrolysis. Two cytoplasmic enzymes were found to digest cleaved signal peptides (87).

VIi. OTHER FACTORS INVOLVED IN PROTEIN TRANSLOCATION Ffh Protein and 4.5S RNA

Thesignal recognition particle (SRP) is a mammalian cytosolic factor which transfers nascent presecretory proteins from the cytosol to the endoplasmic reticulure membrane (124). SRP consists of six distinct proteins and one 7S RNA (125). The 54-kDa subunit, one of the protein components of SRP, directly interacts with signal peptides (67). Recently, the E. coli homologue of the 54-kDa subunit and the 7S RNA of SRP were identified. They were identified as the 48-kDa Ffh protein (13, 102) and 4.5S RNA (95, 96, 100), respectively. Ffh and 4.5S RNA form a complex (95, 100), which interacts with the signal peptide domain of nascent

74

SHIN-ICHI MATSUYAMA and SHOJl MIZUSHIMA

preprolactin and E. coil prolipoprotein (76), suggesting that the complex may be the E. coli homologue of SRP. Genetic analyses have revealed that the genes encoding Ffh and 4.5S RNA are essential for cell growth (21, 93). Precursors of 13-1actamase, alkaline phosphatase, and ribose-binding protein accumulated in the Ffh-depleted cells (93), whereas only the I]-lactamase precursor did so in the cells lacking 4.5S RNA (100). Translocation of these three preproteins was SecB-independent (65, 66). On the other hand, depletion of Ffh or 4.5S RNA did not have a significant effect on the translocation of MBP and LamB, whose export is SecB-dependent (65, 66). The Ffh-4.5S RNA complex may act as a molecular chaperone for a group of preproteins, as SecB does for another group. Components corresponding to Ffh and 4.5S RNA of E. coli have also been identified in Bacillus subtilis (49, 84). They are required for cell growth and the depletion of either B. subtilis Ffh or scRNA caused defective secretion of o~-amylase.

PspAProtein PspA, a peripheral cytoplasmic membrane protein, was originally identified as a protein induced by fl phage infection (20). This protein has a stimulative effect on protein translocation both in vitro and in vivo, although it is not absolutely required (63).

DsbA (PpfA) Protein This is a periplasmic protein which catalyzes intramolecular disulfide bond formation (12, 59). It plays an essential role in the folding of disulfide bond-possessing proteins in the periplasm (6, 59). However its function may not be directly involved in the translocation event.

Ydr Protein A gene of which overexpression suppresses the translocation defect caused by the secFamutation, a dominant negative mutation of the secY gene, has been identified (55). The gene, ydr, seems to encode a 19-kDa peripheral membrane protein. The overproduction of Ydr stabilized the overproduced level of SecY, similar to SecE, suggesting an interaction between Ydr and SecY. However, the role of Ydr in protein translocation remains unknown.

75

Protein Translocation orE. Coli

Chaperone--.-.--)~Preprotein ~ Signalpeptide

,~ ~

'~ATP

....

...

"l'l~"SecE

~'Signa,

SecE [ sock, p12]

gnal l]I ] Si,e~

~-~--__.._d

-

Figure 2. Model for protein translocation. Although SecD, SecF, signal peptidase and signal peptide peptidase are depicted only at a certain stages of translocation, they are assumed to be in the membrane throughout the translocation reaction, p12 = SecG; PMF; proton motive force.

VIII. MODEL FOR PROTEIN TRANSLOCATION Our current model for the translocation of preproteins across the cytoplasmic membrane of E. coli is shown in Figure 2. The first step is the binding of molecular chaperones to nascent polypeptide chains of preproteins being synthesized on ribosomes. This binding event prevents the polypeptide chains from folding into a translocation-incompetent conformation (26, 64, 71, 126). SecB, GroEL, and probably Ffh.4.SS RNA, function as

76


chaperones. There is no evidence to suggest a tight coupling of the translation reaction to the translocation reaction in prokaryotes. Preproteins then interact with SecA with the aid of the signal peptide (2). ATP stimulates SecA-preprotein interaction, possibly by making the SecAconformation suitable for the binding (62). The preprotein binding in turn causes the release of ATP/ADP from SecA (110). We propose that cytosolic SecA is primarily responsible for the interaction with preproteins (82). The signal peptide domain is essential for this interaction, and the positively charged amino acid residues at its N-terminus are involved in the interaction with SecA (2, 48, Moil, H. et al., unpublished data). The initiation complex, comprising a preprotein, SecA, and probably a chaperone, is then moved onto the secretory machinery in the membrane. It is proposed that the preproteins are transferred to SecA on the membrane in a complex with a chaperone (46). It remains unknown when chaperones dissociate from preproteins. SeeA, SexY, SecE, SecG, and SecD occur in the membrane roughly in an equimolar ratio, suggesting that they may constitute the secretory machinery. Interactions between these See proteins have been demonstrated in vivo (15, 16, 77, 79, 85, 103a: K. Nishiyama, S. Mizushima, and H. Tokuda, unpublished data) and in vitro (22, 23, 72, 73) (Figure 3). On the other hand, the results ofstoichiometric studies on SexY, SeeE, and SecG involving reconstitution suggest that these proteins may not exist as a tight complex in the membrane. They may form the complex machinery only in a preprotein-dependent manner. Joly et al. reported recently that SecE and SecY form a stable complex, while band 1 (SecG) is exchangeable (58a). It is unclear how preproteins are transferred from the cytosolic SecA to the machinery in the membrane. They may be transferred from the cytosolic SeeA to the membrane-bound SecA. Alternatively, the preprotein-SecA complex may replace SeeA pre-existing on the membrane. In any event, the transloeation complex comprising a preprotein, SecA, SecY, SecE, SeeD, and SecG can be formed. For the translocation of the first small domain (20--30 amino acid residues) of the preprotein, ATP, but not its hydrolysis, is apparently required, because the signal peptide cleavage that represents an early stage of the translocation reaction can be observed in the presence of AMP-PNP (106). The subsequent process, the translocation of the polypeptide chain of the mature domain, requires both ATP hydrolysis and protonmotive force (PMF) (106, 118). It is proposed that ATP hydrolysis is needed for SecA to dissociate from the translocation complex (106) and PMF is needed to release ADP subsequently formed from SeeA (II1) and to induce transmembrane movement of the protein that has been released from SecA (106). Subsequently, SecA rebinds to the translocation intermediate to allow further translocation. Thus, stepwise translocation can be assumed (106). Stepwise translocation by about 30 amino acid residues was recently observed using mutant proOmpAs possessing a looped structure in different positions of the mature domain (K. Uchida and S. Mizushima, unpublished data). It is also interesting in this respect that SecA was reported to undergo membrane insertion and deinsertion

Protein Translocation orE. Coli

Preprotein~. ~ ~.....

77

~

~ Translocatedprotein

Figure 3. Interaction map of components comprising the protein translocation machinery, pl 2 = SecG

during the translocation reaction (31a, 61a). Although ATP is essentially required for the translocation of the polypeptide chain to a certain stage, a later stage can proceed without ATP hydrolysis when PMF is supplied as an energy source (106,

118,119). Finally, proteins are released from the membrane with the aid of SecD (78) and PMF (37, 121). The precise molecular mechanism of the SecD function remains unknown. It may be involved in the pulling out of the C-terminal domain from the membrane, or simply the release of the completely translocated proteins from the membrane. The interactions between components comprising the translocation machinery are summarized in Figure 3. The interaction map is based on genetic and biochemical evidence. As described above, SecA, SecE, SecY, and p 12 are likely involved in a relatively early step in the translocation process, and interaction among these proteins has been suggested. On the other hand, SecD was shown to participate in a late stage of the translocation reaction (78). The sec titration study also suggested that SecD/SecF follows SecY in the translocation reaction (16). It is interesting in this respect that a stabilization test suggested that SecF interacts with both SecY and SecD (103a). It is probable that SecF connects the early and late steps of the translocation reaction. However, no biochemical evidence supports this view.

ACKNOWLEDGMENTS

We thank Dr. Hajime Tokuda for helpful comments and Ms. Chikako Motoyama for the secretarial support. The work from the authors' laboratories has been supported by grants from the Ministry of Education, Science and Culture of Japan; the Human Frontier Science Program Organization; the Nisshin Foundation; and the Naito Memorial Foundation.

78


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98. Randall, L. L., & Hardy, S. J. S. (1986). Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coil. Cell 46, 921-928. 99. Randall., L. L., & Hardy, S.J.S. (1989). Unity in function in the absence of consensus in sequence: role of leader peptides in export. Science 243, 1156-1159. 100. Ribes, V., ROmisch, K., Giner, A., Dobberstein, B., & Tollervey, D. (1990). E. coli 4.5S RNA is part of a ribonucleoprotein particle that has properties related to signal recognition particle. Cell 63,591-600. 101. Riggs, P. D., Derman, A. J., & Beckwith, J. (1988). A mutation affecting the regulation of a secA-iacZ fusion defines a new sec gene. Genetics 118, 571-579. 102. RSmisch, K., Webb, J., Herz, J., Prehn, S., Frank, R., Vingron, M., & Dobberstein, B. (1989). Homology of 54K protein of signal-recognition panicle, docking protein and two E. coil proteins with putative GTP-binding domains. Nature 340, 478-482. 103. Rothblatt, J. A., & Meyer, D. I. (1986). Secretion in yeast" reconstitution of the translocation and glycosylation of a-factor and invertase in a homologous cell-free system. Cell 44, 619--628. 103a.Sagara, K., Matsuyama, S., & Mizushima, S. (1994). SecF stabilizes SecD and SecY, components of the protein translocation machinery of the Escherichia coil cytoplasmic membrane. J. Bacteriol. 176, 4111-4 116. 104. Schatz, E J., Bleker, K. L., Ottemann, K. M., Silhavy, T. J., & Beckwith, J. (1991). One of the three transrnembrane stretches is sufficient for the functioning of the SecE protein, a membrane component of the E. coil secretion machinery. EMBO J. 10, 1749-1757. 105. Schatz, P. J., Riggs, P. D., Jacq, A., Fath, M. J., & Beckwith, J. (1989). The secE gene encodes an integral membrane protein required for protein export in Escherichia coll. Genes Dee 3, 1035-1044. 106. Schiebel, E., Driessen, A. J. M., Hartl, E-U., & Wickner, W. (1991). z~ttH+ and ATP function at different steps of the catalytic cycle of preprotein translocase. Cell 64, 927-939. 107. Schiebel, E., & Wickner, W. (1992). Preprotein translocation creates a halide anion permeability in the Escherichio coli plasma membrane. ). Biol. Chem. 267, 7505-7510. 108. Schmidt, M. G., Roilo, E. E., Grodberg, J., & Oliver, D. B. (1988). Nucleotide sequence of the secA gene and secA(Ts) mutations preventing protein export in Escherichia coll. J. Bacteriol. 170, 3404-3414. 109. Shun, L. M., Lee, J.-l., Cheng, S., JuRe, H., Kuhn, A., & Dalbey, R. E. ( 1991). Use of site-directed mutagenesis to define the limits of sequence variation tolerated for processing of the M 13 procoat protein by the Escherichio coil leader peptidase. Biochemistry 30, 11775-I 1781. 110. Shinkai, A., Lu, H.-M., Tokuda, H., & Mizushima, S. (1991). The conformation of SecA, as revealed by its protease sensitivity, is altered upon interaction with ATP, presecretory proteins, everted membrane vesicles, and phospholipids. J. Biol. Chem. 266, 5827-5833. 111. Shiozuka, K., Tani, K., Mizushima, S., & Tokuda, H. (1990). The proton motive force lowers the level of ATP required for in vitro translocation of secretory proteins in Escherichia coll. J. Biol. Chem. 265, 18843-18847. i 12. Simon, S. M., Blobel, G. (1992). Signal peptides open protein-conducting channels in E. coll. Cell 69, 677-684. 113. Stader, J., Gansheroff, L. J., & Silhavy, T. J. (1989). New suppressors of signal-sequence mutations, pdG, are linked tightly to the secE gene of Escherichia coll. Genes Dee 3,1 045-1052. 114. Stirling, C. J., Rothblatt, J., Hosobuchi, M., Deshaies, R., & Schekman, R. (1992). Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum. Mol. Biol. Cell 3, 129-142. 115. Sung, M., & Dalbey, R. E. (1992). Identification of protential active-site residues in the Escherichia coil leader peptidase. J. Biol. Chem. 267, 13154-13159.

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PIGMENT-PROTEIN COMPLEX ASSEMBLY IN RHODOBACTER $PHAEROIDES AND RHODOBACTER CAPSULATUS

Amy R. Varga and Samuel Kaplan

I. II.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Reaction Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Composition and Structure . . . . . . . . . . . . . . . . . . . . . . . . . B. Role of the RC-H Subunit . . . . . . . . . . . . . . . . . . . . . . . . . . C. Role of Bacteriochlorophyll . . . . . . . . . . . . . . . . . . . . . . . . . D. Other Gene Products Affecting RC Assembly . . . . . . . . . . . . . . . III. The LHI Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Composition and Structure . . . . . . . . . . . . . . . . . . . . . . . B. Membrane Insertion and Assembly . . . . . . . . . . . . . . . . . . . . . C. Other Gene Products Affecting LHI Assembly . . . . . . . . . . . . . . . IV. The LHII Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Composition and Assembly . . . . . . . . . . . . . . . . . . . . . . . . . B. Other Gene Products Affecting LHII Assembly . . . . . . . . . . . . . .

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Advances in Cell and Molecular Biology of Membranes and OrganeIles Volume 4, pages 85-104. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-924-9 85

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AMY R. VARGA and SAMUEL KAPLAN

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V. The Assembly Model for Pigment-Protein Complex.Abundance and R a t i o . . . VI. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . ". . . . . . . . . . . . . . . . . . . . . . . .

97 98 99 99

I. I N T R O D U C T I O N The anoxygenic photosynthetic bacteria provide a powerful system for the study of membrane biogenesis, protein targeting, and membrane protein complex assembly in prokaryotes. The primary factor in the utility of these organisms lies in the existence of an inducible membrane system of unique composition, termed the intracytoplasmic membrane (ICM). The synthesis of this membrane system is controlled by growth conditions, primarily oxygen tension and light intensity. Under aerobic growth conditions, the synthesis of the ICM is repressed and the cells exhibit a typical Gram-negative bacterial cell structure (Figure 1). Under

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Figure 1. Thin-section electron micrographs of R. sphaeroides. Panel A shows a cell grown under aerobic conditions. The outer membrane (OM) and cell membrane (CM) are clearly seen. Bar = 0.5 pm. Panel B shows a cell grown under photosynthetic conditions. The intracytoplasmic membrane (ICM) vesicles can be seen in this cell. Arrowheads indicate points at which ICM vesicles are connected to the CM. Bar = 0.5 pm.

Assemblyof Pigment-Protein Complexes

87

anaerobic growth conditions, the synthesis of the ICM is induced, forming by invagination from the cell membrane (Figure 1). The ICM remains attached to the cell membrane, but the composition of the ICM is distinct from the cell membrane in that the ICM houses the pigment-protein complexes and electron transport chain components involved in anoxygenic photosynthesis. The pigment-protein complexes that carry out light-harvesting and primary photochemistry are the photochemical reaction center (RC), the primary light-harvesting complex (LHI or B875), and the accessory light-harvesting complex (LHII or B800-850), and they are found exclusively in the ICM. The most intensely studied species within this group of bacteria in recent years have been Rhodobacter (R) sphaeroides and R. capsulatus, and this review will focus on the assembly of pigment-protein complexes in these two species (see 24, 25, 37, 41, 43, 59; for reviews on ICM structure and biogenesis).

II. THE REACTION CENTER A. Compositionand Structure The photochemical reaction center (RC) absorbs light energy directly or via the light-harvesting complexes, performs primary charge separation, and initiates photosynthetic electron transport (51). The RC is a highly ordered integral membrane complex consisting of three polypeptides and 10 noncovalently bound cofactors (51). The RC polypeptides are designated H, M, and Ldue to their relative mobilities in sodium dodecylsulfate polyacrylamide gels (49, 50). The genes for the RC-L and -M polypeptides reside in a multicomponent operon termed the puf operon, and these genes have been cloned and sequenced in both R. sphaeroides and R. capsulatus (74, 75, 77). The third polypeptide of the RC, RC-H, is encoded by an unlinked operon, puhA, that has also been cloned and sequenced (23, 77). The RC-L and -M polypeptides are very hydrophobic, and each has five transmembrane helices, whereas the RC-H subunit has only a single amino-terminal membrane helix and a large cytoplasmic domain (4, 73). There is a high degree of amino acid homology between the R. sphaeroides and R. capsulatus RC-L (78%, 73), RC-M (77%, 73, 77), and RC-H subunits (64%, 73), indicating the close evolutionary relationship between these species. The transcriptional expression of the RC polypeptide-encoding genes is controlled by oxygen (14, 80, 81). All of the cofactors comprising four bacteriochlorophylls (Bchl), two bacteriophephytins (Bphe), two quinones, one carotenoid, and one non-heine iron atom are noncovalently bound by the RC-L and -M subunits (29, 49), and the primary charge separation is carded out by a Bchl dimer referred to as the "special pair" (51). Cross-linking studies have shown that the protein components of the RC are closely associated with H-M-L, H-M, H-L, and M-L cross-linked species detected (34).

88


Studies on the RC have been greatly facilitated by the availability of the crystal structure of the RC complex from R. sphaeroides (3, 4) and a related species, Rhodopseudomonas viridis (21, 22). The detailed structural determinations now available have confirmed the positions of the cofactors relative to the RC-L and -M subunits and indicate that the RC-H subunit does not bind any of the cofactors (5, 76).

B. Role of the RC-H Subunit The role of the RC-H polypeptide in RC structure and function has not been well defined. RC-H does not bind RC cofactors and, in vitro, the RC-H subunit can be removed from the isolated RC, yielding the L-M complex termed RC~ (29), with little attendant loss in photochemical efficiency (2, 18), although RC-H may participate in the formation of the quinone-binding pocket and/or protonation of quinones (3, 18). Regions of RC-H exhibiting conserved amino acid sequence homology among R. sphaeroides, R. capsulatus, and R. viridis subunits (73) (residues 30-42 on the cytoplasmic side of the transmembrane helix and 229-244 near the carboxyl terminus) appear to be involved in the formation of salt bridges between RC-H and -L and RC-H and -M, as well as within the RC-H subunit (4, 11), which may be needed to stabilize the RC complex. Deletion of the carboxyterminal 26 residues of the R. sphaeroides RC-H subunit results in a photosynthetically incompetent phenotype (Lee and Kaplan, unpublished results), indicating that this region may be important in formation of the RC. The RC-H subunit differs from many other integral membrane proteins in that it does not follow the "inside positive" rule as proposed by von Heijne (72), who observed that most transmembrane helices are oriented with the most positively charged amino acids toward the cytoplasmic side of the membrane. The single amino-terminal transmembrane helix of the RC-H polypeptide has several negatively charged amino acids in the conserved region on the cytoplasmic side of the helix (73). This group of negatively charged residues does not appear to be critical for the proper membrane orientation of the helix since they can be deleted, and the helix orientation is maintained (12). In fact, the orientation of the RC-H transmembrane helix can be reversed by inserting positively charged amino acids at the amino terminus (12). The RC-H subunit is detected in the membrane of aerobic R. sphaeroides cells and is found in molar excess in membranes of photosynthetic cells (13). Recently, the synthesis of RC-H and RC-M was examined and RC-H was synthesized in excess relative to RC-M (and -L), resulting in a 3:1 ratio of H:M (71). When the ratio of RC-H to RC-M is disrupted by a mutation that affects the oxygen-regulation of the expression of the RC polypeptides, yielding a RC-H to -M ratio of 1:1.4, loss of excess RC-M is clearly seen (71), indicating that the normal excess of RC-H is important in RC assembly. The amount of stable RC-M, that is, RC-M incorporated into RCs, is most likely limited by the amount of available RC-H polypeptide, and when RC-H is in limiting amounts, the normal productive association between RC-M (and -L) and RC-H is disrupted and excess RC-M is turned over (71).

Assemblyof Pigment-ProteinComplexes

89

An R. sphaeroides mutant lacking the RC-H subunit is photosynthetically incompetent (58). This mutant, termed PUHAI, synthesizes wild-type levels of mRNA encoding the RC-L and -M subunits, abundant Bchl, LHII complexes, and ICM (58), and thus may assemble RC* complexes. In fact, RC* complexes are detected in PUHAI membranes but at levels at least 150-fold lower than RC complexes in wild-type membranes and in amounts insufficient to sustain photosynthetic growth (36). These data on PUHA1 raised the questions of whether RC-H is required for efficient translation of puf mRNA encoding RC-L and -M and whether RC-H is needed for efficient targeting to or assembly of RC-L and -M with cofactors in the ICM. Recent studies indicate that RC-M, and presumably RC-L, is synthesized at essentially wild-type levels in the RC-H-deficient mutant and that the RC-M polypeptide is efficiently targeted to the membranes of PUHA1 (71). The major difference in the behavior of the cofactor-binding RC-M polypeptide in the absence of the RC-H polypeptide is its stability. In PUHAI, RC-M is turned over rapidly with a hf2 of about 30 rain once it reaches the membrane, regardless of the presence of Bchl and ICM (Figure 2), whereas, in wild-type R. sphaeroides 2.4.1. the RC-M subunit in the RC is very stable with a tit2 of greater than 4 hours (71). Conversely, the RC-H polypeptide is not stable in the absence of the RC-L and -M polypeptides, regardless of the presence of Bchl and ICM, and shows a tlf2 of only 6-8 min (71), indicating that specific interactions among all of the RC polypeptides are required for assembly and stability of the RC complex in vivo. Photosynthetic competence of PUHA l can be restored by supplying the puhA gene in trans (58). This complementation also restores the stability of the RC-M and -H polypeptides in photosynthetic cells (Figure 2), although both polypeptides are unstable under aerobic growth conditions as is the case in wild-type cells (Figure 2) (71). The requirement for RC-H in the RC complex appears to be at the level of efficient assembly, promoting interactions between RC polypeptides and cofactors to form the functional complex.

C. Role of Bacteriochlorophyll The oxygen-regulated synthesis of Bchl was observed over 30 years ago (16), when it was demonstrated that high levels of oxygen repressed synthesis of Bchl. The synthesis of Bchl-binding polypeptides is coupled to Bchl synthesis (9, 13, 38, 65), so it was assumed that Bchl-binding proteins were not synthesized in the absence of Bchl synthesis. More recent studies have shown that mRNA encoding Bchl-binding proteins is present in aerobic cells (25, 40, 80, 81) and that the RC-H polypeptide is present in aerobic R. sphaeroides cell membranes (13). Utilizing sensitive radiolabeling techniques, the presence of additional RC polypeptides has been noted in the absence of Bchl, either under highly aerobic conditions in R. sphaeroides (71) or in Bchl- mutants of both R. sphaemides (65) and R. capsulatus (20). The common feature in all of these studies is that RC


90

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2

5

10

20

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.

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4mlul, ~mnnn~ '1.50

180

Chase lime (rain)

Figure 2. Autoradiographs of immunoprecipitated RC-M and -H polypeptides from pulse-chase 3SS-methionine labeled R. sphaeroides wild-type and mutant cells. (A) RC-M immunoprecipitated from PUHA1 grown under anaerobic/dimethyl sulfoxide conditions to induce Bchl synthesisand ICM formation. RC-M is not stable under these conditions. (B) RC-M immunoprecipitated from wild-type cells grown under photosynthetic conditions to induce Bchl synthesisand ICM formation. RC-M is stable under these conditions. (C) RC-H immunoprecipitated from PUHA1 containing the puhA gene in trans grown aerobically so Bchl and ICM synthesis are repressed. RC-H is not stable under these conditions. (D) RC-H immunoprecipitated from Tla cells grown aerobically. This mutant strain produces Bchl under these conditions, and RC-H is stable. (E) RC-H immunoprecipitated from PUHA1 containing the puhA gene in trans and grown under photosynthetic conditions so Bchl and ICM synthesis are induced. RC-H is stable under these conditions. polypeptides are synthesized but are very unstable in the absence of Bchl; RC-H exhibits a tl~ of 5 rain (Figure 2) and RC-M a tn~ of about 30 rain in aerobic R. sphaeroides cells (71). Under photosynthetic growth conditions the turnover of the polypeptides is measured in hours (Figure 2). In contrast, mutant strains that synthesize Bchl under aerobic conditions show significant levels of RC polypep-

Assemblyof Pigment-Protein Complexes

91

tides (strain TA-R; 65), and these polypeptides are stable (Figure 2) (strain Tla; 71), indicating that Bchl binding in the RC complex is critical for complex stability. Studies utilizing site-specific mutagenesis of Bchl-binding sites in the RC of R. capsulatus have shown that significant alterations can be made in the PC, such as substitution of Bphe for one Bchl of the Bchl2 special pair, without severely affecting the cells' ability to grow photosynthetically (10). In most cases the RC is still found in the membrane, with the exception of an RC-Lglul~ mutation (10), although it was not determined if the RC-L polypeptide was inserted into the membrane or if this polypeptide or the RC complex as a whole was unstable due to the mutation.

D. Other Gene Products Affecting RC Assembly The assembly of a structure as complex as the RC might be expected to involve assembly proteins and chaperonins, so regions genetically linked to the pufoperon have been examined to determine their possible roles in RC assembly. The Q open reading frame lies immediately upstream of the pufgenes encoding the LHI structural polypeptides (pufBA) that precede the genes for the RC-L and -M polypeptides (pujLM) (26, 77). Mutants with deletions or interruptions in orfQ show significantly lower levels of Bchl biosynthesis (17, 79). Studies in R. capsulatus indicate that orfQ is part of the puf operon and have shown that the absence of the pufQ gene product does not affect transcription of Bchl biosynthetic enzymes genes (7) nor is it absolutely required for Bchl biosynthesis (1, 52). These results suggest that PufQ may be a membrane-bound Bchl binding protein or catalytically involved in Bchl-protein interactions. There is significant amino acid homology between PufQ and the quinone-binding regions of the RC-L and -M polypeptides (7), implying that PufQ may be involved in redox sensing in the cell and coordinating Bchl with the RC and LHI complexes (7, 52). Site-specific mutagenesis studies in R. sphaeroides indicate that the orfQ gene product is possibly involved in assembly of pigment-protein complexes. A mutation removing the start codon for orfQ yields a photosynthesis-minus phenotype and very reduced levels of Bchl (33). A number of amino acid substitutions in the regions of OrfQ which show homology to RC-L and -M, exhibit specific variations in levels of light-harvesting complexes, although all remain photosynthetically competent. One such mutant, QalaT~ does not contain detectable LHI complex and shows an eight-fold increase in puc-encoded LHII complexes, although no change in puc-specific mRNA is seen (33). These data have led to speculation on the role of OffQ in the heirarchy of pigment-protein assembly, with priority order being RC>LHI>LHII. OrfQ, in conjunction with complex-specific assembly factors (33), may play a role in Bchl binding in pigment-protein complexes. The role of orfQ in light-harvesting complex formation is discussed below. The puff( open reading frame lies immediately downstream of the final structural gene in the pufoperon (pufM) (26, 77). A deletion ofpufX in R. sphaeroides yields

92


a photosynthetically incompetent phenotype, although puf transcription is not blocked and RC polypeptides are synthesized and assembled with cofactors into photobleachable RC complexes (28). Although the PufX polypeptide appears to copurify with the RC-LHI photosynthetic unit, it is not proposed to have a chaperonin function. Rather, the PufX-phenotype is attributed to a block in cyclic electron transfer resulting in impaired generation oftransmembrane potential (27). It appears, therefore, that PufX is not involved in RC assembly but that OrfQ may be involved. More detailed studies of these and other protein-protein interactions, such as those involving RC-H, as well as critical protein-cofactor interactions, will help to determine the order of events involved in RC assembly. In light of the fact that we have demonstrated the presence of the RC-H and RC-M polypeptides in the membranes of aerobically growing R. sphaeroides, albeit with shortened half-lives (71), we would extrapolate and suggest that all of the apopolypeptides corresponding to the three pigment-protein complexes are synthesized under aerobic conditions. As in the case of RC-H and RC-M, the cognate mRNA species for all pigment-protein structural polypeptides are present at low levels in aerobic cells (14, 39, 80), and thus the cell is "primed" to undergo rapid ICM synthesis and photosynthetic growth under appropriate conditions. We also know from studies of the oxygen-regulatory mutant T la (45), which makes RCs and LHII complexes and presumably ICM under highly aerobic growth conditions, that there is no intrinsic block to aerobic assembly of pigment-protein complexes (45, 71). In wild-type R. sphaeroides no Bchl is produced under aerobic conditions despite the presence of low levels of mRNA for Bchl biosynthetic enzymes (33). However, the orfQ gene-specific mRNA is not detected in aerobic cells. Therefore, we envision that the orfQ gene product in its role as an assembly factor is critical to the entire process and in its absence, at the posttranslational level, Bchl would not be inserted into each "pro-complex," and thus polypeptide components would turn over rapidly. Furthernmre, it is possible that the absence of OrfQ and the ability to insert Bchl into nascent complexes leads to an inhibition of the Bchi branch of the tetrapyrrole biosynthetic pathway (l 6). Thus, the analogy can be drawn between the anoxygenic photosynthetic bacteria and green plant chloroplast development. In the latter case, several of the protein components for photosynthetic membrane complexes are present in dark-grown, nonpigmented proplastids, but turn over rapidly in the absence of chlorophyll (Chl) (47). When exposed to light, the final steps in chlorophyll biosynthesis take place and pigment-protein complexes are assembled and remain stable in chloroplast thylakoid membranes (47). Therefore, to extend this analogy, it is not unreasonable to suggest the existence of key anaerobically.induced complex-specific assembly factors for bacteria and light-induced factors for green plants that serve to insert (B)Chl into the developing pigment-protein complexes.


93

III. THE LHI COMPLEX A. Composition and Structure The primary light-harvesting complex, LHI or B875, is in close association with the RC and transfers absorbed light energy to the RC (reviewed in 60). LHI is an integral membrane pigment-protein complex, and the minimal unit consists of oligomers of two low molecular weight polypeptides, termed o~and 13(63, 64, 66, 67), noncovalently bound with two Bchl and two carotenoid pigment cofactors (8). The genes encoding the LHI 0~and 13polypeptides are encoded by the pufoperon, proximal to the genes for the RC L and M polypeptides (39, 77) and are similarly regulated by oxygen at the transcriptional level (14, 81). The LHI complex has not yielded sufficiently ordered crystals for analysis, so the structural details analagous to the RC are not yet available for this pigment-protein complex.

B. Membrane Insertion and Assembly The membrane insertion of the o~ and 13polypeptides and assembly of the LHI complex have been the subjects of several studies in R. capsulatus. In vitro studies have shown that cotranslational membrane association of the LHI a and 13polypeptides is more efficient than post-translational association, suggesting that synthesis of these polypeptides occurs on membrane-bound ribosomes (70). It has been observed that the amino.-temfinal regions of LHI r and 13appear to be involved with membrane insertion of the polypeptides. The amino-termini of LHI r and 13are located on the cytoplasmic side of the ICM (62) and are oppositely charged (63, 64), which is an evolutionarily conserved feature of the LHI polypeptides (Figure 3) (61, 83). The LHI ~ polypeptide has several conserved features that appear to be involved in the proper membrane insertion and interaction of the LHI r and 13polypeptides. The amino terminus of LHI ~ has a structure typical of an uncleaved signal sequence of bacterial membrane proteins (56); typical characteristics include positively charged residues, a short hydrophobic region, and a helix-breaking residue adjacent to the membrane-spanning region of the protein (63). The aromatic residue Trp-8 is highly conserved in LHI o~polypeptides (Figure 3) and appears to be important in insertion of the LHI a polypeptide into the membrane (54). The helix-breaking Pro-13 residue is also highly conserved (Figure 3) and may be required for keeping the LHI 13 partner polypeptide in the membrane (54). The deletion of the charged residues Arg-I 4 and Arg- 15 leads to LHI oc being undetectable in the membranes, as does the deletion of the conserved Trp-8 plus the preceding two residues (53). Alteration of the hydrophobic stretch of amino acids LHI r 7-11 by deletion or insertion of amino acids had some effect on LHI stability, but had a more severe effect on the interaction between LHI o~and [3 such that efficient o~-[3formation was prevented, leading to rapid degradation of LHI [$ and lack of LHI spectral complexes (53). Deletion of the highly conserved LHIo~

94 R.c. LHI ~: R.s. LHI ~:

AMY R. VARGA and SAMUEL KAPLAN I I ADKNDLSFTGLTDEQAQELI~VYMSGLSAFIAVAVL/~LAVMIWRPWF

ADKSDLGYTGLTDEQAQELHSVYMSGLWPFSAVAVLAHLAVYIWRPWF

R.c. LHII~:MTDDK--AGPSGLSLKEAEEIIISYLIDGTRVFGAMALVAHILSAIATPWLG

R.s. LHII~: R.c. LHI ~

R.s. LHI a

R.c. LHIIa

R.s. LHIIQ

DDLNKVWPSGLTVAEAEEVHKQLILGTRVFGGMALIAHFLAAAATPWLG l I MSKFYKIWLVFDPRRVFVAQGVFLFLLAVLIHLILLSTPAFNWLTVATAHGYVAAAQ

MSKFYKINMIFDPRRVFVAQGVFLFLLAVMIHLILLSTPAFNWLEISAAKYNRVAVAE

MNNA-KIWTVVKIPSTGIPLILGAVAVAALIVliAGLLTNTT--WFANYWNGNPMATVVAVAPAQ MTN-GKIWLWKIPTVGVPLFLSAAFIASWILqAVLTTTT--WL-PAYYQGSAAVAAE

F~gure 3. Amino acid sequences of LHI and LHII (x and I~ polypeptides from R. capsulatus (R.c.) and R. sphaeroides (R.s.). Sequences are aligned to show highly

conserved amino acids (boldface type).

Asp- 12, Pro- 13 resulted in LHI a and [$being inserted into the membrane, however, [5 turns over rapidly yielding a LHI-phenotype. The assembly of LHI a and [~ in the membrane appears to depend upon specific interaction and contacts between the amino-termini, particularly LHIaPro-13, which is needed to fix LHI J5 in the membrane; changes in the relative positions of these critical amino acids, or their deletion, affect the tertiary structure of the polypeptides, and proper interaction between o~and [3 cannot take place (55). Just as with the RC polypeptides, the stability of the LHI polypeptides is interdependent; in the absence of one of the polypeptides, the remaining polypeptide is unstable (55). In a mutant that does not synthesize LHI ~, LHI ~ is incorporated into the membranes but is not stable; likewise, in a LHI [$- mutant, LHI a is hardly detectable in the membranes (55). In wild-type cells, LHI ~ appears in the membranes within 10 s of pulse-labeling with radiolabeled amino acids, followed at 40-50 s by the appearance of LHI u, leading to the speculation that LHI [5 performs a chaperone function for LHI a (55).

C. Other Gene Products Affecting LH! Assembly The region upstream of the puhA operon appears to encode a gene product involved in the assembly of the LHI complex in both R. capsulatus and R. sphaeroides. An interruption of this region in R. capsulatus, an open reading frame termed F1696 (Figure 4) (77), yields a mutant that is photosynthetically competent and has the LHII spectral complex but shows a 67% reduction of the LHI complex, although the pufoperon genes encoding the LHI structural polypeptides are intact and puf mRNA levels are normal (6). In R. sphaeroides a homologous region is found upstream ofpuhA (Figure 4). Deletion of the 3' portion of this open reading frame results in an LHI- phenotype that can be complemented by providing the complete open reading frame in trans (58). Additional R. sphaeroides LHI- mutants, RS 103 and Tla, are also complemented by supplying this puhA upstream region in trans (45, 58). Thus, it was suggested that this region upstream of puhA encodes an assembly factor specific for the LHI complex (58).

Assembly of Pigment-Protein Complexes

95

R.s.PucC: M S R I A E H L V R I G P R F L P F A D A A S D Q L P L R K L L R L S L F Q V A V G M A I V L L V G R.C.PucC: R A F A L K N L A R H A P K Y L P F A D V ~ E E V I P L S R L L R L S L F Q I T V G M T L T L L A G R.c.F1696:MLLSRRMIGSLAMTWLPFADA~ETLPLRQLLRLSLFQVSVGMAQVLLLG

50

R.s.PucC: T L N R V M I V E L K V P A S V V G I M A S L P L L F A P F R A L I G F K S D T H V S A L G W R R R R.c.PucC: T L N R V I 4 Z V E L A ~ A S L L S V M L A M P M L F A ~ F R T L I G F K S D T H K S A L G L R R A R.c.FI696:TLMRVMILELGVPALVVAAMISIPVLVAPFRAILGHRSDTYRSALGWKR-

100

R.s.PucC: V P W I Y R G T L A L W G G F A I M P F A L I V L G G Q G Y A E G Q P F W L G V S S A A L A F ~ H V R.c.PucC: - P W I W K G T I Y Q F G G F A I M P F A L L V L S G F G E S V D A P R W I G M S A A A L A F L L V R.c.FI696:VPYLWFGSLWQMGGLALMPFSLILLS--GDQTMGPAWAGEAFAGVAFLMA

150

R,s.PucC: G G G V M T I Q T V G L A L A T D L A P R E D Q P K V V G L M 2 ~ / V L L I S M I F A S I G F G W L L R.c.PucC: G A G V X I V Q T A G L A L A T D L V A E E D Q P K V V G L M T V M L L F G M V I S A L V Y G A L L R.c.F1696:OVGML~4TQTAGLALAADRATEETRPQWALL~'VMFLIGMGISAVIVGWLL

200

R.s.PucC: D P Y Y D A Q L Z K V I S G V A V A V F F L N M I A L W K M E P - - R N R A F T V K P E K E P E F G D H R.c.PucC: A D Y T P G R L Z Q V I Q G T A L A S W L N M A ~ Q E A V S R D R A R Q M E T A E H P T F K E A R.c.F1696:RDFDQITLZRWQGCGAMTLVLNVIALWKQEVM-RPMTKAEREAPRQSFREA

250

R.s.PucC: W R E F I S R E N A L H G L I V I G L G T L G F G M A D V I L E P Y G G E V L S H T V A E T T R L T R.c.PucC: F G L L M G R P G M L A L L T V I A L G T F G F G M A D V L L E P ~ G G Q A L H L T V G E T T K L T R.c. F 1 6 9 6 : W G L L A A E T G A L R L L A T V M V G T L A F S M O D V L L E P Y G G Q V L G L K V G Q T T W L T

300

R.s.PucC: ATFAGGGLVGFWLASWVLGRGFDPLRM-AIrLGAAAGLPGFFAI-HGATEH-TN R.c.PucC: A L F A L G T L A G F G T A S R V L G N G A R P M R W S A G C T D R V - - P G F V A I : M S S L I S Q D G R.c.FI696:AGWAFGALVGFIWSARRLSQGAVAHRVAARGLLVGI-VAFTAVLFSPLFGSKV

350

R.s.PucC: V W V F L L G T L V V G F G G G L F S H G T L T A T M R L A P K E Q V G L A L G A W G A V Q A T A A R,c.PucC: I W L F L A G T F A V G L G I G L F G H A T L T A T M R T A P A D R I G L A L G A W G A V Q A T A A R.c.F1696:--LFFASAMGIOLGSGMFGIATLTVAMMVVVRGHSGIALGAWGAAQATAA

400

R. s. PucC : G V A I A G A G V L R D - - I L Q A M - P D L S G Y G - - P G A P T V A V F A L E A G F L F L T H I V ! L P L 450 R. c. PucC : G L G V A L A G V V R D - - G L V A L - P G T F G S G - - W G P Y N T V F A I E A L ILIVAIAFAVPL R. c. F 1696 :GLAVF I G G A T R D L V A H A A A - A G Y L G S L H S P A L G Y T W Y V T E IGLLF ITLAVLGPL R. s. F1696 : AGKAGHLGALQD-G IGYSS-GTLE IGLLFATL IVLGPL R. s.PucC : LRSALAARRL. R.c.PucC : LKRGG. R. c. F1696 :VRPGSLFPKKPEAGEARIGLAEFPT. R. s. F1696 :VRTT I LSSERP -AGGTRVGLADFPT.

Figure 4. Amino acid sequences of PucC proteins encoded downstream of LHII a and ~ poly~ptides from R. sphaeroides (R.s.) and R. capsulatus (R.c.) aligned with the open reading frame upstream of puhA designated F1696 of R. capsulatus. The

carboxy-terminal portion of the R. sphaeroides F1696 ORF is also shown. Highly

conserved amino acids are shown in boldface type:

The results of Sockett et al. (58) point to the existence of an LHl-specific assembly factor upstream of puhA in R. sphaeroides, and a role for the OrfQ gene product was noted as well. It was shown that the LHI" phenotype of the PUHA1 mutant was complemented in trans by the orfQ gene, although the RC was not restored because the pultA gene was not present (58). These results, coupled with the observations on the effects of single amino acid replacements within OrfQ on the relative abundance of LHI and LHII complexes (see above and 33), point to a role for the orfQ gene product in LHI assembly.

96

AMY R. VARGAand SAMUELKAPLAN IV. THE LHII C O M P L E X

A. Compositionand Assembly The LHII complex is a secondary light-harvesting complex that passes absorbed light energy to LHI and ultimately to the RC (60). The minimal unit of the LHII consists of oligomers of two low molecular weight polypeptides, termed a and ~, noncovalently bound to three Bchl and one carotenoid (15, 57); the isolated R. capsulatus LHII complex also contains a non-pigment-binding polypeptide, designated LHII ~ (30, 31). The genes for the pigment-binding a and ~ polypeptides are encoded by the pucBA operon and have been cloned and sequenced (40, 78), and the gene for the LHII 6, pucE, has also been cloned and sequenced from R. capsulatus (69). There is significant sequence and structural homology between the LHI and LHII a and [3polypeptides (61, 83), indicating that the origin of LHII may have been via gene duplication of the LHI genes. Expression of the puc operon at the transcriptional level is regulated by both oxygen and light (40, 44, 80). Very little is known about the assembly of the LHII complex at the molecular level. It is known that mutations that eliminate carotenoid biosynthesis lead to an LHII- phenotype (42), but the mechanism is unknown. However, it has led to the hypothesis that, unlike the RC or LHI complexes, the LHII complex requires carotenoids for assembly. Mutations that limit levels of Bchl appear to limit the level of LHII complex, and control occurs at both the level of transcription of the puc operon and assembly of the complex (48), but again the mechanism(s) involved is unknown. There is significant sequence and structural homology between LHI and LHII a and ~) polypeptides (Figure 3) (61, 83). The general structure of the amino-terminus of the LHII a polypeptide is homologous to the LHI a polypeptide (Figure 3) so the same rules governing membrane insertion and assembly of the LHI complex may also be applicable to LHII complex assembly. Wood and Kaplan (manuscript in preparation) used a series of water-soluble benzodiazapines to inhibit pigment-protein complex formation in R. sphaeroides. Fluorazapam concentrations of 10-50 gg/ml were able to increasingly inhibit the formation of the LHII complex, and the complex was virtually absent at the higher concentration. Although there is a three-fold effect upon transcription of the puc operon, it is clear that the drug inhibits assembly of the LHII complex; however, it appears to have little effect on growth rate or LHI levels. Other benzodiazapine derivatives affect both LHI and LHII complexes, with the greater effect seen on LHII complex formation. Given the structure of fluorazapam and related compounds and the fact that their generalized effects appear to be minimal, we suggest that these compounds inhibit or interfere with the action of complex~specific assembly factors. For the LHI complex this interference would minimally be the F1696 gene product upstream of puhA (see above) and for the LHII complex this would be pucC gene product (see below).


97

B. Other Gene Products Affecting LHII Assembly The region downstream of the pucBA genes encoding the structural polypeptides of the LHII complex appears to be important in the expression and assembly of the LHII complex in both R. capsulatus and R. sphaeroides (32, 46, 68, 69). An open reading frame immediately downstream of pucA has been designated pucC and encodes a large hydrophobic polypeptide (32, 69), which is highly homologous to the open reading frame F1696 found upstream of puhA and involved in the assembly of the LHI complex (Figure 4) (see above; 6, 69) and which also shows homology to a chlorophyll-binding protein from plants (Moore and Kaplan, unpublished; 24). Deletion of pucC in R. capsulatus shows an LHII- phenotype with a six-fold decrease in transcription from the puc operon, although this transcriptional down-regulation is insufficient to explain the loss of the LHII complex (68). The authors speculate that the pucC gene product participates in the LHII assembly process via a feedback mechanism ensuring that LHII complex synthesis takes place only if all components are present, and the loss of any component of that assembly process, such as PucC, leads to a down regulation in puc expression (68). Additional downstream open reading frames, pucD and pucE (encoding the 8 subunit of the LHII complex), appear to encode gene products that are involved in the stabilization and assembly of the LHII complex, because deletions in these genes lead to destabilization of the LHII complex, in the membrane of R. capsulatus (68). In contrast to R. capsulatus, the R. sphaeroides LHII complex does not contain a 8 subunit, although a low molecular weight polypeptide designated 15A is always absent in LHII- mutants (42, 46); the relationship between this polypeptide and the ~isubunit in R. capsulatus is unknown. The region downstream ofpucBA is required for LHII expression and assembly in R. sphaeroides because when it is interrupted the cells no longer assemble LHII complexes, although the transcript for the puc structural polypeptides is present (46). The R. sphaeroides DNA sequence downstream ofpucBA shows an open reading frame highly homologous to the pucC of R. capsulatus, which is also designated pucC in R. sphaeroides (Figure 4), although no open reading frames homologous to the R. capsulatus pucD and E were found in R. sphaeroides (32). Since the amino acid sequence homology between the R. capsulatus and R. sphaeroides PucC protein is high (56% identity; Figure 4), the function of these gene products in LHII complex assembly is presumed to be similar (32), and perhaps functionally homologous to the F1696 LHI assembly factor found upstream ofpuhA (Figure 4).

V. THE ASSEMBLYMODEL FOR PIGMENT-PROTEIN COMPLEX ABUNDANCE AND RATIO In photosynthetically grown cells, the ratio of LHI to the RC is fixed at approximately 12:1 (82), and this has been referred to as the fixed photosynthetic unit (FPU;

98


25). On the other hand, the level of LHII can vary widely with respect to the FPU and is referred to as the variable photosynthetic unit (VPU; 25). Numerous independent experiments have revealed that in photosynthetically growing cells, the levels of mRNA encoding the apoproteins of the pigment-protein complexes is, in general, in excess to the actual amount of complexes present (19, 33, 44, 48). It appears that the cellular levels of their cognate apoproteins are in excess of the level of assembled complexes present, and thus unused apoproteins are turned over (71). Therefore, the availability of Bchl ultimately determines the cellular abundance of the three pigment-protein complexes, and this may be mediated through the OrfQ protein with the priority of RC>LHI>LHII. Given the above observations and the loose coupling between pigment-protein complex abundance and their cognate mRNA species and apoproteins, we can provide a model that helps to explain these varied observations. The striking resemblance between the pucC and F1696 gene products and their resemblance to green plant chlorophyll-binding proteins (24) suggest that these proteins have similar but not identical roles. Each protein has been implicated in the assembly of a specific complex: F1696 for LHI and pucC for LHII. Therefore, it is evident that the orfQ gene product is involved in both LHI and LHII assembly. We will assume that OrfQ is also involved in RC assembly and that one or more RC-specific assembly factors are present. Given the experimental evidence cited, we propose that OrfQ is capable of interacting with each of the complex-specific assembly factors to assist in the assembly process by inserting Bchl into the "pro-complex." We also assume that the affinity of OrfQ for each assembly complex-specific factor represents a hierarchy of levels with the RC factor(s) at the top and LHII at the bottom. Thus, with limiting Bchl (48), the RC complex will be present, followed by LHI and then LHII complexes. Because of the similarities of the complex-specific factors, they may compensate for one another when participants in the assembly sequence are either over- or under-expressed, depending upon their role in the assembly process. Thus, we imagine that the mutant OrfQ proteins described earlier are altered in amino acids residues that affect their affinitY for one or more complex-specific assembly factors. Therefore, the abundance of each complex and the ratio of one complex to another is broadly fixed at the level of transcription, but it is the posttranslational assembly processes that provides the high level of specificity. The OrfQ protein is clearly critical in this process, and its presence or absence may represent a switch between aerobic and anaerobic expression of the pigment-protein complexes and ICM.

VI. SUMMARY The process of the assembly of the pigment-protein complexes in R. sphaeroides and R. capsulatus is only beginning to be revealed. The role of the RC-H polypeptide as a component critical for the efficiency of association of the pigment-binding


99

RC-L and-M polypeptides with the RC cofactors has been elucidated. The structural role of Bchi in RC complex stability is also being refined because of the availability of the RC crystal structure and the recently developed techniques of site-specific mutagenesis to alter critical Bchl-binding amino acids. The role of assembly proteins in RC assembly is, at present, established at the genetic level, but the gene products that are encoded by these genetic elements are yet to be characterized, and the order of events remains to be demonstrated. The amino acid similarity of both the LHI and LHII pigment-binding polypeptides and, as recently discovered, their putative assembly factors, indicates the close evolutionary relationship of these two light-harvesting complexes, both at the structural level and at the level of assembly. Likewise, the hypothetical existence of RC-specific assembly factors remains to be demonstrated, but in light of the evidence presented, their existence appears quite plausible. Finally, the orfQgene product and its pivotal role in assembly of pigment-protein complexes and subsequent ICM development is clearly testable, and this system is ideal for future exploitation. The genetics and molecular genetic methodologies are established, the protein and cofactor components are readily available. Thus, researchers intent upon an understanding of membrane complex assembly processes have a wealth of potential at their disposal.

ACKNOWLEDGMENT This work supported by USPHS grant GM15590 to S.K.

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proteins LHI and LHII; reacdon center poylpeptides RC-L, RC-M, and RC-H; and enzymes of bacteriochlorophyll and carotenoid biosynthesis in Rhodobacter capsulatus during the shift from anaerobic 9 to aerobic gowOt. 1. Bacteriol. 168, 1180-1188. 81. Zhu, Y. S., & Kaplan, S. (1985). Effects of light, oxygen and substrates on steady state levels of mRNA coding for ribulose 1,5-bisphosphate carboxylase and light-harvesting and reaction center polypeptides in Rhodopseudomonos sphaeroides. J. Bocteriol. 162, 925-932. 82. Zhu, Y. S., Kiley, P. J., Donohue, T. J., & Kaplan, S. (1986). Origin of the mRNA stoichiometry of the puf operon in Rhodobacter splu~eroides. J. Biol. Chem. 261, 10366-10374. 83. Zuber, H., & Brunisholz, R. A. (1991). Structure and function of antenna polypeptides and chlorophyll-woteins complexes: principles and variability, in H. Scheer (Ed.). Chlorophylls. (pp. 627-703). Boca Raton, FL: CRC Press.

IDENTIFICATION AND RECONSTITUTION OF ANION EXCHANGE MECHANISMS IN BACTERIA

Atul Varadhachary and Peter C. Maloney

I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Chemiosmotic Circuits . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mechanisms of Carrier-Mediated Transport . . . . . . . . . . . . . . . . C. Anion-Linked Exchange in Bacteria . . . . . . . . . . . . . . . . . . . . II. Pi-Linked Exchange Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . A. The History of Pi Self-Exchange . . . . . . . . . . . . . . . . . . . . . . B. Current Status of the Pi-Linked Exchange Family . . . . . . . . . . . . . III. Two Key Experiments with Pi-Exchange . . . . . . . . . . . . . . . . . . . . A. Choosing between Antiport and Symport Mechanisms . . . . . . . . . . . B. Stoichiometry and Selectivity . . . . . . . . . . . . . . . . . . . . . . . . IV. Reconstitution of Pi-Linked Exchange . . . . . . . . . . . . . . . . . . . . . . V. Carboxylate-Linked Anion Exchange . . . . . . . . . . . . . . . . . . . . . . A. Oxalobacter formigenes . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Other Indirect Proton Pumps . . . . . . . . . . . . . . . . . . . . . . . . Advances in Cell and Molecular Biology of Membranes and Organeiles Volume 4, pages 105--128. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-$5938-924..9

105

106 106 107 108 109 109 111 113 113 114 116 119 119 121

106

ATUL VARADHACHARY and PETERC. MALONEY

Vl. Other Bacterial Anion Exchange Systems . . . . . . . . . . . . . . . . . . . . VII. Common Themes and Structural Rhythms . . . . . . . . . . . . . . . . . . . VIII. Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

122 123 124 125 125

I. I N T R O D U C T I O N A. Chemiosmotic Circuits Active transport at cellular and organellar membranes is driven by "pumps" or by "carders." Systems in the former category, such as the ion-motive pumps, move their substrates against an electrochemical gradient by linking the transport step to a chemical (or photochemical) transformation. On the other hand, carriers allow substrate movement along an electrochemical gradient, so that if one substrate is taken "uphill," a second substrate must at the same time move in the "downhill" direction. It is the indirect coupling between such primary (pumps) and secondary (carrier) reactions that forms the basis for much of the solute transport observed at bacterial (and other) membranes. In such cases, it is most common to find that a primary proton pump extrudes H +, generating a cytoplasm that is both alkaline and electrically negative relative to the external phase (34, 46), and that these gradients, which together comprise the "proton-motive force," support the activity of a variety of proton-linked carriers. Similar "chemiosmotic circuits" can be built on the movement ofNa + (or other) ions (13, 14, 58), but those that depend on H + seem to be the most common in bacteria and in the major eukaryotic organelles (34, 55). And while in most instances a primary proton pump is responsible for generating a proton-motive force, other arrangements can on occasion serve the same function. In fact, as described later, one can even organize events so that a carder itself serves in this capacity (6, 54). As shown by Figure 1, a surprisingly complex network of membrane transport events can be generated when chemiosmotic coupling ties together the activity of primary and secondary systems. The general principle, of course, extends well beyond this simple illustration, and we now appreciate that the ionic circuits described by Mitchell's chemiosmotic theory drive many different processes, ranging from substrate accumulation to cell motility (42). The availability of such a diverse repertory of driven events, each with its own substrate and kinetic specificity, confers a remarkable flexibility to the cell in its dealings with the external environment, and it is probable that "invention" of this principle was essential to the evolutionary transition from an acellular to a cellular mode of life

(38).

Anion Exchange Mechanisms in Bacteria

107

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Figure 1. Chemiosmotic circuits at the bacterial membrane. All membranes have a collection of pumps and carriers. Those shown here can be found in anaerobes or in facultative organisms growing under anaerobic conditions, where the proton circulation is usually initiated by a proton-translocating ATPase. (Taken from ref. 34)

B. Mechanisms of Carrier-Mediated Transport Carrier-mediated events are traditionally placed in one of three mechanistic groups: (1) that of uniport (also termed facilitated diffusion); (2) symport (or co-transport); or (3) antiport (exchange). Examples of each of these are given in Figure 1. Thus, systems that catalyze uniport (carrier #1 in the figure) allow substrates to move freely along their electrochemical gradient and movement becomes independent of a chemiosmotic circuit, except insofar as the substrate may be charged or behave as a weak acid or base. Uniport systems are well represented in mammalian cells, but are less frequently found in the prokaryotic world, although a few examples have been described (34). Symporters (#2, #4) mediate a reaction in which two or more substrates move in tandem across the membrane; symport is widely spread among bacteria, and this mechanism is discussed in detail with reference to the lactose permease elsewhere in this volume. The remaining mechanistic category is that of antiport. Antiporters move substrates in opposite directions, and in the context of Figure 1 we might distinguish two different roles for such transporters. On the one hand, the activity of proton:cation exchange can effectively "transduce" a proton-motive force into an equivalent cation-motive force. In this way, for example, the presence of a Na§ § antiporter can extrude the Na + that enters by Na+-linked symport, allowing a subsidiary Na + circuit to coexist within the dominant H + circulation (Figure 1). In this sense, then, antiport is used to extrude a substrate. One might alternatively view an exchange system as a way to accumulate desired substrates. The known bacterial anion exchange mechanisms, which are the topic of this chapter, fall into this latter group.

108


C. Anion-Linked Exchange in Bacteria In bacteria, there are now two well-defined families of anion exchange reactions. The Pi-linked exchange family comprises a recently described group of carriers, each one of which accepts inorganic phosphate as a low affinity substrate and some specific organic phosphate as the high affinity ligand. The example, Figure 1 (carrier #5) depicts the activity of UhpT, a Pi-linked antiporter whose physiological action is directed to the transport of glucose 6-phosphate (G6P). This protein catalyzes electrically neutral exchange involving substrate(s) carrying two negative charges. The unusual feature of this protein is that these anionic equivalents can be taken as either a pair of monovalent substrates or as a single divalent species. Such plasticity has (as one might expect) real benefit in the context of bacterial cell biology, since the pH gradient is normally oriented as alkaline inside (see above). Thus, because monovalent sugar phosphate anions would dominate in the relatively acidic external phase, a pair of monoanions would move inward during the first half-turnover. Once within the cell, they would be stripped of their accompanying protons in the relatively alkaline interior, generating a pair of divalent anions. Since G6P is the high affinity substrate, and since two negative charges must move outward during the next half-turnover, it is likely that this latter step is preferentially accomplished by outflow of a single divalent G6P anion (rather than, say, a pair of monovalent Pi anions), leaving behind divalent G6P as a source of carbon and phosphorous. The net reaction, then, is import of 2H § and 1G6P2-, so that the antiport reaction effectively mimics a proton-linked symport, and sugar phosphate accumulation is driven by the pH gradient. This physiological model satisfactorily accounts for the available biochemical data, and it will be of great interest to describe the molecular basis of the rules governing such substrate selectivity and stoichiometry. Since very recent work (64) has identified a portion of the translocation pathway in UhpT, such information may be accessible in the near future. The second main family of bacterial anion exchange is exemplified by the antiport of oxalate with formate, as found in the gram-negative anaerobe, Oxalobacter formigenes. This exchange is catalyzed by a carrier known as OxlT (for oxalate transport) (54) and is of particular interest in the present context, because its activity is part of an "indirect" proton pump (6, .54). OxlT mediates the uptake of divalent oxalate (-OOC-COO-) against monovalent formate (HCOO-), its metabolic derivative. Since the one-for-one "vectorial" exchange carries negative charge inward, and since a "scalar" internal proton is consumed as formate is produced, the ensemble system acts as a proton pump. The antiporter, OxlT, plays a central role in this process and serves as the first of several examples in which a secondary carrier is incorporated into an "indirect" proton pump. The number of such systems is likely to grow much larger as we learn more about how bacteria extract energy from simple organic molecules. In the material that follows, we will review the evidence supporting these views of Pi-linked and carboxylate-linked exchanges, citing experiments that exploit


109

intact cells, membrane vesicles, and even proteoliposomes. A molecular analysis of these systems is now underway, and early results indicate that such exchangers can continue to add important information to mechanisms of antiport and membrane transport.

II. PI-LINKED EXCHANGE MECHANISMS A. The History of Pi Self-Exchange The study of Pi-linked exchanges began in the early 1950s, but it was not until recently that we realized these antiporters could carry both inorganic and organic phosphates. As a result, historical comments must allude to transport of both kinds of substrates. In 1953, Mitchell found that in Staphylococcus aureus (then called Micrococcus pyogenes), inorganic phosphate readily moved between the internal and external pools (41, 43). Full characterization of this exchange represented the first study of bacterial transport, and in doing this, Mitchell accomplished two important tasks. First, he showed that bacteria have an internal aqueous phase separated from the outside world by a lipoidal barrier that excludes large molecules--that is, bacteria have a membrane-bound cytoplasm, just as other cells do. Second, Mitchell exploited the fact that the Pi self-exchange reaction was highly sensitive to heavy metals, such as mercury and mercurials. By a series of clever inhibition experiments, he proved that the number of Pi molecules undergoing transport greatly exceeded the number of Hg atoms needed to inhibit the process. Accordingly, he concluded that the exchange was catalytic and that internalized Pi was not bound to a receptor, directly refuting the "exchange-adsorption" hypothesis for solute accumulation in bacteria. Mitchell also observed that Pi self-exchange was specific for the monovalent anion, HePO41-. This finding had no particular impact at the time, but it later provided our own studies with an important intellectual and experimental tool with which to complete analysis of this system. Figure 2 gives an example of the kind of Pi self-exchange reaction Mitchell studied in S. aureus. (The experiment is actually from our own work with Streptococcus lactis.) Cells harvested from the stationary phase of growth were suspended in a medium containing about 50 I~M Pi. Such cells have an internal Pi pool of 50 raM, but lack internal metabolizable reserves. As a result, ATP levels are low and the proton-translocating ATPase of this anaerobe (e.g., Figure 1) is unable to sustain a proton-motive force. Nevertheless, one can demonstrate membrane transport. Tracer amounts of 32pi are readily taken up by the cell, and at the steady state as much as 50-70% of the label has been internalized, where it is found in the pool of free inorganic phosphate (note that the label is readily chased by excess unlabeled Pi). Moreover, this process requires no metabolic energy; that is, it occurs in the absence of glycolysis, and is insensitive to a protonophore (FCCP) and to an


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Figure 2. The Pi self-exchange reaction in Streptococcus lactis. Accumulation of 32pi was monitored after its addition to cells suspended in 300 mM KCI/20 mM MOP$/K (pH 7). At the arrow, part of the control suspension was given 4 mM Pi; where indicated, parallel suspensions had been treated with 10 pM FCEP (p-trifluoromethoxyphenylhydrazone) or I mM DCCD (N,N'-dicydohexylcarbodiimide). (Taken from ref. 39) inhibitor (DCCD) of the proton-translocating ATPase. These findings therefore establish a kind of paradox. The cell membrane is clearly permeable to Pi, which is free to rapidly move inward or outward, as traced by 32pi. Yet at the same time the membrane sustains a large chemical gradient for Pi; an inside/outside ratio of about 1000:1. How can this gradient be maintained across a permeable membrane in the absence of energy? The only satisfactory answer is that there is a simple one-for-one exchange of internal and external Pi. Mitchell also noted that, while Pi self-exchange was evident in resting cells, in metabolizing cells there was a net accumulation of Pi. From this observation, he hypothesized that the Pi exchange carrier was designed to operate as an energy-dependent Pi uptake system, but that it could cycle idly in the resting state. Some years later, Harold and coworkers continued the study of bacterial Pi transport in another gram-positive cell, Streptococcusfaecalis (23). They observed the expected energydependent system for net Pi transport, but could not demonstrate its exchange component. This apparent conflict was not resolved until much later, when experiments with Escherichia coil made it obvious that bacteria have many different systems that handle Pi. For example, we now know there are systems dedicated to the transport of phosphate alone; in E. coil these are exemplified by the multicomportent, ATP-dependent system known as Pst and by the H+/Pi symporter known as


111

Pit (10, 52, 53, 61). E. coli has two other systems that take Pi with low affinity and sugar phosphates with relatively high affinity (24, 48). These latter systems are now known to mediate the homologous Pi self-exchange reaction, while the Pi-specific porters (Pst, Pit) do not. In retrospect, then, it seems clear that Harold studied one of the dedicated Pi systems, while Mitchell worked with one of the less discriminating sugar-phosphate porters. These sugar phosphate transport systems have also had a long history. It had been known since 1950 (51) that E. coli could use sugar phosphates as sources of carbon and phosphorous, and in 1962, Lin et al., (32) demonstrated that G3P could enter cells without prior hydrolysis. Similar observations were soon made for G6P (19, 22, 48), and the relevant transport systems came to be known as GIpT (for glycerol phosphate transport) and UhpT (describing the uptake of hexose phosphate). In the 1970s, as it became clear that the chemiosmotic theory correctly described membrane events in bacteria, further work seemed to confirm that these transporters fit into the traditional mold as proton symporters (17, 63), and it was not until 1984 that their exchange nature became evident. At that point, independent studies in Streptococcus lactis (e.g. Figure 2) suggested that at least one of these sugar phosphate transporters mediated a Pi self-exchange of the sort described by Mitchell 30 years earlier (39). This idea carried with it the implication that other such systems might also use antiport as their mechanistic base. Further biochemical analysis of the streptococcal system, later extended to E. coli and to S. aureus, now provides definitive evidence supporting the idea of antiport as the operative mechanism of all Pi-linked exchangers (2, 40).

B. Current Status of the Pi-Linked Exchange Family As their name implies, all members of the Pi-linked exchange family take inorganic phosphate as a low affinity substrate, showing Michaelis constants for transport (Kt) on the order of 0.5 mM to 10 raM. In addition, each family member has one or more higher affinity sugar-phosphate substrates with binding constants in the range of 10 I.tM to 100 ~M (40). This family now includes seven known examples, each of which has a different pattern of preferred substrates (Table 1). The examples known in gram-positive cells use hexose phosphates as their natural substrate. The four gram-negative examples take either hexose or triose phosphates, depending on the specific example. In all cases, there is sufficient biochemical work to justify grouping these systems into a single family, but a structural relatedness has been verified only for the gram-negative cases, all of which show significant amino acid sequence homology to one another (__.30-35% amino acid identity) (I 5,

20, 21, 26). A diagnostic feature of this family is the Pi self-exchange reaction described earlier (Figure 2), a reaction that can be documented at all levels of biochemical analysis--in the intact cell, with membrane vesicles and with proteoliposomes. This exchange is relatively specific. No inorganic cations move with Pi during


112

Table 1. Pi-Linked Anion Exchange Systemsa Cell l)~e

Geneb

Primary Organic Substrate

V,~g Ratioc

E. coil

uhpT slpT uhpT pgtP

G6P, M6P G3P G6P PEP G3P G6P, G3P G6P, M6P

5.5

S. lyphimurium

S. aureus 8. iactis

6.5 4.2 4.7

Notes: 'Reviewed in (40). The reactions in S. oureusand S. lactisate presumed to reflect the activity of single systems, since transport-negativemutants show neither homologous nor heterologoes ~change (56, unpublished work). bSystems described by fonr-lemer genetic symbols show close sequence homology (15, 20,

2L 26).

CRatio of maximal velocities for homologous and hetetologons exchanges (2, 3, .56, 60, unpublished work with UhpT).

exchange, and of the inorganic anions tested, only arsenate is known as an alternative substrate; in fact, it appears that these systems do not distinguish between the phosphate and arsenate anions. This tight specificity is accompanied by an equally tight selectivity--in the two examples studied carefully ($. aureus and S. lactis), only monovalent phosphate (arsenate) partakes in exchange (39, 43). More relevant to physiological function is the heterologous exchange of Pi for a sugar phosphate. The identity of the favored sugar phosphate varies with the individual carrier, as does its Michaelis constant for transport; as noted above, it seems generally true that Pi is of relatively low affinity, while the preferred organic phosphate is of much higher affinity. In addition, for reasons that are not clear, the heterologous exchange is slower, usually by about fivefold, than the homologous exchange reaction (Table 1). But perhaps most important, in the cases examined so far, this heterologous exchange proceeds with no net transfer of charge--that is, the exchange is electroneutral. (This behavior contrasts strikingly with that of the carboxylate-linked exchange described later.) Note that in principle, each exchange carrier should also display a sugar-phosphate self-exchange, so that in thinking about the biochemistry and cell biology of these systems, one has to integrate the expected behavior of all three possible modes of operation. In addition, since each substrate (Pi, sugar phosphate) is a weak acid with a pK in the physiological range, and since these carriers might, in principle, select some or all salt forms of its substrates (e.g., monovalent Pi, or both divalent and monovalent sugar phosphate), the analysis of any particular situation can become complex (see below). Fortunately, with adequate control over some of these variables, one may simplify the analysis considerably, and to illustrate this, we have chosen to discuss two key experiments in the next section.


113

III. TWO KEY EXPERIMENTS WITH PI-EXCHANGE A. Choosing between Antiport and Symport Mechanisms

Work with both gram-positive (S. lactis, S. aureus) and gram-negative (E. coil) cells has contributed importantly to the biochemfcal analysis of Pi-linked exchange and to our understanding of mechanism. Among the most important of these experiments have been those that exclude the possibility of symport and positively support the idea of antiport. Recall that for some time sugar phosphate transporters were assumed to operate as symport mechanisms. Work with the lactose carrier of E. coli had established that symporters could display a variety of partial reactions, including a prominent substrate-substrate exchange, in the absence of a driving ion-motive force. Therefore, it was feasible (at least in principle) that the exchanges associated with the UhpT transporters orS. aureus, S. lactis, and E. coil were similar partial reactions on the part of a 2H+/G6P 2- symporter. To evaluate this alternative directly, right-side-out membrane vesicles from E. coli were prepared using sulfate rather than phosphate as the main internal anion. When presented with an oxidizable

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114


energy source, these vesicles accumulated Pi and other substrates by the expected proton-linked reactions (e.g., via Pit, and other carriers), but they could not accumulate G6P (Figure 3A)--even though tests of the cells used to make vesicles had shown vigorous UhpT activity. However, when G6P was added after vesicles had taken up Pi, the capacity to transport G6P was recovered (Figure 3B) (57). This kind of experiment, which can be replicated in S. aureus (56), excludes the model of symport for G6P transport and strongly favors the idea of antiport.

B. Stoichiometry and Selectivity

Variable Stoichiometry Measurement of exchange stoichiometry has also proven instructive--perhaps even essential--to the complete description of Pi-linked exchange. These data provided the basis for both a satisfactory biochemical model of antiport and a way to account for some otherwise puzzling aspects of cell biology. A direct approach to this issue was best done with the UhpT cartier of S. lactis, since in this cell the main sugar phosphatase is completely blocked by low concentrations of the phosphatase inhibitor, orthovanadate. Nevertheless, the conclusions drawn from these studies almost certainly characterize the sugar phosphate carrier proteins of S. attreus and E. coli. When assayed at pH 7, a measurement of net fluxes shows that in S. lactis the macroscopic stoichiometry of heterologous exchange by UhpT is 2:1, Pi:G6P. Since the reaction is electrically neutral, and since Pi-self exchange selects monovalent Pi (in both S. lactis and S. aureus (39, 43), this bulk stoichiometry suggests that at a molecular level the protein moves 2H2PO41- against I G6P 2- (4). In turn, this suggests the protein can bind either a pair of monovalent anions or a single divalent anion. Having established that divalent G6P was a substrate, it was next important to ask whether monovalent G6P could be accepted--bacteria often find themselves in relatively acidic environments where monovalent G6P would dominate (pK = 6.1 ). Accordingly, two relevant parameters were examined at pH 5.2, where HG6P lis enriched. First, kinetic tests revealed a considerably lower rate of exchange but no substantial change in the affinity (IG) of the system for G6P. For this reason, it was concluded that both divalent and monovalent forms of G6P were substrates of UhpT (4). The second test involved determination of the net stoichiometry of heterologous exchange at pH 5.2. Surprisingly, at pH 5.2 the exchange ratio had a value of 1:1 (Pi:G6P), rather than the 2:1 value measured at pH 7 (4) (Figure 4); an intermediate value of stoichiometry was found at an intermediate pH (1:1.5 at pH 6.1). These findings seemed most easily understood after recalling that both monovalent Pi and monovalent G6P anions are acceptable substrates, at least as determined by kinetic tests. Therefore, the 1"1 stoichiometry at acid pH likely reflects the exchange of monovalent salts, and to be consistent with the earlier

Anion ExchangeMechanisms in Bacteria

115

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Figure 4. Variable stoichiometry of Pi-linked exchange in Streptococcus lactis. (A) Membrane vesicles loaded with 50 mM KPi were suspended in 125 mM K2SO4/20 mM MOPS/I( (pH 7) and exposed to 180 laM [14CIG6P, with 0.25 mM Na3VO4 added to block phosphatase activity. (B) After 45 rain, vesicles were isolated by centrifugation, resuspended, and distributed to tubes with assay buffer at pH 7 (v ,T), pH 6.1 (I,c3) or pH 5.2 (A, zx).After a further 10 rain, 3 mM 32pi was added (arrow) and samples taken to evaluate 32p gained (open symbols) and 14C lost (closed symbols). (C) Phosphate gained is correlated with sugar phosphate lost, using symbols as in B. The lines drawn have slopes of 1 (pH 5.2), 1.5 (pH 6.1) and 2 (pH 7). (Taken from ref. 4) finding of two monoanion binding sites (at pH 7), one must only imagine that the bulk ratio of l: 1 (pH 5.2) corresponds to a molecular exchange ratio of 2:2. Clearly, these results suggest that UhpT has an unusual pH-dependent variable stoichiometry, at least with respect to its sugar phosphate substrate. We believe the interpretation of this phenomenon is correctly embodied by the ionophore, A23187, a simple molecule that also shows variability in exchange stoichiometry. A23187 has a pair of carboxyl groups, each of which must be paired with a positively charged substrate before transport can occur. This is accomplished by the binding of either a pair of protons (2H+), or, after a change of conformation, the binding of a single divalent cation (e.g., Ca2+). The ionophore, then, shows a pH-dependent stoichiometry---either 2:2 (2H+:2H+), 2:1 (2H+:Ca2+), or 1:1 (Ca2+:Ca2§ according to the concentrations of each substrate in either aqueous compartment. In the same way, we suggest that UhpT has a pair of binding sites (perhaps a pair of arginine residues) that accept two monovalent anions, and that each of these sites (arginines?) can contribute to a more complex binding pocket that accepts divalent

116


substrate. In this way, the phenomenon of variable stoichiometry for UhpT is attributed to a pH-dependent change in the protonation state of its substrates and not to any special property of the protein itself (4, 40).

The Phenotypeof Proton-LinkedSymport These arguments are relevant to understanding the cell biology of UhpT, for we can now appreciate why these proteins were categorized as proton-linked symporters in the past (17, 63). The analysis of variable stoichiometry has led to the conclusion that UhpT can accept either a pair of monovalent sugar phosphate anions or a single divalent anion, depending on which is the more prevalent (i.e., depending on pH). Therefore, when a pH gradient exists, it is clear that the neutral self-exchange of sugar phosphate will be in a 2:1 ratio, as discussed earlier (Figure 1), and a protein whose mechanistic base is antiport will appear at the macroscopic level to operate as a 2H§ 2- symporter. Note also that the model of symport carries risks that antiport does not. If E. coil growing on G6P were to consume its substrate, a symport mechanism would allow net efflux of internal G6P, whereas an antiport mechanism would not. (This in no way points to antiport as a necessary mechanism, but it does make one feel more comfortable with the general idea.)

Setting the Carbon:PhosphorousRatio Despite its prominence in the discussions outlined above, the self-exchange based on sugar phosphate cannot be the only mechanism used by UhpT. For if that were the case, growth on G6P would seriously distort the normal carbon:phosphorous ratio of40:1 (33). It seems reasonable, therefore, to suggest a concomitant use of the heterologous 2Pi:G6P exchange, allowing the import of sugar to proceed against the simultaneous export of Pi--the net result of 2Pi:G6P antiport. It is not necessary to assume unusual regulatory events to set the balance of these exchange modalities (although such regulation may well occur), for it is easy to see this occurring by mass action alone, as the internal pH and G6P and Pi pools fluctuate in response to the substrate fluxes.

IV. RECONSTITUTION OF PI-LINKED EXCHANGE Any rigorous analysis of antiport systems requires that one have experimental control over the composition of internal and external compartments, and this is most easily accomplished in liposomal systems containing only membrane proteins and simple salts. For that reason, it was important to develop adequate methods of reconstitution for the study of Pi-linked exchange. The techniques we devised for this purpose have greatly aided the study of Pi-linked events and have also had a


117

Table 2. Effect of Osmolytes on Reconstitution of Pi-Linked Exchange a

Test Compound Valine (7%) Methanol (10--20%) None Ethylene glycol (10-15%)

32Pi Transport nmol/mg Protein 15 27 + 5 36 + 9 42 + 16

Proline (12%) Glycine (8%) Glucose (20%)

305 410 425

Glycerol (20%)

530 :k98

Note: =Proteoliposomes loaded with 100 mM KPi were suspended in sulfatebased MOPS-buffered medium, and the Pi self-exchange reaction w a s measured at steady state after a 75-rain incubation with 50 IJM 32Pi.(Data from a larger survey reported in ref. 3.)

positive impact on the more general use of reconstitution as a tool in membrane biology. The general methods we adopted were chosen for their simplicity of use. Thus, we have most often relied on the technique of octylglucoside-dilution to form liposomes. This approach, introduced by Thompson and by Racker and his colleagues (50), exploits the water-soluble non-ionic detergent, octylglucoside. After solubilization by octylglucoside in the presence of exogenous lipid (see below), proteins are incorporated into a liposomal membrane by a dilution step which lowers detergent to a level below its critical micellar concentration (about 25 mM for octylglucoside). The insoluble lipid and protein form small (ca. 15 nm diameter), very stable, unilameilar liposomes with an internal aqueous volume of about 1 ~l/mg phospholipid (3). Our experiments have also drawn on the work of Newman & Wilson, who noted the beneficial effect when solubilization was performed in the presence of excess lipid (45). Despite the general success of reconstitution in other systems, our initial attempts to apply these methods to the Pi self-exchange reaction were disappointing; the activity recovered in proteoliposomes reflected only a small part of that present in the parent membrane vesicles, indicating a significant inactivation. Pronounced inactivation is, in fact, the usual timing at the beginnings of such a study, and one normally expends considerable time and labor in searching for conditions compatible with retention of function. These searches may now be easier, because the solution to this problem for Pi-linked exchange has uncovered an approach of general value. Analysis of this issue for the antiporter of S. lactis led us to conclude that the protein was subjected to an unpredictable all-or-none inactivation during solubilization. On the other hand, when protein was finally reincorporated into a liposomai membrane, after the dilution step, transporters that had survived were

118


able to operate with their normal kinetic properties (3). This prompted us tO search for materials that might protect solubilized membrane protein, and that survey soon led to the discovery that a class of compounds known as "osmolytes" (65) have a particularly beneficial action. We also noted that inactivation of the streptococcal carrier was largely restricted to a brief interval, literally during solubilization, so that these protein stabilants were effective only if added before the detergent (3). The positive effect of osmolytes is described in Table 2. Note that in Pi-loaded liposomes prepared without such intervention, the steady level of 32Pi accumulation is about 20-d0 nmol/mg protein. Traditional stabilants such as methanol, or ethyleneglycol have no effect, but high concentrations of osmolytes, including certain amino acids (proline, glycine, not valine), sugars, and glycerol or higher polyols, lead to a 10 to 20-fold increased recovery of activity. This remarkable effect is not restricted to the Pi-linked exchange proteins, nor is it confined to bacterial systems. Instead, it appears to be a general response on the part of many membrane proteins, prokaryotic and eukaryotic, including ATP-driven pumps, various secondary carriers, and several membrane-bound enzymes (37). As noted earlier, detergent solubilization of many membrane proteins, including the Pi-linked exchangers, yields largely inactive protein, and this presents a major impediment to the characterization and purification of such systems. Fortunately, in many cases this inactivation appears to be largely overcome by appropriate use of osmolytes. Therefore, even though the mechanism by which osmolytes exert their effect remains conjectural (3, 8), the intervention can have a substantial practical value. An example showing the benefits of this approach to reconstitution comes from our attempts to streamline the protocol. Traditionally, membrane proteins are solubilized from membrane vesicles, but because of the labor involved in making vesicles, proteoliposomes are used less frequently than their advantages might warrant. This is especially true if a number of strains are to be screened, since vesicles would have to be prepared for each strain, with the method often having to be optimized for each strain. In an attempt to circumvent this difficulty, we developed a rapid method for reconstitution of membrane proteins directly from intact cells (59, 60). In this technique, cells are digested with lysozyme, osmotically lysed, and the resultant ghosts are then solubilized in the presence of an osmolyte, as already outlined. With this technique, protein can be solubilized from cell cultures as small as a few milliliters, and many different strains can be examined in a single day. Proteoliposomes made in this way are suitable for both quantitative and qualitative studies of antiport. In particular, there is no evidence for parallel reconstitution of the bacterial porins, possibly because of their generally hydrophilic nature, so that the proteoliposomal membrane is capable of sustaining a membrane potential (59). We anticipate that such an approach can become a useful tool for the screening and characterization of large numbers of strains, both wild-type and mutant.


119

V. CARBOXYLATE-LINKED ANION EXCHANGE A. Oxalobacter formigenes As noted in the previous section, the application of reconstitution to the study of antiport systems provides the investigator with an explicit control over internal and external milieux. Perhaps more generally important, the success of osmolytemediated techniques encourages one to use reconstitution as an analytical rather than as a preparative tool. Indeed, this approach was essential in the description and analysis of carboxylate-linked exchange, the first example of which was described using protein reconstituted from membranes of the gram-negative anaerobe, O. formigenes. Early studies of this cell had demonstrated that it uses the decarboxylation of oxalic acid, the simplest dicarboxylic acid, as its sole source of metabolic energy (l, 9). Thus, ifonly for its role in transport of this growth substrate, it seemed that studies of oxalate transport might be of interest. Guided by experiments of reconstitution, we were led to the discovery and eventual purification of a membrane carrier--OxlTwthat contributes in an entirely unexpected way to membrane biology in this cell (6, 54). The carrier is also of interest for biochemical reasons, since the purified material has a specific activity during oxalate self-exchange in excess of 500-700 lamol/min per mg protein, and a turnover number on the order of> 1000Is (6). Among carriers that handle organic molecules, this places OxiT at the head of the line in terms of velocity. Two simple observations form the basis of the model of how OxlT contributes to cell biology. First, work using oxalate-loaded proteoliposomes shows that purified OxlT mediates an oxalate self-exchange, a reaction that proceeds independently of co-ions and that reflects the strict one-for-one antiport of internal and external substrate. Second, this same carrier can accept (with somewhat lower affinity) formate as an alternative substrate. Most important, the heterologous exchange of oxalate and formate is electrogenic, with negative charge moving in the same direction as oxalate. The electrogenic nature of this exchange (which contrasts with the electroneutral behavior of Pi-linked events) is readily demonstrated in a reconstituted system, as shown by Figure 5. In that experiment, proteoliposomes containing purified OxlT were loaded with either the potassium or N-methylglucamine salt of formate, and then suspended in N-methylglucamine or potassium sulfate, respectively, in the presence of labeled oxalate. Of themselves, these maneuvers had no effect on the accumulation of oxalate, but when valinomytin was also present, to impose a membrane potential negative or positive inside, oxalate accumulation was either inhibited (internally negative potential) or accelerated (internally positive potential). These responses are precisely those expected for the transport of divalent oxalate (-O(~-COO-) against monovalentformate (HCCO-). It is the electrogenic nature of the heterologous exchange of oxalate and formate that is essential to the role ascribed to OxlT. Figure 6 depicts this in simplified fashion by showing how the metabolic coupling between OxlT and an internal


120 2O

15

o3

~o t~Q.

! A v

00

A v

, 2

A v

n 4

A

n 6

,

I 8

10

Minutes

Figure 5. OxlT catalyzes the electrogenic exchange of oxalate and formate. During

reconstitution of purified OxlT, proteoliposomes were loaded with 100 mM NMG formate or 200 mM potassium formate. To start each assay, proteoliposomes were diluted from their concentrated stocks into a potassium- or NMG-based buffer, respectively, so that in one case there was an inwardly directed potassium gradient (o,=), while in the other case this gradient was directed outward (o,o). Experimental tubes (solid symbols) received 1 FM valinomycin; control tubes (open symbols) received the ethanol carrier. The reaction was started by addition of 1O0 I~M [14C]oxalate. (Taken from ref. 54)

decarboxylating system allows O.formigenes to sustain a proton-motive force. The complete system has three components---(1 ) OxlT, which catalyzes the electrogenic exchange of divalent oxalate and monovalent formate; (2) a formylCoA transferase (6), which uses formyICoA as a CoA donor for incoming oxalate, thereby releasing the formate whose efflux returns OxlT to its original state; and (3) an oxalylCoA decarboxylase, which acts to decarboxylate oxalylCoA, generating formylCoA and carbon dioxide. (The catalytic role of the formylCoA transferase is not shown in Figure 6.) Since the one-for-one exchange of oxalate and formate carries a single negative charge into the cell, the membrane potential becomes internally negative, and since the decarboxylation reaction consumes a single scalar proton, an internal alkalinity is sustained. In aformal sense, then, a single turnover of the OxlT-transferase-decarboxylase system acts to pump a single proton (H+) out of the cell! Three turnovers of this "pump" move three protons outward, and as these reenter via the proton-translocating ATPase, they provide sufficient energy to synthesize ATP. Therefore, this model also offers a solution to the thermodynamic problem that a decarboxylation of oxalate cannot of itself yield sufficient free energy to make ATP.


121

,§

Oxalate2~..

ANTIPORT --.---eOxalate2- " ~ DIECARBOXYLASE FormatelACID

§ .~. +

~ ALKALINE

CO2

2

3H+

F~ure 6. The indirect proton pump of Oxalobacter formigenes. A proton-motive force arises after entry of divalent oxalate, its internal decarboxylation and exit of monovalent formate. The net entry of one negative charge and the disappearance of one internal proton is equivalent to the outward movement of one positively charged proton. Three such cycles, each with a stoichiometry of 1H+/turnover, would support synthesis of ATP. (Taken from ref. 6) The biochemistry of O. formigenes ensures that this system is always poised for a flux of inwardly flowing oxalate and outwardly moving formate. Such directionality is maintained in two ways. On the one hand, dissemination of the gaseous carbon dioxide limits the availability of product required for the back-reaction. In addition, O. formigenes can express very high levels of both OxlT and the decarboxylase (and, presumably, the CoA transferase). For example, under extreme conditions, when cells are grown with limiting oxalate, so that external substrate is reduced to micromolar levels, OxlT comes to represent at least 5% of membrane protein (54), and the decarboxylase alone can be as much as 10% of cytoplasmic protein (9). Inasmuch as these proteins are central to the lifestyle of this organism (Figure 6), such overexpression would seem to be a sensible investment.

B. Other Indirect Proton Pumps

O. formigenes represents the first explicit demonstration of how vectorial and scalar reactions might be organized so as to construct a new kind of proton pump. But the principle is a general one, and it is sure to emerge in other circumstances.

ATULVARADHACHARYand PETERC. MALONEY

122

Even now, other examples are coming to the surface. For example, we now appreciate that growth by malolactic fermentation (malate + H + - , lactate + CO2) can fit the paradigm, and several lines of evidence suggest there is the required electrogenic net exchange of malate and lactate (47, 49). Similarly, the decarboxylation of histidine to yield histamine follows the same general rules (44), as does decarboxylation of aspartate to yield alanine (Abe and Maloney, unpublished results). Of course, this economical design need not be restricted to simple decarboxylation reactions, and it is possible to imagine the same principle at work in cases where complex intracellular processing is responsible for the scalar consumption of protons. In this context, Archaebacterial methanogenesis might serve as an interesting test case. In such organisms, energy is derived from the metabolism of simple anions (acetate, formate, bicarbonate) to yield even simpler products (methane, water, carbon dioxide, hydrogen gas) (27). If these substrates enter the cell as anions, that is as charged species, one could view the totality of their remaining biochemistry as an ensemble of reactions designed only to consume a single scalar proton for each charge that has entered. This gives the required coupling between vectorial and scalar events, with the end result of an indirect proton pump. While such a suggestion is entirely speculative, it does illustrate the potential utility of the general principle when extended beyond the immediate examples.

VI.

OTHER BACTERIAL A N I O N EXCHANGE SYSTEMS

Despite the common occurrence of anion exchange in eukaryotes, there are still relatively few documented examples of anion exchange in prokaryotes. And while this chapter has emphasized the Pi-linked and carboxylate-linked exchangers, a few others are known. A relatively well-studied example is that of the ATP-ADP translocase of Rickettsia prowazeki. This carrier, which is used by the parasite to gain access to the host adenine nucleotide pool, has now been cloned, sequenced and expressed in E. coil (30). The amino acid sequence of this protein supports its inclusion in the general class of secondary carriers, but does not suggest it belongs to the Pi-linked family described above. A phenotypically similar transporter, catalyzing the exchange of ADP and Pi, has also been described in Rhodobacter capsulatus (11), but this latter example is as yet known only at a biochemical level. And in the same way, biochemical but not genetic evidence has been given for the presence of a dicarboxylate exchange in anaerobically grown E. coli (16). Finally, we note that there are many anion exchange events associated with mitochondria and chloroplasts. This, and the widespread existence of eukaryotic anion exchange, suggests there are equal numbers of prokaryotic examples awaiting identification.


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VII. C O M M O N THEMES AND STRUCTURAL RHYTHMS While the study of anion exchange proteins has its intrinsic value, recent discoveries in the larger field of membrane transport suggest that work on any one of these secondary carriers can have relevance to the entire group. Despite differences in the kinetic and biochemical behavior of these various proteins, an underlying structural theme is emerging. The most convincing evidence to this point arises from theoretical and practical studies of how these proteins are arranged within the membrane. Topological predictions of a large number of secondary carriers, both eukaryotic and prokaryotic, show that their most characteristic feature is the presence of transmembrane segments in multiples of 5-6 (35, 36). In cases where the functioning carrier is known, 10-12 segments appear to comprise the operational unit, whether this is as a dimer of subunits containing 5-6 segments, as in the chloroplast and mitochondrial carriers (7, !8, 29), or as a monomer containing 10-12 segments, as for the UhpT (5) and LacY (12) proteins of E. coli (Figure 7). Some time ago it was argued that transport proteins should function as oligomeric proteins, probably dimers, with the translocation pathway defined as the volume enclosed by the interface between subunits (28, 31). Early work with the mitochondrial exchange proteins supports the idea, but demonstration that LacY and UhpT (and other transporters) can function as monomers appears contradictory. But this conflict can be easily resolved if the larger (monomeric) carriers are viewed as having a substructure allowing them to behave as functional dimers. And, in fact, the argument for an intramolecular dimer is supported (albeit, indirectly) by the finding of sequence homology between the two N- and C-terminal halves of some carders (25). If only for the purpose of designing the next generation of experiments, we offer the UhpT antiporter of bacteria as providing a unique perspective on this issue. As noted before, UhpT should contain a pair of symmetrical binding sites so that the protein can accept either two monovalent substrates or single divalent passenger. Is it unreasonable to suggest that each half of the molecule contains one of these binding sites and that substrate passes by the interface between the N-terminal and C-terminal halves (Figure 7)? Perhaps not. Preliminary experiments have convincingly demonstrated that the seventh transmembrane segment of UhpT comprises part of the transport path (64), and our next experiments will attack this issue more fully by using molecular biology to define the arrangement of those parts of the molecule surrounding this substrate translocation pathway. If this at the same time clearly delineates the region between the N- and C-terminal halves, we will take this as evidence to support the view that UhpT is constructed as, in effect, a covalent heterodimer.

124


NH2

J

COOH CYTOPLASM

i

m PERIPLASM

/

Figure 7. A low resolution view of UhpT: generic structure of a membrane carrier. The schematic shows the topology of UhpT derived by Kadner and his colleagues from hydropathy analysis and gene fusion experiments (26). The N- and C-termini, as well as the central loop, are expected to be exposed to the cytoplasm. Transmembrane segments are indicated by the shaded columns labeled I-XII. New data (64) show that cysteine-265 (circled lies on the pathway taken by substrates moving through Uhpt. This is with the idea that the translocation pathway is defined by the apposition of the N- and C-terminal domains of a covalent heterodimer. VIII.

CHAPTER SUMMARY

Even though anion exchange in bacteria was described as early as 1953, it is only in the last decade that the underlying mechanism has been characterized in a satisfactory way. Work with vesicles and proteoliposomes shows that the chemiosmotic transport of sugar phosphates occurs, not by proton-symport, but by anion exchange, in both gram-positive and gram-negative bacteria. Further analysis shows the existence of a variable stoichiometry that allows homologous sugar phosphate exchange to mediate an unexpected net uptake of substrate. Since this exchange causes the influx of protons along with the sugar phosphate, continued transport depends on maintenance of a pH gradient (alkaline inside), thereby ensuring that the Pi-linked exchangers take part in the overall chemiosmotic circuitry of the cell.


125

The study of Pi-linked exchange has defined not only their physiological and biochemical properties, but has also been the vehicle for development of a particularly simple set of protocols designed to study such proteins in an artificial liposomal system. Such methods, which take advantage of the stabilizing effects of osmolytes (e.g., glycerol), have made it possible to use reconstitution in an analytical fashion, not just as a preparative tool. The value of this methodological strategy is well-illustrated by studies of OxlT, the membrane carrier responsible for the electrogenic exchange of oxalate and formate in O. formigenes. Because this cell had not been subjected to earlier biochemical or genetic tests, it is unlikely that traditional approaches would have so readily led to an appreciation of the central role OxIT plays in establishing the proton-motive force in this anaerobe. This finding is perhaps the major contribution of such work, for OxlT has now become the first of many examples in which generation of a proton-motive force relies, not on a primary proton pump, but on the indirect coupling between secondary carriers and internal metabolism, an organizational scheme that may be of unsuspected antiquity (38). We have also touched on what seems to be an emerging commonality to all membrane carriers, one reflected in the 10-12 transmembrane segments that form the functional unit of UhpT. This strengthens the view that examining bacterial anion exchange might reveal facts and principles more broadly applicable to membrane transport systems.

ACKNOWLEDGMENTS The study of anion exchange in this laboratory is supported by grants from the Public Health

Service (GM24195) and the National Science Foundation (MCB9220823).

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52. Rosenberg, H., Gerdes, R. G., & Chegwidden, K. (1977). Two systems for the uptake of phosphate by Escherichia coil J. Bacteriol. 131,505-511. 53. Rosenberg, H., Gerdes, R. G., & Harold, E M. (1979). Energy coupling of inorganic phosphate in Escherichia coil KI2. Biochem. J. 178, 133-137. 54. Ruan, Z-S., Anantharam, V., Crawford, I. T., Ambudkar, S. V., Seung, Y. R., Allison, M. J., & Maloney, P. C. (1992). Identification, purification and recomfitution of OxlT, the oxalate:formate antiport protein of Oxalobacterformigenes. J. Biol. Chem. 267, 10537-10543. 55. Rudnick, G. (1986). ATP-driven 14+ pumping into intracellular organelles. Annu. Rev. Physiol. 48, 403-413. 56. Sonna, L. A., & Malone),, P. C. (1988). Identification and functional reconstitufion of phosphate:sugar phosphate antiport from Staphylococcus aureus. J. Membr. Biol. 101,267-274. 57. Sonna, L. A., Ambudkar, S. V., & Malone),, P. C. (1988). The mechanism of glucose 6-phosphate transport by Escherichia coil J. Biol. Chem. 263, 6625-6630. 58. Tokuda, H., & Unemoto, T. (1982). Characterization of the respiration-dependent Na+ pump in the marine bacterium Hbrio alginolyticus. J. Biol. Chem. 257, 10007-10014. 59. Varadhachary, A., & Maloney, P. C. (1990). A rapid method for reconstitution of bacterial membrane proteins. Mol. Microbioi. 4, 1407-1411. 0. Varadhachary, A., & Maloney, P. C. (1991). Reconstitution of the phosphoglycerate transport protein of Salmonella typhimurium. J. Biol. Chem. 266, 130-135. 61. WiUsky, G. R., & Malamy, M. H. (1980). Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coll. J. Bacteriol. 144, 366-374. 62. Winkler, H. H. (1973). Energy coupling of the hexose phosphate transport system in Escherichia coli. J. Bacteriol. 116, 203-209. 63. Winkler, H. H. (1973). Distribution of an inducible hexose-phosphate transport system among various bacteria. J. Bacteriol. 116, 1079-1081. 40 Yan, R.-T., & Maloney, P. C. (1993). Identification of a residue in the translocation pathway of a membrane carrier. Cell 75, 37--44. 65. Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., & Somero, G. N. (1982). Living with water stress: evolution of osmolyte systems. Science 217, 1214-1222.

HELIX PACKING IN THE C-TERMINAL HALF OF LACTOSE PERMEASE

H. Ronald Kaback, Kirsten Jung, Heinrich Jung, Jianhua Wu, Gilbert G. PrivY, and Kevin Zen

I. Introduction . II. III. IV. V.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Secondary Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional Interactions Between Putative Intramembrane Charged Residues Use of Site-Directed Fluorescence labeling to Obtain Proximity Relationships S u m m a r y and Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

129 131 .134 . 138 141 143

I. I N T R O D U C T I O N Although the driving force for a variety of seemingly unrelated phenomena (e.g., secondary active transport, oxidative phosphorylation, and rotation of the bacterial flagellar motor) is a bulk-phase, transmembrane electrochemical ion gradient, the molecular mechanism(s) by which free energy stored in such gradients is transduced into work or into chemical energy remains enigmatic. Nonetheless, gene

Advances in Cell and Molecular Biology of Membranes and Organelles Volume 4, pages 129-144. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-$$938-924-9 129

KABACK, )UNG, )UNG, WU, PRIVI~, and ZEN

130

H§

A

B

c

F~ure 1. H§ symport in E. coll. (A) Lactose accumulation in response to A~H+ (interior negative and alkaline) generated either by respiration or ATP hydrolysis. (B) Uphill H + transport in response to an inwardly directed lactose gradient. (C) Uphill H + transport in response to an outwardly directed lactose gradient.

sequencing and analyses of deduced amino acid sequences indicate that many biological machines involved in energy transduction, secondary transport proteins in particular (24, 43), fall into large families encompassing proteins from archaebacteria to the mammalian central nervous system, thereby suggesting that the family members have common basic structural features and mechanisms of action. This contribution will concentrate on recent observations with the lactose (lac) ~ permease of Escherichia coli as a paradigm for secondary transport proteins that catalyze ion-gradient coupled active transport. Accumulation of [3ogalactosides against a concentration gradient in E. coil is catalyzed by lac permease, a hydrophobic polytopic cytoplasmic membrane protein that carries out the coupled translocation of a single [3-galactoside with a single H § (i.e., 13-galactoside/H§ symport or cotransport) (see 31, 32, 33 for reviews). Physiologically, the proton electrochemical gradient across the cytoplasmic membrane (A~x§ is interior negative and/or alkaline, and the permease utilizes free energy

Helix Packing of Lac Permease

131

released from downhill translocation of H + to drive accumulation of [3-galactosides against a concentration gradient (Figure 1). In the absence of (A~H+), lac permease catalyzes the converse reaction, utilizing free energy released from downhill translocation of [$-galactosides to drive uphill translocation of H + with generation of a A~H+ the polarity of which depends upon the direction of the substrate concentration gradient. Lac permease is encoded by the lacy gene, the second structural gene in the lac operon, which has been cloned into a recombinant plasmid (66) and sequenced (6). By combining overexpression of lacY with the use of a highly specific photoaffinity probe (34) and reconstitution of transport activity in artificial phospholipid vesicles (i.e., proteoliposomes) (50), the permease was solubilized from the membrane, purified to homogeneity (51, 22, 70, 72) and shown to catalyze all the translocation reactions typical of the ~3-galactoside transport system in vivo with comparable turnover numbers (46, 69). Therefore, the product of lacY gene is solely responsible for all of the translocation reactions catalyzed by the [3-galactoside transport system.

II. SECONDARY STRUCTURE Circular dichroic measurements on purified lac permease demonstrate that the protein is about 80% helical, an estimate consistent with the hydropathy profile of the permease, which suggests that approximately 70% of its 417 amino acid residues are found in hydrophobic domains with a mean length of 24 :i: 4 residues (21). Based on these findings, it was proposed that the permease is composed of a hydrophilic N-terminus followed by ] 2 hydrophobic segments in a-helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic domains (loops) with a 17-residue C-terminal hydrophilic tail (Figure 2). Support for general features of the model and evidence that both the N and C termini are exposed to the cytoplasmic face of the membrane were then obtained from laser Raman spectroscopy (71), immunological studies (11, 12, 9, 62, 63, 61, 26, 17), limited proteolysis (23, 65) and chemical modification (53). However, none of these approaches differentiates between the 12-helix motif and other models containing l0 (71) or 13 (5) transmembrane domains. Calamia and Manoil (7) have provided elegant, unequivocal support for the topological predictions of the 12-helix model by analyzing an extensive series of lac permease-alkaline phosphatase (lacY-phoA) fusion proteins. Under normal conditions, alkaline phosphatase is synthesized as an inactive precursor in the cytoplasm of E. coli with an N-terminal signal sequence that directs its secretion into the periplasm where it dimerizes to form active enzyme. If the signal sequence is deleted, the enzyme remains in the cytoplasm in an inactive form. When alkaline phosphatase devoid of the signal sequence is fused to the C-termini of fragments of a cytoplasmic membrane protein #1 vivo, enzyme activity reflects the ability of the N-terminal portions of the fusions to translocate the enzyme to the outer surface

1.

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tri P h o E di P h o E -

w

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Figure 5. Different forms of PhoE, detected after its in vitro synthesis. Plasmid piP370 was used to direct the in vitro synthesis of a quasimature PhoE protein, which contains at the N-terminus only two amino acids instead of the signal sequence (28). Lane 1 shows the translation products. The major band below PhoE representschloramphenicol acetyltransferase, which is also encoded by the plasmid. After translation, outer membranes were added to induce trimerization (29), and PhoE was immunoprecipitated with a mAb, recognizing a conformational epitope in PhoE. The immunoprecipitates were incubated at the indicated temperatures in sample buffer before electrophoresis, to assessthe stability of the different PhoE conformations (lanes 2-5). In addition to some denatured PhoE (mPhoE), trimers (triPhoE), dimers (di PhoE) and folded monomers (m" PhoE) can be detected on the autoradiogram. The sample in lane I was boiled before electrophoresis.

161

162

JAN TOMMASSEN and HANS DE COCK

with a defect in the core region of the LPS were less efficient in inducing trimerization (27). Furthermore, the trimers seemed to be associated with LPS, since their mobility during electrophoresis was slightly different, depending on the chemotype of the LPS in the outer membranes used to induce trimerization. The trimers obtained were highly resistant to denaturation in SDS (Figure 5), like the native PhoE produced in vivo. However, they were not inserted into the membranes since they remained in the supernatant after pelleting of the membranes by centrifugation (28). Furthermore, they were not protected against proteases. Insertion in a protease-resistant configuration was observed when small amounts of a detergent (0.06% Triton X-100) were present during trimerization (27). This observation is reminiscent of the results obtained for the insertion of OmpA in large DMPC vesicles, discussed above. Similar to the PhoE porin, the assembly of the related OmpF porin, obtained either by synthesis in an E. coli lysate (112) or by secretion from spheroplasts (111), has been studied in vitro. The results obtained were largely consistent with those described for PhoE. The major discrepancy is that trimerization of the OmpF proteins could not only be induced by outer membranes, but also by LPS preparations, and even by a mixture of an anionic and a nonionic detergent. Similarly, the renaturation of OmpF, isolated from the outer membrane and denatured in guanidium hydrochloride, by a combination of SDS and octyl-pentaoxyethylene, has been reported (38). An explanation for this discrepancy is not obvious at the moment. Anyhow, these results suggest that LPS is neither required for the folding nor for the trimerization of the OmpF molecules. However, it is still possible that the LPS plays an important role in the in vivo situation and that SDS functions as the substitute for LPS in vitro. Furthermore, the OmpF secreted by spheroplasts was reported to trimerize more efficiently than the OmpF or PhoE proteins synthesized in vitro in an E. coli lysate (112). There are several possible explanations for this difference: (1.) the in vitro synthesized proteins contained short N-terminal extensions of a few amino acid residues that may have interfered with the trimerization process, (2.) trimerization may have been inhibited by binding of cytoplasmic chaperones like SecB (30), which were present in the lysates used to direct the in vitro synthesis of these proteins, and (3.) secretion across the cytoplasmic membrane might be accompanied by conformational changes favoring trimerization. Indeed, the spheroplast-secreted OmpF porin appeared to be different from the in vitro synthesized OmpF in its reactivity with antibodies recognizing conformational epitopes (112).

D.

Sorting Signals in Outer Membrane Proteins

In general, the membrane-spanning segments of a polytopic inner membrane protein are hydrophobic and can insert into the membrane, independent of the other membrane-spanning segments. In contrast, the membrane-spanning segments of OMPs are ~-strands with only one hydrophobic side (see Section II.B). Conse-

Outer Membrane Proteins in E. coli

163

quently, OMPs are compatible with the lipidic environment of the membrane only after the formation of the entire ~barrel structure containing a hydrophobic exterior. It is, therefore, not surprising that large deletions (removing > 30 amino acid residues) in the mature domain of PhoE prevented the normal incorporation of the mutant proteins into the outer membrane and led to their periplasmic accumulation (14). Such large deletions can be expected to interfere with the folding of the protein into the [3-barrel configuration. Similarly, large deletions in the membrane-embedded N-terminal part of OmpA prevented the normal incorporation of this protein in the membrane (66), whereas deletions in the C-terminal periplasmic tail of this protein (see Figure IB) were tolerated (18). Although the entire conformation of OMPs is apparently required for their correct localization, some portions of these proteins might be more important to the correct conformation than others, and in addition, specific sorting signals might still be present to allow interaction with the appropriate membrane. A priori, one could expect that the J-strands are more important for the assembly of the proteins than the surface exposed loops, since there are restrictions on both the [3-structure and on the hydrophobicity of residues in these segments. Comparison of the primary structures of the porins OmpF, OmpC, and PhoE (8.5) show that the sequences of the membrane-spanning segments are much better conserved than those of the exposed loops. A similar observation was made when the primary structures of the OmpA proteins of different Enterobacteriaceae were compared (I 7). Furthermore, insertions (2, 4) and deletions (/) were well tolerated in the exposed loops of PhoE, as well as of other OMPs (e.g., refs. 22, 41, 67). However, the [3-strands also tolerated mutations. In the case of PhoE, hydrophobic residues in the J-strands that are exposed to the fatty acyl chains of the lipids or to the subunit interface of the trimers could be replaced by hydrophilic or even charged residues without affecting the assembly of the protein (119). However, two of such substitutions in a single [3-strand dramatically affected the efficiency of outer membrane incorporation. Furthermore, the first membrane-spanning segment of PhoE could be completely deleted (e.g., mutant protein A3-30, in ref. 16) and still, the mutant protein was correctly inserted in the membrane as evidenced by the binding of PhoE-specific bacteriophage and mAbs to intact cells. However, part of the total amount of the mutant protein produced accumulated in the periplasm, showing that the efficiency of outer membrane insertion was reduced. The first membrane-spanning J-strand of PhoE does not face the lipids, but is located at the subunit interface within the trimers (2.5). Consistently, no trimers of this mutant protein were detected, indicating that either the stability of the trimers is drastically reduced or that the protein inserts into the membrane as a monomer. According to the model for the localization of OMPs, depicted in Figure 3, these proteins adopt their tertiary structure in the periplasm before inserting into the outer membrane. Consequently, a putative sorting signal might be a conformational one and therefore difficult to detect by comparison of the primary structures of diverse OMPs. Nevertheless, a few short stretches of amino acid residues with vague

164


sequence homology were detected in the primary structures of the porins, LamB and OmpA (92). Probably, most of these sequences do not correspond to sorting signals since the homologies became insignificant when more OMP sequences became available, or because deletion analysis in OmpA or PhoE showed that they were not involved in outer membrane localization. For the most pronounced similarity region, sequence comparisons have recently been updated (91), and it appeared that only a single glycine residue, corresponding to Gly- 144 in PhoE, was completely conserved in this segment. Gly-144 is located in a periplasmic turn in the PhoE structure (25). In the OmpA model (134), the corresponding residue is located in an entirely different position near the cell surface, which makes a common function for these residues unlikely. Gly-144 of PhoE was replaced by a leucine residue (31). Indeed, outer membrane localization was somewhat defective, especially at higher growth temperatures. However, in the in vitro assembly system for PhoE (described in Section V.C), the mutant protein appeared to be defective in folding (31). Therefore, we believe that Gly-144 of PhoE is not part of a sorting signal, but is important for protein folding. A more significant similarity was recently detected at the C-termini of OMPs (118). Comparison of the last ten amino acid residues of diverse OMPs revealed the presence of potential amphipathic ~strands with hydrophobic residues at positions 1 (Phe), 3 (preferentially Tyr), 5, 7, and 9 from the C-terminus. In the porins this segment corresponds to the last membrane-spanning segment (25), and its deletion in PhoE had a detrimental effect on assembly in the membrane (15). This consensus sequence was found in the vast majority of bacterial OMPs, including E. coli proteins with widely diverse functions and OMPs of other Gram-negative bacteria (118). In a few cases, including LamB, Trp instead of Phe was found at the C-terminal position. However, like Phe, Trp is a hydrophobic aromatic residue and is therefore expected to functionally replace the Phe. In OmpA and its analogs of other bacteria, the consensus sequence is not found at the ultimate C-terminus, which is extending into the periplasm (Figure 1B), but at the C-terminus of the membrane embedded part (i.e., residues 161-170). The consensus sequence was not detected at the C-termini of OMPs that are involved in the secretion of macromolecular compounds like PapC, which is involved in the secretion of pill subunits (95), or TolC (93) known to be involved in the secretion of o~-haemolysin (139). Interestingly, the consensus sequence is located in a region in OmpA that was earlier implicated in sorting to the outer membrane (66). As mentioned in the beginning of this section, large deletions in OmpA prevented the correct incorporation of the protein into the outer membrane. However, immunoelectron microscopy revealed that the mutant proteins were still associated with the outer membrane, unless the deletions covered an area between residues 154 and 180. When this region was deleted, the mutant proteins accumulated in the periplasm (66). These results suggested that the area between residues 154 and 180 (including the consensus sequence between residues 161-170) contains an outer membrane


165

sorting signal. Moreover, of many missense mutations created in ompA, only a mutant protein containing two amino acid substitutions in the consensus sequence was totally blocked in outer membrane assembly (65). Since the C-terminal Phe is the most pronounced feature of the consensus sequence, this residue was used as a target for extensive site-directed mutagenesis in PhoE (118). High-level expression of all mutant proteins obtained was lethal to the cells, and the efficiency of outer membrane incorporation was dramatically affected. The mutational effect was the least severe when the Phe was replaced by another aromatic residue and was most severe when this residue was deleted. In the latter case, hardly any PhoE could be detected in the outer membrane, and the protein accumulated as aggregates in the periplasm (117). In vitro the folding and the trimerization of this mutant protein were virtually unaffected, but the insertion in isolated outer membranes was drastically reduced. When the expression level of the mutant proteins carrying substitutions for or deletion of the C-terminal Phe was reduced, the detrimental effects on growth were alleviated and more mutant protein was correctly assembled into the outer membrane (117). This result suggests that the C-terminal Phe is not absolutely required for the correct assembly of the protein, but does contribute to the efficiency of the process. Apparently, there is a kinetic partitioning between outer membrane incorporation and aggregation of periplasmic assembly intermediates. At high levels of expression, more protein will aggregate and become assembly-incompetent, whereas at low expression levels the tendency to aggregate will be reduced, resulting in increased outer membrane incorporation. The C-terminal Trp in Lamb may play a similar role as the Phe in PhoE, since deletion of the last two amino acid residues of LamB has been reported to drastically affect outer membrane incorporation (56).

VI.

CONCLUSIONS A N D FUTURE PROSPECTS

Whereas the molecular details of the mechanism of the transport of proteins across the cytoplasmic membrane are beginning to emerge, even the basic concepts of the last steps in the biogenesis of OMPs are far from clear. The proteins probably fold into their tertiary structure after passage through the inner membrane (Figure 3). After folding into a [~-barrel, the proteins have a hydrophobic exterior, compatible with insertion in the lipidic environment of the membrane. It is not yet clear where folding takes place. The process probably does not occur free in the periplasm, but is associated at the periplasmic face of one of the membranes, since amphiphiles were usually observed to induce folding in vitro. Although refolding of denatured OMPs was observed in the absence of any other proteins, this does not exclude the possibility that chaperones or enzymes are involved in this Process in vivo. Several other proteins have been reported to renature spontaneously in vitro, whereas their folding is guided by other proteins in vivo (e.g., ref. 40). The possible involvement of periplasmic proteins in the folding of OMPs will be an important topic of future

166


research. Many OMPs, including the porins, are devoid of cysteine residues, and in these cases the involvement of protein disulfide isomerases can be excluded. Nevertheless, the expression of OmpF was affected at the transcriptional level in a dsbA mutant lacking a periplasmic disulfide isomerase (99). On the other hand, native OmpA does contain a disulfide bond and its formation was apparently retarded in a dsbA mutant (8). However, since the two cysteines of OmpA are located in the C-terminal periplasmic extension, no effect on outer membrane localization would be expected. The cis-trans isomerization of peptidylpropyl bonds is frequently a rate-limiting step in protein folding. An enzyme catalyzing this reaction has been detected in the periplasm of E. coli (54), but the involvement of this enzyme and of putative chaperones in the folding of OMPs remains to be investigated. Another important aspect for future research will be to gain insight in the mechanism of selective insertion of OMPs in the outer membrane and not in the inner membrane. There are some indications that LPS is involved in this process, but the effect of LPS could be indirect. The role of the observed outer membrane particles and of lipid packing will have to be investigated. Furthermore, if the C-terminal consensus sequence (118) indeed represents a sorting signal, a receptor for this signal can be expected to exist and should be identified.

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132. Van der Ley, P., Struyv6, M., & Tommassen, J. (1986). Topology of outer membrane pore protein PhoE of Escherichia coli. Identification of cell surface-exposed amino acids with the aid of monoclonal antibodies. J. Biol. Chem. 261, 12222-12225. 133. Verldeij, A., van Alphen, L., Bijvelt, J., & Lugtenberg, B. (1977), Architecture of the outer membrane of Escherichia coli K I2. II. Freeze fracture morphology of wild type and mutant swains. Biochim. Biophys. Acta 466, 269-282. 134. Vogel, H., & Jithnig, E (1986). Models for the structure of outer-membrane proteins of Escherichia coil derived from Raman spectroscopy and prediction methods. J. Moi. Biol. 190, 191-199. 135. Von Heijne, G. (1986). Net N-C charge imbalance may be important for signal sequence function in bacteria. J. Mol. Biol. 192, 287-290. 136. Von Heijne, G. (1990). The signal peptide. J. Membr. Biol. 115, 195-201. 137. Voorhout, W., de Kroon, T., Leunissen-Bijvelt, J., Verkleij, A., & Tommassen, J. (1988). Accumulation of LamB-LacZ hybrid proteins in intracytoplasmic membrane-like structures in Escherichia coil KI2. J. Gen. Microbiol. 134, 599-604. 138. Vos-Scheperkeuter, G. H., & Witholt, B. (1984). Assembly pathway of newly synthesized LamB protein an outer membrane protein of Escherichia coli K-12. J. Mol. Biol. 175, 511-528. 139. Wandersman, C., & Delepelaire, P. (1990). TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc. Natl. Acad. Sci. USA 87, 4776-4780. 140. Watanabe, M., & Biobel, G. (1989). SecB functions as a cytosolic signal recognition factor for protein export in E. coli. Cell 58, 695-705. 141. Weiss, J. B., & Bassford, P. J. Jr. (1990). The folding properties of the Escherichia coil maltose-binding protein influence its interaction with SecB in vitro. J. Bacteriol. 172, 3023-3029. 142. Yamada, H., & Mizushima, S. (1980). Interaction between major outer membrane protein (0-8) and lipopolysaccharide in Escherichia coil KI2. Eur. J. Biochem. 103, 209-218. 143. Yamaha, K., & Mizushima, S. (1988). Introduction of basic amino acid residues after the signal peptide inhibits protein translocation across the cytoplasmic membrane of Escherichia coll. Relation to the orientation of membrane proteins. J. Biol. Chem. 263, 19690-19696.


STRUCTU RE-F U NCTION RELATIONSHIPS IN THE MEMBRANE CHANNEL PORIN

Georg E. Schulz

I. Porin Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Porin Location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Orientation in the M e m b r a n e . . . . . . . . . . . . . . . . . . . . . . . . B. Packing in the Crystal . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The Aromatic Girdles . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Folding Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Permeation Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Pore Eyelet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. A n Electric Separator . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Voltage Dependence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Porin Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments ................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Cell and Molecular Biology of Membranes and Organelles Volume 4, pages 175-187.

Copyright 91995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-924.9

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176 177 177 180 I g0 181 183 183 183 185 185 186 186

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GEORG E. SCHULZ I.

P O R I N STRUCTURE

Protozoa have to protect themselves against adverse environments. For this purpose, Gram-negative bacteria have an outer membrane containing channels that are permeable to nutrients. These channels are f o r n ~ by porins that are usually hemotrimeric proteins with subunit sizes ranging from 30 to 50kDa and solute exclusion limits of around 600 Da (1, 2). The channels are quite permeable for polar solutes, but exclude nonpolar molecules of comparable sizes. Porins have been subdivided into specific and nonspecific types. For a particular solute, specific porins show a comparatively large diffusion rate at low concentrations and saturation effects at high concentrations. In contrast, nonspecific porins act like inert holes, their diffusion rates being proportional to the solute concentration. A general description of the porin architecture was derived from electron microscopy studies (3). The first porin crystals suitable for X-ray analysis were obtained from Escherichia coil (4). The first atomic structure was that of the major porin of Rhodobacter capsulatus (5-9). An analysis of other crystals using molecular replacement with the R. capsulatus porin structure showed that the 16-stranded [~-barrel fold is present in a number of porins (I0). Three more porin structures are now known in atomic detail (11); they confirm the general picture derived from the first structure characterization. After careful protein preparation the major porin of R. capsulatus yielded particularly good crystals (5). As shown by amino acid sequence analysis (6), one subunit consists of 301 amino acids. The crystal structure has been solved at 1.8 resolution and refined to a crystallographic R-factor of 18.6% using a 97% complete data set (9). The model of one subunit contains all polypeptide atoms, 274 water molecules, 3 calcium ions, 3 detergent molecules (octyltetraoxyethylene, CsE4), and 1 bound ligand. Since this ligand could not be identified, it was modeled as C8E4. Each porin subunit consists of a very large 16-stranded antiparallel [~-barrel and 3 short r All ~strands are connected to their next neighbors. The loops at the bottom end of the barrel are short and smooth. In contrast, the top end of the barrel is rough and contains much longer loops. The longest loop has 44 residues and runs into the interior of the barrel where it constricts the pore size to a small eyelet (9). The chain fold of trimeric porin (Figure 1) shows that the [~-barrel height in the trimer center is lower than the barrel height facing the membrane. This center forms a three-pronged star composed of all three subunits resembling the hub and spokes of a wheel. The mobilities of the atoms in this central star are the lowest of the entire molecule as determined by the crystallographic temperature factors (9). The interior of this star is nonpolar, 18 phenylalanines (6 from each subunit) interdigitate tightly, while the surface is polar. The star contains all six chain termini paired in salt bridges. Trimeric porin can thus be described as a rigid central core constructed like

The Membrane Channel Porin

177

4

Figure 1. Stereoview of the Ca-backbone chain fold of trimeric porin taken from ref. (7). The view is from the external medium.

a water-soluble protein that is surrounded by three ~-barrel walls fencing off the membrane.

II. PORIN LOCATION A. Orientation in the Membrane A cut through the center of a pore shows the shape of the channel as illustrated in Figure 2. The aggregation to trimers connects the rear ~-barrel walls at the height levels indicated by dashed lines. The central part is low, giving rise to a common channel formed by all three subunits in the upper half. The three pore eyelets limiting the diffusion are located between the barrel wall I close to the molecular threefold-axis and the 44-residue loop 3 inside the barrel. The longitudinal position of the porin in the membrane is clearly defined by the nonpolar ring with a height of 24 A that surrounds the trimer and fits the nonpolar moiety of the membrane. The rough upper end of the J3-barrel (Figure 1) contains the larger loops with numerous charged side chains, whereas the smooth lower barrel end has only small loops with few polar residues. As shown by binding studies with antibodies and phages, the large polar loops face the external medium (12). Accordingly, the external medium is at the top of Figures 1 through 5. This orientation agrees well with other features of the outer surface of the trimer (Figure 3). While the nonpolar surface moiety faces the nonpolar interior of the lipid bilayer, the upper polar part with its numerous ionogenic side chains is most

178

GEORG E. SCHULZ

10,~'

l

Figure 2. Shapeof a porin subunit represented by a sliced 6 ,~ resolution density map calculated from the high resolution model (8). The density level is at 1 o; the distance from the viewer is indicated by shading. The sectional areas 1,2,3,4,5 and 6 belong to the ~-barrel wall at the trimer interface, the ~barrel wall facing the membrane, the pore size-defining 44-residue loop inside the I~-barrel, a smallish domain facing the external medium, the nonpolar moiety of the membrane, and the LPS core, respectively. As indicated by bars, there is no free space between sectional areas 2 and 3. likely connected to the lip~olysacchaddes (LPS) forming the external layer of the bacterial outer membrane. Presumably, the numerous Asp and Glu side chains are glued by divalent cations (like calcium and magnesium) to the carboxylate groups of the LPS cores. This network integrates the podn efficiently in the tough bacterial protection layer of crosslinked LPS molecules, avoiding a fragile protein-membrane interface. Furthermore, Figure 3 shows two girdles of aromatic residues along the upper and lower border lines between polar and nonpolar residues. The upper girdle contains mostly tyrosines pointing with their hydroxyl groups to the upper polar moiety of the membrane, while the lower girdle has phenylalanines pointing to the nonpolar membrane interior and tyrosines pointing to the polar part of the periplasmic membrane layer. These patterns are obviously significant. They were also observed in three recently elucidated porin structures (11). Moreover, there are four type-If' ~turns at the lower smooth end of the barrel. These four turns point with their peptide amides toward the membrane where they can form hydrogen bonds to the phosphodiesters of the pedplasmic layer of the bacterial outer membrane.

r e 3. Projection of the outer surface of the &barrel onto a cylinder as taken from ref. (9. All Imps at the periplasmic side are indicated. S u l c e areas A,B,C, and D contain the ionogenic side chains connecting to the LPS core, the nonpolar side chains facing the nonpolar interior of the membrane, the lefthand parts of the interface and the righthand parts, respectively. lonogenic atoms are emphasized by squares and polar atoms by circles. The aromatic girdles are obvious.

180

GEORG E. SCHULZ B. Packing in the Crystal

The exceptionally well ordered crystals of the R. capsulatus porin (9) have a packing s,;heme shown in Figure 4. It resembles the crystal packing of the photoreaction center (13). The polypeptides form only polar contacts. Apart from the subunit interface, there exists merely one contact type (i.e., between head and tail) giving rise to a trimer arrangement like that in a cubic closest packing. This contact contains five hydrogen bonds and one salt bridge and is obviously strong. The closest lateral distance between trimers is 15 ,~, and it occurs between nonpolar surfaces. Any conceivable contact through detergent molecules should therefore be very weak and should provide only a minor contribution to the crystal packing energy. The molecular packing in the crystal corresponds to a stack of lipid bilayers containing porins, suggesting that such bilayers can also be formed in the crystal. This hypothesis could be tested by measuring the electric conductivity of the crystal along and perpendicular to the threefold rotation axis. Such measurements can also be done after changing the detergent in the crystal (possibly to lipids) or after crystallizing with other detergents, which would yield information about the detergent(lipid)-protein contact.

C. The Aromatic Girdles Since the Tyr(Trp)-Phe-pattern (Figure 3) has been observed in all of the four known porin structures (9,11), it most likely reflects a function. I suggest that these bulky, aromatic side chains prevent conformational damage of the protein on

.

.

.

.

.

.

I

Figure 4. Crystal packing arrangement of trimeric porin molecules, the molecular threefold axes (&) are crystallographic. The space group R3 requires only one contact type to form a three-dimensional network. This contact (,-) is head to tail and polar. There is no lateral contact between the nonpolar surfaces (cross-hatche~, which are presumably covered by detergent.

181


.

.

.

.

~/ _

Jo,

i'-

.

"

!

~ . _. p o l a r

,

Figure S. Sketch describing the suggested shielding r61e of phenylalanines and tyrosines during relative movements of protein and membrane.

mechanical movements in the membrane as indicated in Figure 5. Any transversal wave in the membrane or any knocking at a porin trimer immersed in the membrane causes rocking movements that would expose nonpolar protein surface to a polar membrane layer and polar protein surface to the nonpolar membrane interior. Both contact types give rise to a large surface tension, which is likely to scramble the polypeptide conformation. Since the aromatic side chains rotate around their Ca-Cp-bonds much faster than the trimer can rock, they should be able to shield the respective surfaces against the wrong counterpart (Figure 5).

D. Folding Pathway Considering the chain fold of the homotrimer with the central rigid star, the prongs of which are connected by the three ~-barrel walls, a folding pathway can be suggested: At first, the three-pronged star folds like a water soluble protein. Since this star contains all chain termini, the remaining chain parts form three large loops of about 200 amino acid residues, each suspended between the three prongs. On contact with the nonpolar membrane interior, the three 200-residue loops arrange themselves to the actually observed most simple ~-barrel topology with all strands antiparallel and connected to their next neighbors. This folding process is straight~ forward as it requires no chain crossing over, or wrapping around. On folding, the nonpolar side chains of the 15-barrel residues orient themselves to the outside and face the nonpolar moiety of the membrane. Subsequently, the large 44-residue loop is inserted into the barrel. This loop supports the barrel at its center against the membrane pressure, and it defines the pore size.

Figure 6. Stereo view of a pore eyelet as taken from ref. (9).The pore eyelet is lined by negatively charged side chains at its upper rim and positively charged ones at the lower rim. There exists a strong transversal electric field between these charges as indicated by the rigidity ofthe neighboringarginines at the lower rim (see text). Water molecules (x)and bound ca2+(e)are given. The molecular threefold axis is indicated (A).

183


III.

P E R M E A T I O N PROPERTIES

A. The Pore Eyelet The eyelet of the channel is lined by ionogenic groups that segregate into positively and negatively charged rims (Figure 6). The positive rim is closer to the trimer center and consists of half a dozen Arg, Lys and His side chains. The negative rim is further to the circumference of the trimer; it contains about a dozen Asp and Glu residues located mostly in the 44-residue loop that is inserted into the ~-barrel. These abundant negative charges are partially compensated by two bound Ca 2+ions that tighten the rim structure appreciably. As a consequence, the removal of these Ca 2§ should change permeabilities. Actually, it was previously observed that the analyzed porin permits the diffusion of ATP only after the bacterium has been treated with EDTA (I 4).

B. An Electric Separator The juxtaposed positive and negative rims cause a transversal electric field across the eyelet. The field strength is best estimated from two arginines participating in the positive rim. These arginines are at van der Waals distance in good electron density, demonstrating that they are rigidly positioned in spite of their repelling positive charges (the crystals are at pH 7.2, the pK of Arg is 12.4). Such an arrangement seems only possible if the charges are fixed by a strong electric field. An X-ray structure analysis at 1.8 A resolution allows the assignment of reasonably well fixed water molecules (9). The eyelet contains quite a number of them (Figure 6). They orient their dipoles in the electric field (Figure 7). In the center of the eyelet there are mobile water molecules confined to a rather small cross section of about 4 A by 5 A. They form a hole through which a molecule with the cross section of an alky! chain should be able to permeate. The diffusion of a small polar solute is illustrated in Figure 8A. The solute is oriented by the transversal electric field formed by the charged rims of the eyelet and will remain oriented over the whole diffusion distance. The solute is oriented in the pore eyelet like a substrate on an enzyme. This reduces the entropy of the diffusion process in a fashion similar to that for a chemical reaction by binding to an enzyme surface. Without tumbling, the activation energy barrier for permeation is lowered and the diffusion of polar solutes is appreciably accelerated. Although the eyelet cross section that is free of fixed water molecules suffices for the penetration of alkyl chains, these still cannot permeate because they are blocked by the strong transversal electric field. The eyelet with the opposing charges at its rim like a charged capacitor that stores an electric energy, which is proportional to the high dielectric constant of 80 of oriented water molecules (Figure 8B) multiplied by the volume. Any incoming molecule with a lower dielectric constant (the respective value for an alkyl chain is about 2), that is, with

184

GEORG E. SCHULZ

/

Figure 7. Sketch of the pore eyelet indicating a ring of bound water molecules that are oriented in the transversal electric field. The rim of negatively charged side chains is strengthened by two strongly (Ca) and one weakly (Ca') bound Caz+. The depth of the pore eyelet is about 6 A. For a hydrated ion, always half the hydration shell has an energetically unfavorable orientation in the electric field. a smaller dipole than water, causes a decrease of the stored capacitor energy and therefore generates a force expelling the intruder. Consequently, the transversal field acts as an electric separator, facilitating the permeation of polar solutes while blocking off nonpolar ones. A special situation seems to arise for ions. When diffusing through the pore, half their hydration shell enters into an energetically unfavorable orientation (Figure 7). In the R. capsulatus porin, them exists a possible separate pathway for positive ions at one corner of the eyelet. This pathway is outside the electric field and completely

A

B

\

/

-c

Figure 8. Diffusion through the pore eyelet. (A) Sketch of a permeating zwitter ion ~alanine that remains oriented in the field. Its permeation is facilitated by entropy reduction. (B) Sketch of the pore eyelet as a capacitor explaining the high activation energy barrier for the permeation of nonpolar solutes.


185

lined by carbonyl and carboxyl oxygens. It may explain the observed, although low, cation selectivity of this porin. Since this channel is blocked by a weakly bound Ca 2+ ion, its importance can be tested by measuring cation currents as a function of Ca 2+ .concentration.

C. VoltageDependence There are observations indicating that some porins incorporated into black lipid films reduce their electric conductivity upon application of a voltage of approximately 100 mV across this film (2) (i.e., voltage closure occurs). Such a behavior can be understood when considering the shape of the pore (Figure 2). The membrane channel has a rather large cross section over its whole length except for the short distance of about 6 A at the diffusion-defining eyelet at the center. This implies that any voltage across the membrane (say 100 mV) lies essentially across the eyelet with its small cross section (certainly more than 90 mV). This is a strong longitudinal electric field. Since the energy gained by moving a single charge (e.g., the protonated e-amino group ofa lysine)over 100 mV is 10 kJ/mol and thus equivalent to the energy of a hydrogen bond, the strong longitudinal electric field along the eyelet can tear off any charged group that can be moved over the 6 ,~, length of the eyelet (which is possible for lysine). The applied voltage thus disrupts the eyelet structure and diminishes the permeability. In conclusion, the pores are vulnerable to such high voltages. This does not exclude, however, that some porins possess lids that move onto and close the pore in a longitudinal electric field.

IV. PORIN SPECIFICITY Porins have been subdivided into 2 classes, specific and nonspecific ones. The structurally known R. capsulatus porin has been previously classified as nonspecific. In the crystal structure, however, this porin is ligated by a small molecule (Figure 9). Moreover, efficient binding of tetrapyrrols to this porin has been reported (15). In the crystal, the bound ligand cannot be identified from the density. The binding site is formed between the 44-residue loop inserted into the barrel (where it forms the eyelet) and a smallish domain protruding to the external medium. Facing the external medium this binding site can pick up solutes at very low concentrations. After being bound close to the eyelet (Figure 9), the solute may subsequently dissociate and diffuse through the pore at a considerable rate. Binding and permeation should follow Michaelis-Menten kinetics and should show saturation effects as observed within the class of specific porins. Accordingly, the R. capsulatus porin previously assigned to the class of nonspecific porins is actually both specific and nonspecific, depending on the solute. Generalizing this observation, it is suggested that many porins belong to both classes, but that the specific solutes have been detected in only a very few cases.

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GEORG E. SCHULZ

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