Current Topics in Membranes and Transport VOLUME 36
Protein-Membrane Interactions
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Current Topics in Membranes and Transport VOLUME 36
Protein-Membrane Interactions
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Current Topics in Membranes and Transport Edited by Joseph F. Hoffman
Gerhard Giebisch
Deparhnent of Cellular and Molecular Physiology Yale University School of Medicine New Haven, Connecticu
Deparmtenr of Cellular and Molecular Physwbgy
Yale University School of Medicine New Haven, Connecticui
VOLUME 36 Protein-Membrane Interactions Guest Editor Toni Claudio Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven, Connecticut
Volume 36 is part of the series from the Yale Department of Cellular and Molecular Physiology
I#( San Diego
ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers
New York Boston London Sydney Tokyo Toronto
This book is printed on acid-free paper.
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COPYRIGHT 0 1990 BY ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.
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Contents
Contributors, ix Preface, xiii Yale Membrane Transport Processes Volumes, xv
PART 1.
PROTEIN INSERTION INTO AND ACROSS MEMBRANES
Chapter 1. Mitochondrial Protein Import: Specific Recognition and Membrane Insertion of Precursor Proteins NIKOLAUS PFANNER AND WALTER NEUPERT
I. 11. Ill. IV. V.
Introduction, 3 Receptor Proteins on the Mitochondria1 Surface, 5 The “General Insertion Protein,” 8 Role of Receptors and General Insertion Protein, 10 Summary and Perspectives, 11 References, I 1
Chapter 2. An Enzymological Approach to Membrane Assembly and Protein Secretion BILL WICKNER Text, 15
Chapter 3. Protein Translocation in Yeast JOANNE CROWE AND DAVID I. MEYER
I. Introduction, 19 11. Yeast as a Model System, 20 111. The Importance of Preprotein Conformation, 21 IV. Binding to the Membrane and Translocation Are Separable Events, 22 V. Analysis of pt l l - A Translocation Mutant, 24 References, 25
V
vi
CONTENTS
PART II.
STRUCTURE OF PROTEINS IN THE MEMBRANE
Chapter 4. Architectural Editing: Regulating the Surface Expression of the Multicomponent T-cell Antigen Receptor RICHARD D. KLAUSNER, JENNIFER LIPPINCOTT-SCHWARTZ, AND JUAN S. BONIFACINO I. 11. 111. IV. V. VI. VII.
Introduction, 31 Structure and Composition of the T-cell Antigen Receptor, 33 Subunit Interactions within the T-cell Receptor, 36 Stoichiometry within the Complex, 39 Assembly of the T-cell Receptor Complex, 40 Fate of Newly Synthesized T-cell Receptor Chains, 43 Summary, 47 References, 48
Chapter 5. The Photosynthetic Reaction Center from the Purple Bacterium Rhodopseudomonas viridis: Aspects of Membrane Protein Structure HARTMUT MICHEL AND JOHANN DEISENHOFER
I. Introduction, 53 11. Results and Discussion, 54 111. Conclusions, 68 References, 68
Chapter 6. Bacteriorhodopsin Folding in Membranes: A Two-Stage Process D. M. ENCELMAN, B. D. ADAIR, I. F. HUNT, T. W. KAHN, AND J.-L. POPOT I. Introduction, 71 11. Bacteriorhodopsin, 73 111. Bacteriorhodopsin Fragments Contain Stable Transbilayer Helices, 73
IV. V. VI. VII.
Links and Retinal Are Not Required for Folding, 74 Polar Interactions in Helix-Helix Associations, 75 Packing Effects, 76 Summary, 77 References. 77
PART 111.
PROTEIN MOBILITY IN MEMBRANES
Chapter 7. Molecular Associations and Membrane Domains MICHAEL EDIDIN I. Models of Membrane Organization, 81 11. The Study of Large-Scale Molecular Mobility in Cell Surface Membranes, 83
vi i
CONTENTS 111. A Basis for the Organization of Morphologically Polarized Cell Surfaces, 90 IV. Concluding Remarks, 93 References, 93
Chapter 8. Actin-Membrane Interactions in Eukaryotic Mammalian Cells THOMAS P. STOSSEL I. The Actin System and Membrane Function, 97 11. The Erythrocyte Cytoskeleton: Paradigm or Distraction in Approaching the Interaction between the Eukaryote Plasma Membrane and Actin System?, 98 111. The Actin System in Membrane Stabilization and Retraction, 99 IV. The Actin System in Membrane Propulsion, 101 V. The Actin System and Membrane Propulsion and Retraction, 104 References, 106
Chapter 9. Biogenesis and Cell Surface Distribution of Acetylcholine Receptors Stably Expressed in Fibroblasts TONI CLAUD10
I. Introduction, 109 11. Acetylcholine Receptor-Fibroblast Cell Lines, 110 111. Properties of Acetylcholine Receptors Expressed in Acetylcholine Receptor-Fibroblast Cells, 111 IV. Properties of Individually Expressed Subunits, 112 V. Posttranslational Modifications, 113 VI. Conclusions, 113 References. 114
Chapter 10. Control of Organelle Movements and Endoplasmic Reticulum Extension Powered by Kinesin and Cytoplasmic Dynein MICHAEL P. SHEETZ, SANDRA L. DABORA, ERIC STEUER, AND TRINA A. SCHROER I. Introduction, 117 11. Membranous Organelle Transport on Microtubules, 118 111. Interaction of Motors with Organelles to Produce Motility, 121 References, 126
PART IV. SIGNALING AND COMMUNICATION Chapter 11. G Protein-Coupled Receptors: Structure and :unction of Signal-Transducing Proteins ERIC M. PARKER AND ELLIOTT M. ROSS I. Introduction, 131 11. Mechanism of G-Protein Activation by Agonist-Liganded Receptors, 111. General Structure of G Protein-Coupled Receptors, 134
32
viii
CONTENTS
1V. Structure of the Ligand-Binding Domain, 136 V. Structure of the G Protein-Binding Domain, 138 VI. Receptor-Mimetic Peptides as Models for the G Protein-Binding Domain, 140 References, 141
Chapter 12. Mechano-Sensitive Ion Channels in Microbes and the Early Evolutionary Origin of Solvent Sensing CHING KUNG, YOSHIRO SAIMI, AND BORIS MARTINAC
I. Introduction, 145 11. A Stretch-Activated Ion Channel of Escherichia coli, 146 111. A Stretch-Activated Ion Channel in Yeast, 147 IV. Touch Receptors and Channels of Paramecium, 149 V. Mechano-Sensitive Channels and the Concept of Solvent Senses, 150 References. 152
Chapter 13. Selection of an afl T-cell Antigen Receptor in Vivo and Expression in Vitro in a Soluble Form M. M. DAVIS, B. FAZEKAS DE ST.GROTH, L. J. BERG, A. LIN, B. DEVAUX, C. SAGERSTROM, J. F. ELLIOTT, AND P. J. BJORKMAN
I. Introduction, 155 11. Positive Selection, 156 111. Negative Selection, 157 IV. Soluble T-cell Receptor Heterodimers, 158 References, 159
Chapter 14. Perforin and the Mechanism of Lymphocyte-Mediated Cytolysis ECKHARD R. PODACK AND MATHIAS G . LICHTENHELD I. Introduction, 161 11. Physicochemical and Functional Properties of Perforin, 162
111. Sequence of Murine and Human Perforin, 163 IV. Homology of Perforin to Complement Proteins: The Perforin Family, 167 V. Lack of Homologous Restriction of Perforin, 168 VI. Expression of Perforin mRNA in Vitro and in Vivo, 169 VII. The Contribution of Membrane Pores to DNA Degradation, 170 VIII. Conclusions, 171 References, 171 Note Added in Proof, 175
index, 177
Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.
B. D. Adair, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 0651 1 (71)
L. J. Berg, Howard Hughes Medical Institute and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305 (I 55)
P. J. Bjorkman, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305 (155) Juan S. Bonifacino, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 (31)
Toni Claudio, Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510 (109)
Joanne Crowe, Department of Biological Chemistry and Molecular Biology Institute, School of Medicine, University of California, Los Angeles, Los Angeles , California 90024 ( 19) Sandra L. Dabora, Department of Cell Biology and Physiology, Washington University Medical School, St. Louis, Missouri 631 10 (117)
M. M. Davis, Howard Hughes Medical Institute and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305 (155)
Johann Deisenhofer, Howard Hughes Medical Institute and Department of Biochemistry, University of Texas, Southwestern Medical Center, Dallas, Texas 75235 (53) B. Devaux, Howard Hughes Medical Institute and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305 (155) ix
CONTRIBUTORS
X
Michael Edidin, Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 (81)
J. F. Elliott, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305 (155)
D. M. Engelman, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 0651 1 (71)
B. Fazekas de St. Groth, Howard Hughes Medical Institute and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305 (155)
J. F. Hunt, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 0651 1 (71)
T. W. Kahn, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 0651 1 (7 1)
Richard D. Klausner, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 (31)
Ching Kung, Laboratory of Molecular Biology and Department of Genetics, University of Wisconsin-Madison, Madison, Wisconsin 53706 (145) Mathias G. Lichtenheld, Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33101 (161)
A. Lin, Howard Hughes Medical Institute and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305 (155)
Jennifer Lippincott-Schwartz, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 (31)
Boris Martinac, Laboratory of Molecular Biology and Department of Genetics, University of Wisconsin-Madison, Madison, Wisconsin 53706 (145)
David I. Meyer, Department of Biological Chemistry and Molecular Biology Institute, School of Medicine, University of California, Los Angeles, Los Angeles, California 90024 (19)
Hartmut Michel, Max-Planck-Institut fur Biophysik, D-6000 Frankfurt/M 7 1, Federal Republic of Germany (53)
Walter Neupert, Institut fur Physiologische Chemie, Universitat Miinchen, D-8000 Munchen 2, Federal Republic of Germany (3)
CONTRIBUTORS
xi
Eric M. Parker, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 (131) Nikolaus Pfanner, Institut fur Physiologische Chemie, Universitat Miinchen, D-8000 Munchen 2, Federal Republic of Germany (3) Eckhard R. Podack, Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33101 (161) J.-L. Popot, Institut de Biologie Physico-Chimique, CollZge de France, 7523 1 Paris, France (71) Elliott M. Ross, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 (131) C . Sagerstrom, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305 (155)
Yoshiro Saimi, Laboratory of Molecular Biology and Department of Genetics, University of Wisconsin-Madison, Madison, Wisconsin 53706 (145) Trina A. Schroer, Department of Cell Biology and Physiology, Washington University Medical School, St. Louis, Missouri 631 10 (1 17) Michael P. Sheetz, Department of Cell Biology and Physiology, Washington University Medical School, St. Louis, Missouri, 631 10 (1 17) Eric Steuer, Department of Cell Biology and Physiology, Washington University Medical School, St. Louis, Missouri 63110 (117) Thomas P. Stossel, Department of Medicine, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02 114 (97) Bill Wickner, Molecular Biology Institute and Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, California 90024 (15)
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Membrane-associated proteins mediate the full range of communications between a cell and its environment. The aim of this book is to explore some of the unique properties of membrane proteins which allow them to be intermediaries in cell-cell interactions and to interact with their environment. The contributions have been organized into four major sections. Part I covers membrane protein synthesis, targeting, and translocation into and across membranes. Part I1 treats structures of proteins in membranes. Problems associated with heterooligomeric subunit assembly, the folding of membrane proteins within the lipid bilayer, and the first successful crystallization of a membrane protein are described. In Part 111 the ability of membrane proteins to move or be moved in the membrane as well as the control of organelle movements are discussed. Part IV describes the involvement of membrane proteins in trans-membrane signal transduction. The conference from which this book was derived was sponsored by the Department of Cellular and Molecular Physiology of the Yale University School of Medicine. Many distinguished scientists participated, among whom was Dr. Hartmut Michel who learned during the first coffee break that he had won the Nobel Prize in Chemistry. I would like to thank all the participants, especially those who contributed to this volume. I gratefully acknowledge the following organizations for their generous financial support: Biogen Research Corporation, Johnson & Johnson, Miles, Inc., G. D. Searle Research & Development, Smith Kline & French Laboratories, The Squibb Institute for Medical Research, and The Upjohn Company. TONICLAUDIO
xiii
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Yale Membrane Transport Processes Volumes Emile L. Boulpaep (ed.). (1980). “Cellular Mechanisms of Renal Tubular Ion Transport”: Volume 13 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. William H. Miller (ed.). (198 1). “Molecular Mechanisms of Photoreceptor Transduction”: Volume 15 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. Clifford L. Slayman (ed.). (1982). “Electrogenic Ion Pumps”: Volume 16 o f Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Joseph F. Hoffman and Bliss Forbush 111 (eds.). (1983). “Structure, Mechanism, and Function of the Na/K Pump”: Volume 19 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York . James B. Wade and Simon A. Lewis (eds.). (1984). “Molecular Approaches to Epithelial Transport”: Volume 20 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Edward A. Adelberg and Carolyn W. Slayman (eds.). (1985). “Genes and Membranes: Transport Proteins and Receptors”: Volume 23 o f Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, Orlando. Peter S. Aronson and Walter F. Boron (eds.). (1986). ‘“a+-H+ Exchange, Intracellular pH, and Cell Function”: Volume 26 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, Orlando. Gerhard Giebisch (ed.). (1987). “Potassium Transport: Physiology and Pathophysiology”: Volume 28 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, Orlando. William S. Agnew, Toni Claudio, and Frederick J. Sigworth (eds.). (1988). “Molecular Biology of Ionic Channels”: Volume 33 of Current Topics in Membranes and Transport (J. F. Hoffman and G. Giebisch, eds.). Academic Press, San Diego. xv
XVI
YALE MEMBRANE TRANSPORT PROCESSES VOLUMES
Stanley G. Schultz (ed.). (1989). “Cellular and Molecular Biology of Sodium Transport”: Volume 34 of Current Topics in Membranes and Transport (J. F. Hoffman and G. Giebisch, eds.). Academic Press, San Diego. Toni Claudio (ed.). (1990). “Protein-Membrane Interactions”: Volume 36 of Current Topics in Membranes and Transport (J. F. Hoffman and G. Giebisch, eds.). Academic Press, San Diego.
Part I
Protein Insertion into and across Membranes
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 36
Chapter I
Mitochondrial Protein Import: Specific Recognition and Membrane Insertion of Precursor Proteins NIKOLAUS PFtWNER AND WALTER NEUPERT Institut fur Physiologische Chemie Universitiit Miinchen 0-8000Miinchen 2 , Federal Republic of Germany
I.
Introduction Receptor Proteins on the Mitochondria1 Surface A. Functional Characterization of Receptor Sites B. Identification of Receptors C. Import of Precursor Proteins Bypassing Receptor Sites 111. The “General Insertion Protein” IV. Role of Receptors and General Insertion Protein V. Summary and Perspectives References 11.
I. INTRODUCTION Eukaryotic cells are divided into numerous membrane-bounded compartments (“organelles”), each of which contains a unique and specific set of proteins. Most of the organellar proteins are synthesized as precursor proteins on cytosolic polysomes and thus have to be transported to their functional destination (Wickner and Lodish, 1985). The question of how proteins are directed to their specific target membrane and how they are translocated into and across organellar membranes poses a central theme of modem cell biology. The cytosolic precursor proteins carry specific targeting sequences that are assumed to bind to complementary structures (“receptors”) on the surface of organelles. The translocation
Copyright 0 1990 by Academic Press, Inc. All nghts of reproduction in any form reserved.
4
NIKOLAUS PFANNER AND WALTER NEUPERT
into and across the membranes may be mediated by proteinaceous components and/or lipids of the membranes. The biogenesis of organellar proteins is even more complex in case of mitochondria. Two membranes (outer and inner mitochondrial membranes) limit two soluble compartments, namely, the intermembrane space and the matrix. Whereas some mitochondrial proteins are coded for by mitochondrial genes and are synthesized in the matrix, >90% of the proteins are coded for by nuclear genes and are imported from the cytosol (Pfanner and Neupert, 1987a; Attardi and Schatz, 1988; Hart et al., 1989). It was first shown for mitochondrial protein import that translocation of precursor proteins across membranes is not mechanistically coupled to synthesis of the protein on ribosomes, both in vivu and in vitro (Hallermayer and Neupert, 1976; Harmey et al., 1976, 1977; Hallermayer et al., 1977). Many precursor proteins carry amino-terminal extension sequences ( “presequences”) of about 20-80 amino acid residues. Presequences contain signal information for translocation into mitochondria (Honvich et al., 1985; Hurt and van Loon, 1986). With some precursor proteins, targeting sequences were also found in nonamino-terminal (carboxyl-terminal) regions of the polypeptide (Pfanner et al., 1987b,c; Smagula and Douglas, 1988). The precursor proteins are recognized by specific receptors on the mitochondrial surface and are then inserted into the outer membrane (see later). Further translocation into the inner membrane occurs predominantly at sites of close contact between the mitochondrial outer and inner membranes (“contact sites”) (Schleyer and Neupert, 1985; Schwaiger et al., 1987; Rassow et a l . , 1989). Hydrophilic (proteinaceous) components apparently represent essential parts of contact sites (Pfanner et al., 1987a; Vestweber and Schatz, 1988). Presequences are proteolytically cleaved by the processing peptidase in the mitochondrial matrix (Hawlitschek et al., 1988), the proteins are sorted to their final intramitochondrial location (Hart1 et ul., 1987), and are often assembled into multisubunit complexes (Schmidt et al., 1983; Lewin and Norman, 1983). Protein import requires energy in (at least) two different forms. Adenosine triphosphate is involved in unfolding of precursor proteins in the cytosol and/or in release of precursor proteins from cytosolic cofactors (Pfanner et al., 1987d, 1988a; Eilers and Schatz, 1988; Murakami et al., 1988). The electrical potential (AT)across the inner mitochondrial membrane is needed for the initial transfer of precursors into or across the inner membrane (Pfanner and Neupert, 1985; Schleyer and Neupert, 1985). This article focuses on the problems of specific recognition and membrane insertion of mitochondrial precursor proteins. Harmey et al. (1977) had proposed that “some mechanism of selective recognition of precursor proteins by the mitochondria” exists. A detailed functional analysis in recent years has led to the characterization of receptor sites and of a membrane insertion site for precursors
1. MITOCHONDRIAL PROTEIN IMPORT RECEPTORS
5
and thereby provided the basis for identification of components of the mitochondria] protein import apparatus.
II. RECEPTOR PROTEINS ON THE MITOCHONDRIAL SURFACE A. Functional Characterization of Receptor Sites Pretreatment of isolated mitochondria with proteases diminished subsequent import of in vitro-synthesized precursor proteins (Gasser et al., 1982; Argan et al., 1983). A mild pretreatment with proteases inhibited not only import but also binding of precursors to mitochondria (Riezman et al., 1983; Zwizinski et al., 1984). The mitochondrial membranes were shown to remain intact under these conditions, suggesting that proteinaceous surface components are involved in import of precursor proteins. These components are assumed to perform the function of receptor sites. The import of various precursors exhibited a differential sensitivity toward pretreatment of mitochondria with specific proteases, supporting a model in which several distinct receptor sites exist on the mitochondrial surface (Zwizinski et al., 1984). Precursor proteins bind to the mitochondrial surface in the absence of a membrane potential across the inner membrane. After reestablishing a membrane potential, the precursor proteins are imported from the binding sites without prior release from the membranes (Zwizinski et al., 1983; Riezman et al., 1983; Pfanner and Neupert, 1987b; Pfanner et al., 1987d). This type of binding is termed specific (productive) binding and depends on the presence of surface proteins (“receptors”) (Zwizinski et al., 1984), whereas nonproductive (unspecific) binding also occurs to protease-pretreated mitochondria. Unspecific binding may occur to lipids of the outer membrane. The precursor of the mitochondrial outer membrane protein porin was prepared in large amounts (Pfaller et al., 1985) and bound to isolated mitochondria such that the mitochondrial import sites for porin and other precursor proteins (see Section 111) were saturated by the porin precursor. The precursor of the inner membrane protein ADP/ATP carrier could still bind to its proteinaceous surface sites (receptor); that is, binding of ADP/ATP carrier to its receptor was not competed for by porin (Pfaller et al., 1988). Since the affinities of those two precursors for interaction with mitochondria are in a similar range (Pfaller and Neupert, 1987; Pfaller et al., 1988), porin and ADP/ATP carrier seem to use distinct receptor sites. In summary, functional characterization suggests the following properties of specific mitochondrial import receptors. (i) Receptors are exposed on the mito-
6
NIKOLAUS PFANNER AND WALTER NEUPERT
chondrial surface. (ii) A specific receptor recognizes only a subset of precursor proteins; it is not involved in import of all precursor proteins. (iii) Receptors are required for specific binding of precursors to the mitochondrial surface.
B. Identification of Receptors In the past no receptor protein for import of mitochondrial precursor proteins could be identified despite numerous efforts in several laboratories. This led to speculations that the initial steps of mitochondrial protein import may not involve receptor proteins on the mitochondrial surface (Roise et al., 1986; Hurt and van Loon, 1986), although functional evidence clearly suggested the existence of specific receptor sites (summarized in Section &A; Pfanner et al., 1988b). We have started a systematic and basic approach with the aim of identifying the mitochondrial import receptors. Since the mitochondrial outer membrane contains only a relatively small number of proteins (-25), we tried to produce monospecific antibodies against each of these proteins and to test the effect of these antibodies on the import of precursor proteins. Outer membrane vesicles were isolated from mitochondria of the fungus Neurospora crassa. The proteins were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to nitrocellulose. Twenty-five distinct bands were excised and used for generation of polyclonal antisera in rabbits. We obtained monospecific antisera against many of the outer membrane proteins (Sollner et al., 1989). In a first screening we investigated the effect of immunoglobulin G (IgG), which was prepared from the antisera, on the import of ADP/ATP carrier and porin in the assumption that these two precursor proteins use different receptor sites (see Section 11,A). Immunoglobulin G was prebound to mitochondria, the mitochondria were reisolated, and the import of in vitro-synthesized precursors was tested. Most of the IgG had no significant effect on import of precursor proteins, including IgG against porin, the major protein of the outer membrane. Immunoglobulin G directed against a mitochondrial outer membrane protein of 19 kDa (MOM19) inhibited the import of porin, but not of ADP/ATP carrier. Thus MOM19 which is exposed on the mitochondrial surface appeared to be a possible candidate for a mitochondrial import receptor (Sollner et al., 1989). Fab fragments directed against MOM 19 were prebound to mitochondria and the import of precursors to the four mitochondrial subcompartments was studied (Table I). The import of porin (outer membrane), cytochrome cl, and Fe/S protein of the bc, complex (intermembrane space side of the inner membrane), subunit 9 of F,F,-ATPase (inner membrane), and subunit p of the F,F,-ATPase (matrix side of the inner membrane) were strongly inhibited. The import of ADP/ATP carrier (inner membrane) was practically unaffected. A series of controls excluded unspecific effects of IgG and Fab fragments against MOM19 (antiMOM 19), such as inactivation of precursor proteins or cytosolic cofactors and
7
1. MITOCHONDRIAL PROTEIN IMPORT RECEPTORS
TARGETING PATHWAYS
Protein
OF
TABLE I MITOCHONDRIAL PRECURSOR PROTEINS
Functional destination
Present in N-terminal prokaryotic targeting ancestor sequence
Receptor
Membrane insertion
Cytochrome cl Intermembrane space Fe/S protein Intermembrane space Fo-ATPase subunit 9 Inner membrane Fl-ATPase subunit p Matrix Porin Outer membrane
Yes Yes Yes Yes
Yes
?
?
ADP/ATP carrier
Inner membrane
No
No
MOM72b GIP
Cytochrome c
Intermembrane space
Yes
?
No surEndogenous face reactivity of ceptor precursor
UMitochondrial outer membrane protein of 19 kDa. bMitochondria1 outer membrane protein of 72 kDa. General insertion protein.
inhibition of later transport steps (translocation from the outer into the inner membrane, the membrane potential-dependent step, and proteolytic processing of precursor proteins) (Sollner et al., 1989). As a further control, the import of cytochrome c was tested. The precursor, apocytochrome c, spontaneously inserts into the outer mitochondrial membrane (Rietveld et al., 1985; Stuart et al., 1990) and does not use a protease-accessible surface receptor (Nicholson et al., 1988). As expected, anti-MOM19 did not inhibit the import of apocytochrome c. Inhibition of protein import by anti-MOM19 occurred at the level of specific binding of precursors to the mitochondrial surface. Thus MOM19 fulfills all the functional criteria established for a mitochondrial import receptor: it is exposed on the mitochondrial surface; it is involved in the import of a subset of precursor proteins, and it is required for specific binding of precursors to mitochondria. We conclude that the outer membrane protein MOM19 is identical to (or closely associated with) a specific import receptor (Sollner et al., 1989). With similar procedures we found IgG and Fab fragments against a mitochondrial outer membrane protein of 72 kDa (MOM72). Anti-MOM72 selectively inhibited the import of ADP/ATP carrier, but not of other mitochondrial precursor proteins tested. The inhibition of import occurred at the level of specific binding of ADP/ATP carrier to the mitochondrial surface, whereas other import steps were unaffected (Sollner et al., 1990). We conclude that MOM72, which is exposed on the mitochondrial surface, represents a specific import receptor for ADP/ATP carrier and probably similar precursors (Table I).
a
NIKOLAUS PFANNER AND WALTER NEUPERT
C. Import of Precursor Proteins Bypassing Receptor Sites Blocking of receptor sites by specific antibodies or degradation of receptors by treatment of mitochondria with proteases strongly reduced the import rates of mitochondrial precursor proteins (Pfaller et al., 1989; Sollner et al., 1989). This suggests a crucial role of receptor sites for the efficiency of protein import. A residual import of precursors, however, can also occur when the surface receptors are blocked or degraded. This low efficient import still exhibits several basic features of mitochondrial protein import, including dependence on ATP and membrane potential A* and translocation via contact sites (Pfaller et al., 1989). Precursor proteins are obviously able to bypass surface receptors and enter the mitochondrial import pathways at a later stage. The very low efficiency suggests that bypass import does not significantly contribute to the import processes under physiological conditions (Pfanner et al., 1988~). The existence of bypass import, at least under certain experimental conditions (high amounts of precursor proteins), led to a series of very surprising findings. Nonmitochondrial “targeting” sequences, such as a chloroplast signal sequence (Hurt et al., 1986) or sequences of a cytosolic protein (Hurt and Schatz, 1987), could direct proteins into mitochondria albeit with a low efficiency (summarized in Pfanner er al., 1988~).The main common property of these nonmitochondrial signals was the abundance of positively charged amino acid residues, which appears to be an essential requirement for the A*-dependent insertion into the inner membrane. Import directed by nonmitochondrial signals was not affected by pretreatment of mitochondria with proteases. These “targeting” signals apparently bypass the surface receptors (Pfaller et al., 1989). Mitochondria1 import receptors therefore specifically interact with authentic mitochondrial targeting sequences; receptors are responsible for the selectivity of protein import. Yeast mitochondria with disrupted outer membrane are able to translocate precursor proteins directly across the inner membrane (Ohba and Schatz, 1987a; Rassow, Pfanner, and Neupert, in preparation). The nature and function of these inner membrane import sites is unknown. They might for example be related to the translocation sites that are (permanently or transiently) present in contact sites between both membranes (Schwaiger et al., 1987). Further studies are required to decide how specific these import sites are and if they contain components for recognition of mitochondrial precursor proteins.
111.
THE “GENERAL INSERTION PROTEIN”
After interaction with specific surface receptors, mitochondrial precursor proteins are inserted into the outer membrane. Studies on the import pathways of
1. MITOCHONDRIAL PROTEIN IMPORT RECEPTORS
9
ADP/ATP carrier and porin suggested the existence of a new functional component for protein translocation across membranes, a membrane insertion site (Pfanner and Neupert, 1987b; Pfaller and Neupert, 1987). Precursor proteins that are inserted into the outer membrane are not accessible to specific antibodies or to low concentrations of proteases added to the mitochondria, in contrast to precursors that are bound to the surface receptors (Sollner et ul., 1988), suggesting that the membrane insertion sight is buried in the outer membrane. The membrane insertion sites are saturable; the determined number of sites is practically identical for ADP/ATP carrier and porin and is in a similar range as the number of receptor sites (Pfaller and Neupert, 1987; Pfaller et al., 1988). Precursor proteins inserted into the outer membrane are extractable from the membranes by “hydrophilic perturbants” or “protein denaturants” such as carbonate ions or urea (Pfanner and Neupert, 1987b; Pfaller and Neupert, 1987). The precursor proteins thus may be inserted into a proteinaceous membrane environment. The precursor of porin competed for the import of nearly all other mitochondrial precursor proteins tested, including cytochrome cl, Fe/S protein, F,-ATPase subunit 9, F,-ATPase subunit p, and ADP/ATP carrier (Table I). Competition of import specifically occurred at the level of insertion of precursors into the outer membrane (Pfaller et al., 1988) and not for the interaction with receptor sites (see Section 11,A). We concluded that the various precursor proteins competed for interaction with the same component of the protein import apparatus, namely, a common membrane insertion site. The only precursor protein the import of which was not competed for was apocytochrome c (Table I); this fits well with the bulk of evidence suggesting that cytochrome c uses a very unique import pathway (Nicholson et al., 1988). The common membrane insertion site, termed the general insertion protein (GIP), has not been identified so far. The receptors MOM19 and MOM72 can form a high molecular weight complex that contains two other outer membrane proteins. One of these proteins, termed MOM38 (molecular weight 38 K), exhibits the properties expected of GIP (Pfaller, Sollner, Griffiths, Pfanner, and Neupert, in preparation). A recent finding on protein import into yeast mitochondria also may be of interest for the identification of GIP. Antibodies directed against 45-kDa mitochondrial proteins inhibit import of precursor proteins when bound to mitochondria that had been pretreated with proteases (Ohba and Schatz, 1987b). Where the antibodies bound to intact mitochondria, the inhibition of import was only marginal; the antibodies obviously do not block receptor proteins that are exposed on the mitochondrial surface. Since the inactivated component(s) appears to be protected against proteases, it may be buried in the outer membrane and has to be “freed” from other proteins by the pretreatment with proteases in order to be accessible to the antibodies. We speculate that a component that is recognized by the anti-45-kDa antibodies is related to GIP (Pfaller et
10
NIKOLAUS PFANNER AND WALTER NEUPERT
al., 1988). Vestweber el al. (1989) reported that these antibodies also recognized a protein of 42 kDa, termed ISP42 (import site protein). Inhibition of import was found to be caused by the anti-ISP42 antibodies. Moreover, a fraction of precursor proteins that was accumulated in contact sites was cross-linked to ISP42. MOM38 of Neurospora crassa and ISP42 of yeast thus may be related to GIP.
IV.
ROLE OF RECEPTORS AND GENERAL INSERTION PROTEIN
Receptor proteins on the mitochondrial surface are responsible for the specificity and selectivity of protein uptake (see Section I1,C). They recognize mitochondria] targeting sequences and strongly enhance the import rates of those precursors. Precursor proteins with amino-terminal targeting sequence interact with the receptor MOM19, whereas ADP/ATP carrier, a precursor with several internal targeting sequences (but no N-terminal signal), interacts with MOM72 (Table I). Treatment of mitochondria with the protease elastase generates an 17-kDa fragment of MOM19 that still mediates import of F,-ATPase subunit f3, whereas import of other MOM 19-dependent precursor proteins is inhibited (Sollner et al., 1989). Distinct segments of MOM19 may thus be responsible for interaction with the various precursor proteins. This offers the possibility for characterization of functional sites of this import receptor. Most mitochondrial proteins that were found to require MOM19 for import have equivalents in bacteria and thus were probably already present in the prokaryotic ancestors of mitochondria (Table I) (the evolutionary origin of porin is unknown). According to the endosymbiont hypothesis, after endocytosis of the prokaryotic cell, the (now) mitochondrial genes for these proteins were transferred to the nucleus (see Hart1 et al., 1987, for a discussion). An amino-terminal targeting sequence directed the proteins back to mitochondria. We propose that MOM19 was used as surface receptor for those precursor proteins. On the other hand, proteins exist that were most likely not present in the prokaryotic ancestor. The ADP/ATP carrier was probably established in the eukaryotic cell (Klingenberg, 1985). Its targeting sequences are not located at the amino terminus of the precursor protein (Pfanner et al., 1987b; Smagula and Douglas, 1988), and the precursor uses a different surface receptor, MOM72 (Sollner et al., 1990). The receptors MOM19 and MOM72 then transfer the precursor proteins to the GIP in the outer membrane. The receptors themselves may possess some activity for membrane insertion of precursor proteins and thereby facilitate the action of GIP. Alternatively, receptors may only be able to bind precursor proteins, and the insertion into the outer membrane is solely performed by GIP (possibly in cooperation with lipids of the outer membrane). As described earlier, MOM19, MOM72, and probably GIP can be detected in a protein complex in the outer membrane (Pfaller et al., in preparation). The receptors and GIP may not func-
1. MITOCHONDRIAL PROTEIN IMPORT RECEPTORS
11
tion as independent entities in the outer membrane. Their possible assembly into a multisubunit complex may help in coordination of their activities; it may even be a prerequisite for some of their functions. Beyond GIP, the import pathways diverge; some precursors assemble into the outer membrane (porin), whereas most precursors move on to contact sites between both membranes and then to the other mitochondrial subcompartments (Pfaller et al., 1988; Hart1 et al., 1989). Since translocation of proteins across the mitochondrial membranes occurs predominantly at contact sites, receptors and GIP may be concentrated in contact site regions of the outer membrane to ensure efficient and rapid translocation of precursor proteins. It might well be that, in addition, receptors and GIP are distributed over the entire mitochondrial surface in order to increase the probability for the initial high-affinity binding of precursor proteins and to collect the precursors for transfer to contact sites. This implies lateral diffusion of receptors and/or GIP in the outer membrane. V.
SUMMARY AND PERSPECTIVES
Functional characterization of initial steps of mitochondrial protein import provided the tools for identification of two mitochondrial outer membrane proteins, MOM 19 and MOM72, as specific receptors for precursor proteins. Bound precursor proteins are transferred to a common membrane insertion site in the outer membrane, the “general insertion protein” (GIP). Future research will address the role of functional domains of the receptors and the type and specificity of interaction with precursor proteins. The existence of (at least) two distinct membrane-bound receptors for precursor proteins appears to be of relevance for protein translocation across membranes in general; it may have implications on other organelles such as chloroplasts and the endoplasmic reticulum. A GIP for the entry of precursor proteins into a membrane is most likely not only present in mitochondria, but may also be found in several other biological membranes. For instance, the SecY (PrlA) protein, an integral protein of the cytoplasmic membrane of Escherichia coli that is involved in export of proteins (Watanabe and Blobel, 1989; Wickner, 1989), might have a similar role. ACKNOWLEDGMENTS We thank R . A. Stuart for critical reading of the manuscript. We are grateful to our colleagues for their many contributions to this work. REFERENCES Argan, C., Lusty, C. J., and Shore, G. C. (1983). Membrane and cytosolic components affecting transport of the precursor for ornithine carbamyltransferase into mitochondria. J . Biol. Chem. 258, 6667-6670. Attardi, G . , and Schatz, G. (1988). Biogenesis of mitochondria. Annu. Rev. Cell Biol. 4, 289-333.
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Eilers, M., and Schatz, G. (1988). Protein unfolding and the energetics of protein translocation across biological membranes. Cell 52, 481-483. Gasser, S. M., Daum, G., and Schatz, G. (1982). Import of proteins into mitochondria: Energydependent uptake of precursors by isolated mitochondria. J . Biol. Chem. 257, 13034-13041. Hallermayer, G., and Neupert, W. (1976). Studies on the synthesis of mitochondrial proteins in the cytoplasm and on their transport into the mitochondrion. In “Genetics and Biogenesis of Chloroplasts and Mitochondria” (T. Biicher, W. Neupert, W. Sebald, and S. Werner, eds.), pp. 807-812. North-Holland Publ., Amsterdam. Hallermayer, G., Zimmermann, R., and Neupert, W. (1977). Kinetic studies on the transport of cytoplasmically synthesized proteins into the mitochondria in intact cells of Neurosporu crussu. Eur. J . Biochem. 81, 523-532. Harmey, M. A,, Hallermayer, G., and Neupert, W. (1976). In vitro synthesis and transport into mitochondria of cytoplasmically translated proteins. In “Genetics and Biogenesis of Chloroplasts and Mitochondria” (T. Biicher, W. Neupert, W. Sebald, and S . Werner, eds.), pp. 8138 18. North-Holland Publ., Amsterdam. Harmey, M. A,, Hallermayer, G., Korb, H., and Neupert, W. (1977). Transport of cytoplasmically synthesized proteins into the mitochondria in a cell free system from Neurosporu crussu. Eur. J . Biochem. 81, 533-544. Hartl, F.-U., Ostermann, J., Guiard, B., and Neupert, W. (1987). Successive translocation into and out of the mitochondrial matrix: Targeting of proteins to the intermembrane space by a bipartite signal peptide. Cell 51, 1027-1037. Hartl, F.-U., Pfanner, N., Nicholson, D. W., and Neupert, W. (1989). Mitochondria1protein import. Biochim. Biophys. Actu 988, 1-45. Hawlitschek, G., Schneider, H., Schmidt, B., Tropschug, M., Hartl, F.-U., and Neupert, W. (1988). Mitochondrial protein import: Identification of processing peptidase and of PEP, a processing enhancing protein. Cell 53, 795-806. Horwich, A. L., Kalousek, F., Mellman, I., and Rosenberg, L. E. (1985). A leader peptide is suficient to direct mitochondrial import of a chimeric protein. EMBO J . 4, 1129-1 135. Hurt, E. C., and Schatz, G. (1987). A cytosolic protein contains a cryptic mitochondrial targeting signal. Nature (London) 325, 499-503. Hurt, E. C., and van Loon, A. P. G. M. (1986). How proteins find mitochondria and intramitochondrial compartments. Trends Biochem. Sci. 11, 204-207. Hurt, E. C., Soltanifar, N., Goldschmidt-Clermont M., Rochaix, J.-D., and Schatz, G. (1986). The cleavable pre-sequence of an imported chloroplast protein directs attached polypeptides into yeast mitochondria. EMBO J . 5 , 1343-1350. Klingenberg, M. (1985). Principles of carrier catalysis elucidated by comparing two similar membrane translocators from mitochondria, the ADPIATP carrier and the uncoupling protein. Ann. N . Y . Acud. Sci. 456, 279-288. Lewin, A. S., and Norman, D. K. (1983). Assembly of F,-ATPase in isolated mitochondria. J . Biol. Chem. 258, 6750-6755. Murakami, H., Pain, D., and Blobel, G. (1988). 70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria. J . Cell B i d . 107, 205 1-2057. Nicholson, D. W., Hergersberg, C., and Neupert, W. (1988). Role of cytochrome c heme lyase in the import of cytochrome c into mitochondria. J . Biol. Chem. 263, 19034-19042. Ohba, M., and Schatz, G. (1987a). Disruption of the outer membrane restores protein import to trypsin-treated yeast mitochondria. EMBO J . 6, 21 17-2122. Ohba, M . , and Schatz, G. (1987b). Protein import into yeast mitochondria is inhibited by antibodies raised against 45-kd proteins of the outer membrane. EMBO J . 6, 2109-21 15.
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Pfaller, R., and Neupert, W. (1987). High-affinity binding sites involved in the import of porin into mitochondria. EMBO J. 6, 2635-2642. Pfaller, R., Freitag, H., Harmey, M. A., Benz, R., and Neupert, W. (1985). A water-soluble form of porin from the mitochondrial outer membrane of Neurosporu crussu; Properties and relationship to the biosynthetic precursor form. J. Biol. Chem. 260, 8188-8193. Pfaller, R., Steger, H. F., Rassow, J., Pfanner, N., and Neupert, W. (1988). Import pathways of precursor proteins into mitochondria: Multiple receptor sites are followed by a common membrane insertion site. J . Cell Biol. 107, 2483-2490. Pfaller, R., Pfanner, N., and Neupert, W. (1989). Mitochondria1 protein import: Bypass of proteinaceous surface receptors can occur with low specificity and efficiency. J. Biol. Chem. 264, 34-39. Pfanner, N., and Neupert, W. (1985). Transport of proteins into mitochondna: A potassium diffusion potential is able to drive the import of ADP/ATP carrier. EMBO J. 4, 2819-2825. Pfanner, N., and Neupert, W. (1987a). Biogenesis of mitochondrial energy transducing complexes. Curr. Top. Bioenerg. 15, 177-219. Pfanner, N., and Neupert, W. (1987b). Distinct steps in the import of ADP/ATP carrier into mitochondria. J . Biol. Chem. 262, 7528-7536. Pfanner, N., Hartl, F.-U., Guiard, B., and Neupert, W. (1987a). Mitochondria1 precursor proteins are imported through a hydrophilic membrane environment. Eur. J. Biochem. 169, 289-293. Pfanner, N., Hoeben, P., Tropschug, M., and Neupert, W. (1987b). The carboxyl-terminal two thirds of the ADP/ATP carrier polypeptide contains sufficient information to direct translocation into mitochondria. J. Biol. Chem. 262, 14851-14854. Pfanner, N., Miiller, H.,Harmey, M. A., and Neupert, W. (1987~).Mitochondrial protein import: Involvement of the mature part of a cleavable precursor protein in the binding to receptor sites. EMBO J. 6, 3449-3454. Pfanner, N., Tropschug, M., and Neupert, W. (1987d). Mitochondrial protein import: Nucleoside triphosphates are involved in conferring import-competence to precursors. Cell 49, 8 15-823. Pfanner, N., Pfaller, R., Kleene, R., Ito, M., Tropschug, M., and Neupert, W. (1988a). Role of ATP in mitochondrial protein import: Conformational alteration of a precursor protein can substitute for ATP requirement. J. Biol. Chem. 263, 4049-4051. Pfanner, N., Hartl, F.-U., and Neupert, W. (1988b). Import of proteins into mitochondria: A multistep process. Eur. 1. Biochem. 175, 205-212. Pfanner, N., Pfaller, R., and Neupert, W. (1988~).How finicky is mitochondrial protein import? Trends Biochem. Sci. 13, 165-167. Rassow, J., Guiard, B., Wienhues, U., Herzog, V., Hartl,F.-U., and Neupert, W. (1989). Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites. J. Cell Biol. 109, 1421- 1428. Rietveld, A., Ponjee, G. A. E., Schiffers, P., Jordi, W., van de Coolwijk, P. J. F. M., Demel, R. A., Marsh, D., and de Kruijff, B. (1985). Investigations on the insertion of the mitochondrial precursor protein apocytochrome c into model membranes. Biochim. Biophys. Acru 818, 398409. Riezman, H., Hay, R., Witte, C . , Nelson, N., and Schatz, G. (1983). Yeast mitochondrial outer membrane specifically binds cytoplasmically synthesized precursors of mitochondrial proteins. EMEO J. 2, 1113-1118. Roise, D. Horvath, S. J., Tomich, J. M., Richards, J. H., and Schatz, G. (1986). A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipid bilayers. EMBO J . 5, 1327-1334. Scbleyer, M., and Neupert, W. (1985). Transport of proteins into mitochondria: Translocational intermediates spanning contact sites between outer and inner membranes. Cell 43, 339-350.
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Schmidt, B . , Hennig, B., Zimmermann, R., and Neupert, W. (1983). Biosynthetic pathway of mitochondrial ATPase subunit 9 in Neurosporu crussa. J . Cell Biol. 96, 248-255. Schwaiger, M., Herzog, V., and Neupert, W. (1987). Characterization of translocation contact sites involved in the import of mitochondrial proteins. 1. Cell Biol. 105, 235-246. Smagula, C. S . , and Douglas, M. G. (1988). ADP-ATP carrier of Saccharomyces cerevisiae contains a mitochondrial import signal between amino acids 72 and 111. J . Cell. Biochem. 36,323-328. Sollner, T., Pfanner, N., and Neupert, W. (1988). Mitochondria1protein import: Differential recognition of various transport intermediates by antibodies. FEES Left. 229, 25-29. Sollner, T., Grifiths, G., Pfaller, R., Pfanner, N., and Neupert, W. (1989). MOM19, an import receptor for mitochondrial precursor proteins. Cell, in press. Sollner, T., Pfaller, R., Grifiths, G., Pfanner, N., and Neupert, W. (1990). A mitochondrial import receptor for the ADPlATP carrier. Submitted. Stuart, R. A,, Nicholson, D. W., and Neupert, W. (1990). Early steps in mitochondrial protein import: Receptor functions can be substituted by membrane insertion activity of apocytohchrome c. Celf 60, in press. Vestweber, D., and Schatz, G. (1988). Mitochondria can import artificial precursor proteins containing a branched polypeptide chain or a carboxyl-terminal stilbene disulfonate. J . Cell Biol. 107, 2045-2049. Vestweber, D., Brunner, J., Baker, A,, and Schatz, G. (1989). A 42k outer-membrane protein is a component of the yeast mitochondria1 protein import site. Nature (London) 341, 205-209. Watanabe, M., and Blobel, G. (1989). Site-specific antibodies against the PrlA (SecY) protein of Escherichia coli inhibit protein export by interfering with plasma membrane binding or preproteins. Proc. Natl. Acad. Sci. U.S.A. 86, 1895-1899. Wickner, W. (1989). Secretion and membrane assembly. Trends Biochem. Sci. 14, 280-283. Wickner, W. T., and Lodish, H. F. (1985). Multiple mechanisms of protein insertion into and across membranes. Science 230, 400-407. Zwizinski, C., Schleyer, M., and Neupert, W. (1983). Transfer of proteins into mitochondria: Precursor to the ADP/ATP carrier binds to receptor sites on isolated mitochondria. J . Biol. Chem. 258, 4071-4074. Zwizinski, C . , Schleyer, M., and Neupert, W. (1984). Proteinaceous receptors for the import of mitochondrial precursor proteins. J . Biol. Chem. 259, 7850-7856.
CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 36
Chapter 2 An Enzymological Approach to Membrane Assembly and Protein Secretion BILL WICKNER Molecular Biology Institute and Department of Biological Chemistry University of California, Los Angeles Los Angeles, California 90024
Despite almost two decades of study, little is known at a mechanistic level of how proteins assemble into, or across, biological membranes. This problem has been intensively studied for mitochondria, endoplasmic reticulum, chloroplasts, and the bacterial cell surface. In mammalian systems, in vivo studies have been difficult, and most work has focused on addition of intact organelles to in vitro protein synthesis reactions. Microorganisms offer several major advantages for the study of this fundamental process. They have advanced genetics, they can readily be grown in large culture for biochemistry, and their growth on minimal medium allows isotopic labeling for in vivo studies. Bacterial secretion studies have employed each of these major avenues, and today are the most advanced toward mechanistic understanding of protein translocation. Genetic identification of temperature-sensitive mutants that affect secretion (sec mutants) or of mutants that suppress leader sequence mutants, and are thereby more permissive for protein localization @rl mutants), has led to the identification of several genes that are essential for the membrane transit of most proteins. In vivo studies, in which growing cells are pulse-labeled and then “chased” with a chemical excess of nonradioactive amino acids, has established that translocation is not coupled to translation in this organism. Indeed, the “battle” over whether bacterial translocation is obligately cotranslational or not set the stage for subsequent studies in yeast and mammalian systems that have shown that translocation is, in general, never coupled to translation in Nature (or Science or Cell!). With the experimental resolution of the process into the distinct steps of translation and translocation, it became possible to study the energetics 15 Copyright 0 1990 by Academc Press, Inc. All rights of reproduction in any form reserved.
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BILL WICKNER
of the process. Bacterial protein export, as with mitochondria1 protein import, requires a membrane electrochemical potential in vivo. After translocation, leader peptidase, an inner membrane protein, cleaves the amino-terminal leader sequence from exported proteins. Two years ago, the only available pure component of this system was leader peptidase. Thus, the biochemistry of this process was not yet studied. To begin this study, we have undertaken an enzymological approach, beginning with the in vivo translocation reactions of Tai, Blobel, and their colleagues, and moving toward a reconstitution of protein translocation that will employ defined, purified components. We began by isolating a precursor form of a secreted protein. We chose proOmpA, the precursor form of the major outer membrane protein A. The isolation of proOmpA was hampered by its limited solubility. Elliott Crooke, who at the time was a student in my laboratory, chose the bold approach of solubilizing the proOmpA in 8 M urea, reflecting a faith that the proOmpA would be renaturable for membrane assembly once it was purified and the urea was removed. When Crooke purified the proOmpA and removed the urea by dialysis, he was dismayed to find that the proOmpA was totally inactive for translocation. However, he found that the addition of a soluble protein extract to the proOmpA prior to dialysis allowed the dialyzed proOmpA to be active for membrane assembly. This provided the assay for “trigger factor,” a protein that stabilizes the proOmpA in an active confirmation for membrane assembly. Trigger factor is just one of the three known Escherichia coli “chaperone” proteins; the others are GroEL and SecB protein. The relative roles of each in protein export will require a genetic analysis; this should be facilitated by the recent isolation of the trigger factor gene (B. Guthrie and W. Wickner, unpublished). Biochemically, each is able to form a complex with proOmpA. This complex is isolable and stable, yet can readily release the proOmpA to assembly across the membrane. Studies are under way to try to determine the structure of proOmpA in these complexes, that is, whether the proOmpA has a unique tertiary structure or is unfolded at the tertiary-structure level. In addition, it will be important to determine the basis of specificity of these and other chaperone proteins. The ability to combine genetics and biochemistry in yeast and E . coli should make these the premier organisms for such studies. In our experiments, the only cytosolic proteins that are needed for translocation are the chaperones and the preproteins themselves. Proceeding to the membrane, the SecA protein is essential for translocation. Thanks to the studies of Don Oliver, this protein was available in large quantities and had been shown to be a peripheral membrane protein of 102,000 Da. We found that the pure SecA protein would restore activity to membranes that had been inactivated for translocation by N,-ATP, and proceeded from this observation to the finding that SecA itself is an ATPase. Most strikingly, this ATPase activity is subject to a remarkable group of allosteric controls that we are only just now sorting out.
2. AN ENZYMOLOGICAL APPROACH
17
SecA protein is a fully water-soluble protein, with little tendency to aggregate at even high protein concentrations. Nevertheless, SecA binds to membrane vesicles or even to liposomes as a peripheral membrane protein. We have not yet been able to demonstrate any high-affinity binding to a saturable site, although this may simply reflect current limitations on our assay conditions. The ATP hydrolytic activity of SecA is stimulated up to 100-fold by the simultaneous presence of proOmpA (or other precursor proteins) and inner membrane vesicles. We term this activity “translocation ATPase.” What are the characteristics of this translocation ATPase? Omission of any one of Mg, SecA protein, proOmpA, or inner membrane vesicles results in a complete loss of the translocation ATPase activity. The membranes contribute at least two essential components, the lipid and the SecY protein. The proOmpA itself must be in a “competent” state for translocation (either freshly diluted from urea or in complex with chaperone) in order to function for translocation. This assay is thus clearly coupled to each of the elements of protein transit across membranes. However, there is not a tight, stoichiometriccoupling of the moles of ATP hydrolyzed to the moles of proOmpA translocated, and in this sense we view our current translocation ATPase assay as “uncoupled.” We have been able to exploit this reaction to advance our understanding in two directions. Membrane vesicles have been solubilized in detergent and reconstituted to form proteoliposomes that support translocation ATPase. The detergent extract contains solubilized SecY, and this SecY is required for the reconstitution. The secY protein is being purified in a functional state based on this assay. A second manner in which we have exploited the translocation ATPase assay began with a humble Mg concentration curve. Roland Lill in the laboratory found that, at low Mg level, the SecA ATPase can be stimulated to high levels of activity by proOmpA and lipids, in the absence of SecY or other integral membrane proteins. This activity depends on the proOmpA as directly as the SecYdependent translocation ATPase. In addition, we have observed that the leader peptide plus the membrane protein can substitute for the intact precursor protein (R. Lill, unpublished observations)! Thus, the SecA protein can directly recognize the preprotein. Many challenges remain in the study of protein translocation. How is the energy of ATP hydrolysis and the membrane electrochemicalpotential coupled to the translocation event? What is the chemical role of the SecY protein, and of other integral membrane proteins? Does the precursor protein pass through the bilayer directly or through a proteinaceous transport pore? I am confident that the resolution and functional reconstitution of the integral membrane proteins that support translocation will be essential to answer these mechanistic questions. My laboratory and many others are devoted to this approach.
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CLRRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 36
Chapter 3
Protein Translocation in Yeast JOANNE CROWE AND DAVID I . MEYER Department of Biological Chemistry and Molecular Biology Institute School of Medicine University of California, Los Angeles Los Angeles, California 90024
I.
Introduction
11. Yeast as a Model System 111. The Importance of Preprotein Conformation
IV. Binding to the Membrane and Translocation Are Separable Events A. Binding to the Membrane B. Translocation V. Analysis of ptll-A Translocation Mutant A. In Vivo Analysis B . In Vitro Analysis References
1.
INTRODUCTION
In eukaryotes, proteins that are destined for secretion, lysosomes, the plasma membrane, and organelles of the secretory pathway must be transported from their site of synthesis to their ultimate location in the cell. The first step in this process is the targeting of the nascent protein to the rough endoplasmic reticulum (ER), followed by translocation into or across the membrane. Analysis of this process in vitro was made possible by the development of the heterologous cellfree translocation assay (Blobel and Dobberstein, 1975), the use of which resulted in the characterization of several participating components. These include the signal recognition particle (SRP) (Walter et al., 1981; Walter and Blobel, 1981a,b), its receptor, the docking protein (Meyer et a l . , 1982; Gilmore et al., 1982), signal peptidase-purified as a complex (Evans 'et a l . , 1986)-the putative signal sequence receptor (SSR) (Wiedmann et al., 1987), and a protein 19 Copyright 6 1990 by Academc Press, Inc. All nghts of reproduction in any form reserved.
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JOANNE CROWE AND DAVID I . MEYER
associated with the docking protein (Tajima et al., 1986). The binding of ribosomes to the ER appears to depend on an unidentified membrane protein (Hortsch et a l . , 1986). There are probably several other ER proteins involved in the translocation process; however, their isolation has not yet been possible using conventional biochemical techniques in an in vitro system. Proteins specific to the rough ER have not so far been efficiently fractionated, making identification of participants by reconstitution of translocation in vitro very difficult.
II. YEAST AS A MODEL SYSTEM The yeast Saccharomyces cerevisiae carries out secretion and membrane biogenesis in a fashion analogous to mammalian cells and possesses all the relevant organelles: rough ER, Golgi apparatus, and secretory vesicles. In addition, it has a well-characterized, easily manipulated genome, and a convenient methodology exists for the isolation of genes of interest. The combination of in vivo and genetic analyses of yeast translocation mutants with the in vitro analysis of these mutants using the yeast cell-free translocation assay (Rothblatt and Meyer, 1986; Hansen et a l . , 1986; Waters and Blobel, 1986) represents a powerful system for characterizing both membrane and cytosolic components involved in translocation. This approach has led to considerable understanding of the events in this process (summarized in Table I). An important advantage of the yeast cell-free assay, which has been exploited in many studies (reviewed in Zimmerrnann and Meyer, 1986), is the uncoupling of translocation from translation. The precursor of the yeast mating pheromone, prepro-a-factor, can be translocated across yeast rough microsomes in the total absence of ongoing protein synthesis, although certain cytosolic factors are required for this process (Rothblatt e f a l . , 1987; Sanz and Meyer, 1988; Chirico et TABLE I EVENTSIN PROTEIN TRANSLOCATION IN YEAST" Location Cytosol ER Cytosolic face ER Membrane ER Lumen
Process
Translation Conformation preservation Recognition Binding to the membrane Translocation Processing, glycosylation
OThe process of translocation in yeast can be divided into several separate events, each occurring at a precise intracellular location.
3. PROTEIN TRANSLOCATION IN YEAST
21
al., 1988; Deshaies et al., 1988). A more refined assay, in which affinity-purified precursor proteins-rapidly diluted out of urea-can cross yeast rough microsomes in the absence of cytosolic factors, has allowed further dissection of the translocation process in vitro (Sanz and Meyer, 1988; Crooke et al., 1988a). 111. THE IMPORTANCE OF PREPROTEIN CONFORMATION There is considerable evidence that preprotein conformation and its maintenance play an important role in translocation across a variety of different membranes (reviewed in Meyer, 1988). For certain bacterial preproteins, such as proOmpA, a cytosolic component known as trigger factor is needed for translocation in vitro across inverted vesicles (Crooke and Wickner, 1987). It has been shown to maintain the protein in a more protease-sensitive state, indicative of a more relaxed or “open” conformation. The product of the bacterial SecB gene appears to perform a similar function to trigger factor, albeit acting on different precursor proteins, both in vivo and in vitro (Randall and Hardy, 1986; Collier et al., 1988). Even in the absence of these cytosolic proteins, the presence of a signal sequence on the precursor of the bacterial maltose-binding protein has been shown to play a role in the retardation of folding (Park et al., 1988). Cytosolic factors identified as members of the hsp70 family of heat shock proteins have been postulated to act as preprotein “unfoldases” or preservers of conformation (Pelham, 1986; Rothman and Kornberg, 1986). These proteins, which bind ATP, have been shown in yeast, both in vitro and in vivo, to be necessary for translocation into the ER, and also to be involved in import into mitochondria and vacuoles (Chirico et al., 1988; Deshaies et al., 1988; Murakami et al., 1988). They have also been shown to stimulate translocation of preproteins across mammalian rough ER in vitro (Zimmermann et al., 1988). Several studies have examined the effect of precursor folding on translocation. Mitochondria1 membranes, which are able to translocate even large, branched molecules (Vestweber and Schatz, 1988a,b), show dramatically reduced translocation efficiencies when the preprotein is artificially held in a folded conformation (Eilers and Schatz, 1986, 1988). The use of denatured precursor proteins enhanced the efficiency of posttranslational translocation across mitochondrial, bacterial, and yeast ER membranes in vitro (Sanz and Meyer, 1988; Eilers et al., 1988; Crooke et al., 1988b). In this assay, precursors that have been denatured in 8 M urea are competent for translocation when the urea is rapidly diluted into a solution of membranes in buffer only. This competence is lost as the time between dilution and the addition of membranes increases. The translocation competence can be preserved over longer periods of time if the preproteins are diluted from the urea into a solution of appropriate cytosolic factors. This provides an
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JOANNE CROWE AND DAVID I. MEYER
experimental system for the analysis of the requirements of posttranslational translocation. Using this simplified version of the yeast in vitro system, Sanz and Meyer (1988) have shown that SRP, when present during the refolding of urea-denatured proOmpA or prepro-a-factor, can replace all the cytosolic factors required for posttranslational translocation competence in yeast, as was also shown in bacteria (Crooke et al., 1988a). It can also stimulate the translocation of purified proOmpA across pancreatic microsomes. In addition, they demonstrated that proOmpA incubated with SRP showed enhanced sensitivity to protease, suggesting that it is in a more relaxed conformation than native proOmpA. These data imply that, among other things, SRP enhances the translocation competence of the preprotein by maintaining it in a more open or unfolded state. Since SRP is the only cytosolic factor required for translocation across mammalian microsomes, it is likely that it fulfills this function in addition to its well-established role in targeting proteins to the rough ER. These dual functions put SRP into the family of proteins described as “molecular chaperones” (Ellis, 1987), and it will be interesting to see which of its subunits is responsible for each activity. Although the yeast hsp70 heat shock proteins stimulate translocation in vitro (Chirico et al., 1988; Deshaies et al., 1988; Murakami et al., 1988) they require the presence of other cytosolic components to perform this function. Moreover, unlike SRP, neither the hsp70 proteins, SecB protein, nor trigger factor have yet been shown to interact with signal sequences or to be directly involved in the translocation process. A yeast equivalent of mammalian SRP has not yet been found. When these additional yeast factors are eventually isolated, the cloning of their genes will allow a more detailed analysis of their role in translocation in vivo .
IV. BINDING TO THE MEMBRANE AND TRANSLOCATION ARE SEPARABLE EVENTS Translocation has been shown to be a two-step process for both yeast (Sanz and Meyer, 1989) and Neurospora (Addison, 1988) microsomes, for bacterial membranes (Thom and Randall, 1988), and also for nuclear-encoded proteins crossing mitochondria1 membranes (Eilers and Schatz, 1988; Pfanner er al., 1988). Whether this extends to mammalian ER remains to be seen. The mammalian system, in particular, is difficult to elucidate because of the inability of microsomes to translocate most proteins posttranslationally. However, there is some evidence to suggest that it might also be a multistep process. An engineered protein with a C-terminal loop has been shown to bind to mammalian microsomes, and only to be translocated upon the reduction of the disulfide bonds maintaining the loop (Miiller and Zimmerman, 1988). In addition, a possible
3. PROTEIN TRANSLOCATION IN YEAST
23
SSR has been found in mammalian membranes (Wiedmann et al., 1987), but the protein and its function remain to be characterized. Similarly, a putative outer membrane receptor for transit peptides has been found in chloroplast membranes (Pain et al., 1988). Sanz and Meyer (1989) have used a further refinement of the posttranslational assay using purified precursors diluted out of 8 M urea to study translocation across yeast microsomal membranes. They found that this process could be divided into two sequential events in vitro,separable by the withholding of ATP, that were amenable to biochemical as well as genetic analysis. In this assay, the labeled precursor protein was diluted into buffer containing yeast microsomes in the absence of any cofactors. The membranes were pelleted by centrifugation through a sucrose cushion to remove any unbound precursor, and the pellet was resuspended and divided into two aliquots. From the first sample, the amount of specifically bound labeled precursor could be determined, and from the second, after addition of ATP and further incubation, the proportion of bound precursor that was translocated and processed to the mature form could be measured. A. Binding to the Membrane Prior to translocation, in the absence of ATP, both proOmpA and prepro-afactor bind with high affinity to specific receptors on the cytoplasmic face of the microsomal membranes. The binding is saturable, with a dissociation constant of 7.5 x 10- M, and can be competed for by other precursor proteins, but not by mature proteins, which suggests a possible involvement of signal sequences. It is strongly inhibited by predigestion of the membranes with papain, a process known to inhibit translocation. Moreover, proOmpA that had been incubated in buffer for 15 hr at O'C, and was consequently incompetent for translocation (Sanz and Meyer, 1988), was also unable to bind to the membranes. This indicates that conformations of the precursor is important in the binding step. Whether it is important for further steps in the translocation process remains to be determined. 8. Translocation Addition of ATP to the membrane aliquot that had bound precursor proteins allowed translocation and processing to the mature form. The translocation could be inhibited by pretreatment of the membranes with N-ethylmaleimide, urea, or papain. However, since only papain also inhibited binding of the precursor, translocation may be mediated by another protein or set of proteins distinct from that mediating binding. One of these must be an ATPase, since translocation requires the hydrolysis of ATP. Further anaIysis of these proteins, or other membrane components involved in
24
JOANNE CROWE AND DAVID 1. MEYER
translocation, is very difficult using conventional biochemical methods in vitro. Luminally disposed proteins, or those embedded in the lipid bilayer, would be difficult to modify chemically or enzymatically, and are probably inaccessible to antibodies. The study of yeast translocation mutants provides an alternate way to identify and characterize these components.
V.
ANALYSIS OF ptI7-A
TRANSLOCATION MUTANT
Translocation mutants in microbial systems can be selected by screening mutagenized cells for the incorrect localization of an artificial fusion protein (Beckwith and Ferro-Novick, 1986; Ito, 1986; Deshaies and Schekman, 1987). In wild-type yeast cells this protein is secreted, while in the mutant it accumulates in the cytoplasm. The temperature-sensitive protein translocation mutant ptll was isolated in this way by Toyn and co-workers (1988). They used a fusion of the prepro region of the MFcll gene to T R P l , a gene that encodes phosphoribosyl anthranilate (PRA) isomerase, a cytoplasmic enzyme essential for tryptophan biosynthesis. In a trpl strain this fusion protein is efficiently translocated out of the cytoplasm and growth is permitted only on tryptophan-supplemented media. A translocation mutant in this strain, however, will accumulate enough of the fusion protein in the cytosol to reverse the Trp- phenotype to Trp+ . The mutants can then be easily isolated by screening for growth in the absence of added tryptophan. The translocation phenotype of the ptll mutation, which has been localized to chromosome 15, has been examined by Toyn et al. (1988) both in vivo and in vitro.
A. In Viwo Analysis The mutant ptll exhibits a temperature-sensitive, pleiotropic accumulation of presecretory proteins in the cytoplasm. The behavior of three secretory proteins having different final destinations was examined in this mutant. These were prepro-a-factor, which is secreted into the medium, and the precursors of the vacuolar hydrolase carboxypeptidase Y (CPY) and a 33-kDa cell wall glycoprotein (Sanz et al., 1987). More than 95% of all three proteins remains unprocessed at the nonpermissive temperature of 37°C. Protease digestion after gentle lysis showed these unprocessed precursor forms to be accumulated in the cytoplasm. Pulse-chase experiments showed that the defect was at the level of entry into the ER, and that transport further along the secretory pathway was unaffected in this mutant. We have already referred to the ability of yeast to translocate full-length preproteins across microsomal membranes in vitro. It was unknown, however, if translocation could occur posttranslationally in vivo. The use of p t l l , which
3. PROTEIN TRANSLOCATION IN YEAST
25
effectively accumulates precursor proteins in the cytoplasm, provided a definitive way to answer this question. The mutant was pulse-labeled at the restrictive temperature of 37°C in order to establish a cytoplasmic accumulation of radiolabeled preproCPY. When the cells were shifted to the permissive temperature of 22"C, the labeled cytoplasmic form of CPY could be seen to chase into the glycosylated ER and Golgi forms. This indicates that in yeast, a protein at least as large as 50-60 kDa can be efficiently translocated posttranslationally across the ER in vivo.
6. In Who Analysis Studying a translocation mutant in a cell-free system allows the localization of the defect to either the membrane or the cytosolic compartment. Wild-type membranes, when combined with cytosol derived from either wild-type or mutant cells, translocate pure proOmpA when it is rapidly diluted from 8 M urea at both 22" and 37°C. When membranes from ptll are used, however, they show reduced translocation of proOmpA at 22"C, and are virtually translocation-incompetent when assayed at 37°C. This phenotype could be reversed simply by shifting the assay temperature back to 22"C, indicating that the defect is in the ptll membranes, and is both temperature-sensitive and reversible in vitro. When ptll membranes are analyzed using the two-step binding assay described previously, they were able both to bind and to translocate proOmpA at 22°C. However, while translocation was completely blocked when assayed at 37"C, binding of the preprotein to the mutant membranes was unaffected. Taken together these data indicate that the product of the PTLI gene is most likely a membrane component directly involved in translocation, but not preprotein binding. Isolation of the wild-type allele of this gene by complementation of the mutant phenotype, will permit the use of biochemical studies to characterize further the role played by its protein product, Ptllp, in the translocation process. Similar analyses of other mutants will hopefully lead us to an even deeper understanding of the whole translocation process. REFERENCES Addison, R. (1988). Translocation of a fragment of invertase across microsomal vesicles isolated from Neurospora crassa requires the hydrolysis of a nucleotide triphosphate. J . Biol. Chem. 263, 1 4281 - 14287. Beckwith, J., and Ferro-Novick, S . (1986). Genetic studies on protein export in bacteria. Cur. Top. Microbiol. Immunol. 125, 5-27. Blobel, G . , and Dobberstein, B. (1975). Transfer of proteins across membranes. 11. Reconstitution of functional rough microsomes from heterologous components. J . Cell Biol. 67, 852-862. Chirico, W. J., Waters, M. J., and Blobel, G. (1988). 70K heat shock related proteins stimulate protein translocation into microsomes. Nature (London) 332, 805-8 10. Collier, D. N., Bankaitis, V. A., Weiss, J-. B., and Bassford, P. J. (1988). The antifolding activity of SecB promotes the export of the E . coli maltose-binding protein. Cell 53, 273-283.
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Crooke, E., and Wickner, W. (1987). Trigger factor: A soluble protein that folds proOmpA into a membrane-assembly competent form. Proc. Natl. Acad. Sci. U.S.A. 84, 5216-5220. Crooke, E., Guthrie, B., Lecker, S., Lill, R., and Wickner, W. (1988a). ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle. Cell 54, 1003-1011. Crooke, E., Brundage, L., Rice, M., and Wickner, W. (1988b). ProOmpA spontaneously folds into a membrane assembly competent state which trigger factor stabilizes. EMBO J. 7, 1831-1835. Deshaies, R.J., and Schekman, R. W. (1987). A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum. J. Cell B i d . 105, 633-645. Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, E. A , , and Schekman, R. W. (1988). 70kD stress protein homologues facilitate translocation of secretory and mitochondria1 precursor polypeptides. Nature (London) 332, 800-805. Eilers, M., and Schatz, G. (1986). Binding of a specific ligand inhibits import of a purified precursor protein into the mitochondria. Nature (London) 322, 228-232. Eilers, M., and Schatz, G. (1988). Protein unfolding and the energetics of protein translocation across biological membranes. Cell 52, 481-483. Eilers, M., Hwang, S., and Schatz, G. (1988). Unfolding and refolding of a purified precursor protein during import into isolated mitochondria. EMBO J. 7, 1139- 1145. Ellis, J. (1987). Proteins as molecular chaperones. Nature (London) 328, 378-379. Evans, E. A,, Gilmore, R., and Blobel, G. (1986). Purification of microsomal signal peptidase as a complex. Proc. Natl. Acad. Sci. U.S.A. 83, 581-585. Gilmore. R., Walter, P., and Blobel, G . (1982). Protein translocation across the endoplasmic reticulum. I. Detection in the microsomal membrane of a receptor for the signal recognition particle. J. Cell Biol. 95, 463-469. Hansen, W., Garcia, P., and Walter, P. (1986). In vitro protein translocation across the endoplasmic reticulum: ATP-dependent post-translational translocation of the prepro-a-factor. Cell 45, 397406. Hortsch, M., Avossa, D., and Meyer, D. I. (1986). Characterisation of secretory protein translocation: Ribosome-membrane interaction in the endoplasmic reticulum. J. Cell Biol. 103, 241253. Ito, K. (1986). Genetic control of protein secretion and localisation. Adv. Biophys. 21, 267-280. Meyer, D. I. (1988). Preprotein conformation: The year’s major theme in translocation studies. Trends Biochem. Sci. 13, 471-474. Meyer, D. I., Krause, E., and Dobberstein, B. (1982). Secretory protein translocation across membranes-the role of the “docking protein.” Nature (London) 297, 647-650. Miiller, G . , and Zimmerman, R. (1988). Import of honeybee prepromelittin into the endoplasmic reticulum: Energy requirements for membrane insertion. EMBO J. 7, 639-648. Murakami, H., Pain, D., and Blobel, G. (1988). 70-kD heat shock related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria. J. Cell Biol. 107, 2051-2057. Pain, D., Kanwar, Y. S., and Blobel, G. (1988). Identification of a receptor for protein import into chloroplasts and its localization to envelope contact zones. Nuture (London) 331, 232-236. Park, S . , Liu, G . , Topping, T. B., Cover, W. H., and Randall, L. L. (1988). Modulation of the folding pathways of exported proteins by the leader sequence. Science 239, 1033-1035. Pelham, H. R. B. (1986). Speculations on the functions of the major heat shock and glucose-related proteins. Cell 46, 959-961. Pfanner, N., Hartl, F.-U., and Neupert, W. (1988). Import of proteins into mitochondria: A multistep process. Eur. J. Biochem. 175, 205-212. Randall, L. L., and Hardy, S. J. S. (1986). Correlation of competence for export with lack of tertiary
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structure of the mature species: A study in vivo of maltose-binding protein in E. coli. Cell 46, 921-928. Rothblatt, J. A., and 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. Rothblatt, J. A,, Webb, J. R., Ammerer, G., and Meyer, D. I. (1987). Secretion in yeast: Structural features influencing the post-translational translocation of prepro-a-factor in vifro. EMBO J. 6 , 3455-3464. Rothman, J. E., and Kornberg, R. D. (1986). An unfolding story of protein translocation. Nafure (London) 322, 209-210. Sanz, P., and Meyer, D. I. (1988). Signal recognition particle (SRP) stabilizes the translocationcompetent conformation of presecretory proteins. EMBO 1.7, 3553-3557. Sanz, P., and Meyer, D. I. (1989). Secretion in yeast: Preprotein binding to a membrane receptor and ATP-dependent translocation are sequential and separable events in vitro. J. Cell Biol. 108, 2101-2106. Sanz, P., Herrero, E., and Sentandreu, R. (1987). Secretory pattern of a major integral mannoprotein of the yeast cell wall. Biochim. Biophys. Actu 924, 93-103. Tajima, S., Lauffer, L., Rath, V. L., and Walter, P. (1986). The signal recognition particle receptor is a complex that contains two distinct polypeptide chains. J. Cell Biol. 103, 1167- 1178. Thorn, J. R., and Randall, L. L. (1988). Role of the leader peptide of maltose-binding protein in two steps of the export process. J. Bucteriol. 170, 5654-5661. Toyn, J., Hibbs, A. R., Sanz, P., Crowe, J., and Meyer, D. I. (1988). I n vivo and in vifro analysis of prll, a yeast rs mutant with a membrane-associated defect in protein translocation. EMBO J. 7, 4347-4353. Vestweber, D., and Schatz, D. (1988a). Mitochondria can import artificial precursor proteins containing a branched polypeptide chain or a carboxyl-terminal stilbene disulfonate. J. Cell Biol. 107, 2045-2049. Vestweber, D., and Schatz, G. (1988b). A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites. J. Cell Biol. 107, 2037-2043. Walter, P., and Blobel, G. (1981a). Translocation of proteins across the endoplasmic reticulum. 11. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in vitru-assembled polysomes synthesising secretory proteins. J. Cell Biol. 91, 55 1-556. Walter, P., and Blobel, G. (1981b). Translocation of proteins across the endoplasmic reticulum. 111. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J . Cell Biol. 91, 557-561. Walter, P., Ibrahimi, I., and Blobel, G. (1981). Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in virro assembled polysomes synthesizing secretory proteins. J . Cell Biol. 91, 545-550. Waters, M. G . , and Blobel, G. (1986). Secretory protein translocation in a yeast cell-free system can occur post-translationally and requires ATP hydrolysis. J . Cell Biol. 102, 1543-1550. Wiedmann, M., Kurzchalia, T. V.,Hartmann, E., and Rapoport, T. A. (1987). A signal sequence receptor in the endoplasmic reticulum membrane. Nature (London) 328, 830-833. Zimmermann, R., and Meyer, D. I. (1986). 1986: A year of new insights into protein translocation across membranes. Trends Btochem. Sci. 11, 512-515. Zimmermann, R., Sagstetter, M., Lewis, M. J., and Pelham, H. R. B. (1988). Seventy-kilodalton heat shock proteins and an additional component from reticulocyte lysate stimulate import of M13 procoat protein into microsomes. EMBO J . 7, 2875-2880.
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Part II
Structure of Proteins in the Membrane
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CURRENT TOPICS IN MEMBRANES AND TRANSFORT, VOLUME 36
Chapter 4
Architectural Editing: Regulating the Surface Expression of the Multicomponent T-cell Antigen Receptor RICHARD D . K U U S N E R , JENNIFER LIPPINCOTTSCHWARTZ, AND JUAN S . BONIFACINO Cell Biology and Metabolism Branch National Institute of Child Health and Human Developmen1 National Institutes of Health Bethesda, Maryland 20892
1. Introduction 11. Structure and Composition of the T-cell Antigen Receptor A. Clonotypic Subunits B. Nonpolymorphic Subunits 111. Subunit Interactions within the T-cell Receptor IV. Stoichiometry within the Complex V. Assembly of the T-cell Receptor Complex VI. Fate of Newly Synthesized T-cell Receptor Chains A. Getting to the Cell Surface B. Getting Newly Synthesized Chains Out of the Endoplasmic Reticulum VII. Summary References
1.
lNTRODUCTlON
As we learn more about the structure of membrane proteins, it is becoming clear that a growing number of proteins are complex assemblies of multiple chains. For many, these complexes are the result of the specific assembly of oligomers of identical subunits. These may be covalently linked, as in the case of the transferrin receptor (reviewed in Harford et al., 1990) or the insulin receptor (reviewed in Rosen, 1987) or, perhaps more commonly, represent tight non31
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RICHARD D. KLAUSNER ET AL.
covalent associations as in a number of viral membrane glycoproteins. These include vesicular stomatitis virus (VSV) G protein (Kreis and Lodish, 1986), the influenza hemagglutinin (Wilson et al., 1981), and the Rous sarcoma virus (RSV) envelope glycoprotein (Einfeld and Hunter, 1988). Other membrane complexes, such as the T-cell antigen receptor (Samelson et al., 1985), the IgE receptor (Metzger et al., 1986), and surface major histocompatibility complex (MHC) molecules (reviewed in Kaufman et al., 1984), are formed by the association of two or more different subunits. How often what is considered to be a monomeric chain in fact exists as an oligomer is not clear. Apparently, the more investigators look for oligomers, the more they find them. One impediment to the identification of the oligomeric structure of integral membrane proteins is the tendency of oligomers to dissociate upon detergent solubilization when they are not covalently linked to each other. This can be avoided with techniques that maintain, as near as possible, the native oligomeric state of integral membrane proteins including the use of relatively nondenaturing, nonionic detergents, and/or the addition of lipids during solubilization to optimize lipid/detergent ratios. Over the past several years, as our knowledge of the number of membrane proteins that are a part of multichain complexes increases, a variety of observations has led to the realization that there is an intimate relationship between the intracellular assembly of these complexes and the expression on the surface of the cell. The ability of the cell to prevent the expression on the surface of incompletely assembled complexes implies the existence of an important underlying mechanism that can (1) recognize and distinguish assembled from unassembled complexes and ( 2 ) differentiate the fate of fully assembled complexes from either partially assembled, incorrectly assembled, or unassembled membrane proteins. We will refer to this overall cellular process as “architectural editing.” We use this term because our current understanding is that it is something about the structure of the complexes (i.e., their architecture) that the cell recognizes. The editing refers to the fact that once the architecture is recognized as being either correct or incorrect, the cell then can determine the subsequent fate of the complex. We have been studying the T-cell antigen receptor as a model for a multisubunit membrane complex, the intracellular fate of which is determined by its oligomeric structure. We now recognize that this receptor is an extremely complex hetero-oligomer. At least six different gene products encode the receptor, which is made up of at least seven transmembrane proteins (Samelson et al., 1985). The structural complexity of this receptor has provided us with the opportunity to examine the role of architectural editing in determining cell surface expression of fully assembled receptor complexes. Many of the observations that have been made with the T-cell antigen receptor have found support and/or precedent in simpler oligomeric structures, such as viral glycoproteins. In addi-
4. REGULATING EXPRESSION OF THE T-CELL RECEPTOR
33
tion, and perhaps because of the complexity of this receptor, it has enabled us to recognize a wider repertoire of editing possibilities than has been previously recognized. Before discussing how architectural editing determines the expression of correctly assembled cell surface complexes, it is necessary to introduce the structural components that make up this receptor.
II. STRUCTURE AND COMPOSITION OF THE T-CELL ANTIGEN RECEPTOR
A. Clonotypic Subunits The function of the T-cell antigen receptor is to recognize antigen and, through that recognition, to initiate the biochemical events that lead to T-cell activation. The T-cell antigen receptor is one of two types of antigen receptors that allow the immune system to recognize a myriad of different proteins. The other type, present on B cells, is surface immunoglobulin (Ig). Because of the ready availability of Ig in the serum as a soluble protein, the determination of Ig structure was one of the great advances of immunology (reviewed in Davies and Metzger, 1983; Amzel and Poljak, 1979). All Ig molecules have a characteristic domain structure referred to as the immunoglobulin or Ig domain. This domain is formed by the folding together of two sets of @pleated sheets. Immunoglobulins can be divided into several different types of Ig domains. The amino-terminal domains are called the variable regions, and the more carboxy-terminal domains are constant regions. This is a manifestation of the genetically determined diversity of the amino acid composition of the different domains. It is the variability of the amino acid composition of the variable domains that gives the immune system its astoundingly broad repertoire for the recognition of a very large number of different antigens. Any clone of B cells expresses on its surface an Ig molecule with a carboxy-terminal extension that allows it to cross the membrane and thereby be anchored as an integral membrane protein. The Ig receptor expressed on any particular clone of B cells has a unique amino acid composition in its variable region that gives it its antigen specificity. The recognition element of the T-cell antigen receptor is composed of two Iglike chains that have been referred to as Ti chains (see reviews in Davis and Bjorkman, 1988; Wilson et al., 1988; Allison and Lanier, 1987). As with the Bcell receptor, the T-cell antigen receptor is different for every clone of T cells. It has been calculated that the genetic mechanisms that give rise to the extraordinary diversity of Ti allows the T-cell population to recognize up to 10l8 determinants. There are two types of pairs of Ti chains that can provide the recognition components of T-cell antigen receptors. The most abundant and well studied are the Ti-a and -p chains. Subsequent to the recognition and discovery of the Ti-a@
34
RICHARD D. KLAUSNER ET AL.
heterodimers, two other Ig-like chains, Ti-y and -6 were described (reviewed in Brenneq er al., 1988). It is most likely that Ti-yS serves the function of recognizing antigen but its role has not been definitively determined. For the remainder of this chapter, we will be referring to the predominant and better studied T-cell receptor that contains Ti-a and -p chains. The a and p chains each have core protein molecular weights of -30,000. Multiple N-linked glycosylations raise their M , values to between 40,000 and 55,000. As with Ig, they contain both amino-terminal variable and carboxy-terminal constant domains. The a and p chains are linked to each other by a single disulfide. Each chain has a hydrophobic domain near the carboxy terminus that serves as the transmembrane domain. What is unusual about the transmembrane domains of the a and p chains is that they contain one or two positively charged groups in the region predicted to cross the lipid bilayer. Each of the chains has short charged cytoplasmic tails composed of 5-10 amino acid residues.
B. Nonpolymorphic Subunits Studies in the early 1980s pointed to the possibility of the physical association
on the surface of the T cell of a group of membrane proteins referred to as the CD3 complex. The connection between CD3 and the T-cell receptor was first suggested by the observation that monoclonal antibodies against one of the chains of CD3 were capable of either stimulating or blocking T-cell function (van Wauwe et al., 1980; Reinherz et al., 1980, 1982). In the intervening years, it has become clear that CD3 is, in fact, a collection of subunits of the T-cell receptor. The evidence that has led to this recognition includes (1) the ability to coimmunoprecipitate Ti chains using anti-CD3 antibodies and the ability to coprecipitate CD3 chains using anti-Ti antibodies (Reinherz er al., 1983; Borst er al., 1983a,b; Samelson et al., 1985); (2) the ability completely and stoichiometrically to comodulate CD3 and Ti from the surface of T cells using cross-linked antibody against either of the components (Reinherz et al., 1982; Meuer et al., 1983); (3) the finding that in variant or mutant T cells, failure to synthesize Ti chains resulted in the complete abrogation of surface expression of CD3 (Weiss and Stobo, 1984; Schmitt-Verhulst et al., 1987; Chen et al., 1988); and (4) the ability chemically to cross-link Ti to CD3 on the surface of intact T cells (Allison and Lanier, 1985; Brenner er al., 1985). For historical reasons, CD3 was thought of as one complex of membrane proteins fundamentally distinct from the T-cell receptor. Unfortunately, this view persists in the literature. We now know, however, that the functional T-cell receptor is made up of the clonotypic chains referred to as Ti and nonpolymorphic chains that include three CD3 components. This does not simply represent the association of two distinct sets of surface molecules, but rather the assembly of these components into a single macromolecular receptor complex.
4. REGULATING EXPRESSION OF THE T-CELL RECEPTOR
35
The CD3 complex was initially described biochemically in human T cells (Borst et al., 1983a,b; Kanellopoulos et al., 1983). It consists of three chains: y, 6, and E. The gene for each of these chains has been cloned so each chain is structurally well defined (van den Elsen et al., 1984; Krissansen et al., 1986; Gold et al., 1986). y and 6 are glycoproteins with core M, values of approximately 17,000-18,OOO. Glycosylation results in M, for 6 of -20,000 and for y of 26,OOO-28,000. The chain is a nonglycosylated 20-kDa protein. Each of these chains possesses relatively long intracellular and extracellular domains, the latter of which bear homology to members of the Ig gene superfamily. In addition, the three chains have significant sequence homology to each other. That they are structurally related is reflected in their genomic organization. In both human and mouse, the genes encoding these three chains are clustered within 30-300 kb of each other (Tunnacliffe et al., 1987; Clevers et al., 1988). The y and 6 genes are within 2 kb of each other and may, in fact, share transcriptional control elements (Georgopoulos et al., 1988). The murine CD3 chains are highly homologous to their human counterparts (van den Elsen et al., 1985; Krissansen et al., 1987; Gold et al., 1987; Haser et al., 1987). One distinction is that whereas the human y is more highly glycosylated than human 6, the opposite holds in murine T cells. In the latter, the M, of mature and completely processed 6 is 25,000-26,000, while the y chain has M,of approximately 20,000-21 ,OOO. In addition to the CD3 chains, there are two other nonpolymorphic chains that are part of the T-cell receptor complex. The 5 chain is a 16-kDa nonglycosylated transmembrane protein originally described in murine T cells (Samelson et al., 1985). Its structure is quite distinct from the other constant chains of the receptor (CD3). Whereas the CD3 chains contain large extracellular, Ig-like domains, 5 contains approximately eight amino acids for an extracellular domain (Weissman et al., 1988a). All of the nonpolymorphic chains are transmembrane proteins with single membrane-spanning regions (van den Elsen et al., 1984; Gold et al., 1986; Krissansen et al., 1986; Weissman el al., 1988a). Interestingly, all of them contain single negative-charge groups in their transmembrane regions. The 5 chain exists as a disulfide-linked homodimer in the vast majority of T-cell receptors. 6 bears no sequence or structural homology to the CD3 chains and, in fact, the gene is found on chromosome 1 in both mouse and human and is thus unlinked to the CD3 cluster (Weissman et al., 1988b; Baniyash et al., 1989). For these reasons, we do not consider the 5 chain part of the CD3 complex. We have subsequently described yet another chain of the T-cell receptor complex, which we have termed q (Baniyash et al., 1988; Orloff et al., 1989). AS of the time of this writing, the gene encoding has not been identified and thus we know significantly less about its structure than any of the other chains. The q chain is a 22-kDa protein that, like 5, is basic. Biochemical characterization has revealed that q is nonglycosylated and we have detected no posttranslational modifications of it (Orloff et al., 1989). It is immunologically related to 5 in that
36
RICHARD D. KLAUSNER ET AL.
certain antipeptide antibodies raised against peptides contained within 5 will directly recognize q. However, other anti-t; antibodies do not recognize q. In addition, amino acid composition studies have demonstrated that q and 5, despite their being immunologically related, are distinct proteins (Orloff et al., 1989). A peculiar characteristic of q is the fact that it exists as part of the T-cell receptor in a covalent linkage to a 5 monomer (Baniyash et al., 1988). Thus, even on cloned T cells, there seem to be two populations of receptors. A minority (-20% on many T cells) contain the (q heterodimer while the majority contain only the 55 homodimer. We do not know yet whether these two dimers are mutually exclusive or whether a single receptor complex can have both O. 1%) of ionic detergents such as deoxycholate or sodium dodecyl sulfate (SDS). If, however, immune precipitates are washed with progressively higher concentrations of denaturing ionic detergents, one observes a hierarchy of subunit dissociation. Using anti+ antibodies, for example, as one raises the concentration of ionic detergent, one sees the simultaneous loss of a,p, and 5 with the continued coprecipitation of the full CD3 complex. With increasing concentrations, one tends to lose 6 followed by y and finally the target antigen, in this case, the E chain (R. D. Klausner et al., unpublished observations). Using a particular anti-5 polyclonal antiserum, we have found that the complex can be dissociated such that the CD3 triplet is no longer coprecipitated but the Ti a p heterodimer is (Bonifacino et al., 1988a). This suggests that arc{ can exist as a partially stable complex, independent of CD3.
TABLE I PARTIAL T-CELLRECEPTOR COMPLEXES A. Murine T Cells
Complex Cell line
Characteristics
apy6e0.5 pm) and the saltatory (oscillatory) nature of the organelle movements, both of which might be explained by the effect of the dense cytoskeleton in the cell. The in vitro assays for organelle motility thus appear to provide a valid means for studying the molecular mechanism of organelle motility and perhaps its regulation.
B. Microtubule Differentiation and Cell Polarity In locomoting cells, establishment of cell polarity can be correlated with the posttranslational modification of microtubules (Gundersen and Bulinski, 1988) and in many cases the movement of the microtubule-organizing center relative to the nucleus (Singer and Kupfer, 1986). This might induce asymmetry within the cell by way of specific transport of certain components to one portion of the cell. Perhaps modification of microtubules and their associated proteins may affect the transport of organelles to different sites in the cell. The role that microtubule
120 Organelles
MICHAEL P. SHEETZ ET AL. Centrosome (MTOC)
Soluble Factors
Cytosol Fraction or Purified Motor Protein
FIG. 2. In vifro organelle motility assays are performed by mixing the potassium iodide-washed organelles with purified microtubules (polymerized from centrosomes for directionality measurements) and an appropriate motor fraction (see Schroer et al., 1989, for details). The components are mixed on a glass coverslip, and microtubules and organelles are visualized by video-enhanced light microscopy. The number of individual organelle movements per unit time per video field are counted as the measure of the amount of motility (video field area and the concentration of microtubules are kept constant).
structure plays in the transport of organelles is unknown at present; however, our understanding of actin-based motility systems provides a basis for speculation about certain factors that might influence motility on microtubules.
C. Paradigms for Control of Motility from Actin-Based Systems In our studies of actin-based motility in vifro we found that motility could be affected by the binding of two kinds of actin-binding proteins. Certain proteins were found to coat actin filaments and prevent the movement of myosin-coated beads on the same filaments. These included the troponin-tropomyosin complex (Vale et al., 1985b) and N-ethylmaleimide (NEM)-inactivated myosin subfragment I (Meusen and Cande, 1979). A second class of proteins induced the formation of an ATP-insensitive linkage between the myosin-containing object and the filament and blocked movement. For example, beads coated with a mixture of myosin and brush border spectrin moved slowly or not at all (M.P.
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Sheetz and M. Mooseker, unpublished observations). Similarly, the presence of inactive myosin heads (i.e., by NEM) could slow movement (Jones and Sheetz, 1986). We have not examined the effects of analogous proteins on microtubulebased organelle movement.
111. INTERACTION OF MOTORS WITH ORGANELLES TO PRODUCE MOTILITY The in virro reconstitution of organelle movements on purified microtubules has led to a better understanding of the motile complex formed by an organelle and a motor. The simplistic notion that a motor enzyme binds directly to a membrane receptor and by itself causes movement appears to be incorrect, since we have found it impossible to produce organelle motility with purified motors alone (Schroer et al., 1988, 1989). Purified motors will stimulate the movement of impure organelles (Vale et al., 1985b); however, washing organelles with 0.6 M potassium iodide removes all latent organelle motor activity, and no motility is seen in the presence or absence of highly purified motors. Two similar models have been developed from our studies of kinesin- and dynein-dependent organelle motility. The specific inactivation of cytoplasmic dynein (a minus-enddirected motor) has been shown to inhibit organelle movement toward the minus ends of microtubules in a variety of in virro systems including fibroblast extracts (Schroer et al., 1989), squid axons (Schnapp and Reese, 1989), and fish chromatophores (Haimo et al., 1989). In the fibroblast studies the fraction of inactivated dynein closely correlated with the degree of inhibition of organelle motility. We do not know whether or not a single dynein molecule (a multimeric complex of heavy and light chains) on an organelle surface is sufficient for movement. However, we observed that the velocity of organelle movement in the presence of mixtures of active and inactivated dynein is considerably slower than normal, suggesting that multiple dynein molecules may contribute to movement. The movement of the organelles could not be restored using purified dynein alone but required additional soluble component(s) of cytoplasm. The “accessory factors” copurified with the microtubule motors in a microtubule affinity purification step, and further purification is currently under way. To study the role of kinesin (a plus-end-directed motor) in organelle motility, we have used an antikinesin antibody affinity column to subtract kinesin form a supernatant that supported organelle movement (Schroer et al., 1988). After kinesin was removed, organelle movement was inhibited by 70% and motility could not be restored by the readdition of kinesin. The degree of inhibition was greater than expected (less than 50% of the organelle movements driven by the starting supematant were toward the plus ends of microtubules), suggesting that dynein-dependent organelle motility was also inhibited. Organelle motor activity
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could be recovered from the antibody affinity column by elution with 0.5 M potassium chloride and 10 mM ATP. Under these conditions only a small fraction of the kinesin and cytoplasmic dynein in the preparation were eluted, but both anterograde and retrograde organelle movements were seen. Our results suggested that additional soluble proteins were required for kinesin-dependent organelle motility and that these proteins might bind to kinesin and, therefore, bind to the antikinesin column (see Fig. 3). In addition, the results suggest that the accessory proteins required for cytoplasmic dynein motility also bind to kinesin and that the amount of the accessory factors and not the amount of the motors may be limiting in producing translocating organelles. ORGANELLE MOTOR COMPONENTS Kinesin [Antemgrade Motor ? I SALT-WASHED ORGANELLES
(A)
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FIG.3 . Summary diagram of the current in vitro motility experiments showing that the organelle translocation complex contains not only a motor protein but also at least one accessory component. In contrast, the latex beads will move with only the motors bound to their surface.
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A. Model of the Organelle Translocation Complex The motility of organelles is unlike the motility of latex spheres because highly purified motors are sufficient to drive the movement of inert particles but not organelles. Kinesin will bind to membranes without causing motility (Schroer and Sheetz, 1989), suggesting that the accessory factors play an important role with the motors in producing the motile organelle translocation complex. Although little is known about the interactions of motors and the accessory factors, we can certainly speculate about the molecular nature of the organelle translocation complex and how it might be regulated. We suggest the following testable model for organelle motility. The basic elements of this model are the two microtubule motors (kinesin and cytoplasmic dynein), the accessory protein(s), and the organelle membrane-binding sites (motor receptors). In this model the membrane-binding site determines the directionality of organelle transport, the accessory proteins regulate the activity of the motor proteins in the binding site, and the motors themselves provide the force for movement when assembled into an active complex. An additional component that may be present under certain circumstances is a stabilizing linkage, which would serve to attach the organelle to the cytoskeleton in order to maintain its position (synapsin possibly plays this role for synaptic vesicles: Benfenati et al., 1989). There are many possible sites for regulation and, therefore, it is best to discuss this model in terms of specific types of organelle movements.
6. Endoplasmic Reticulum Extension The extension of the ER within cultured cells to form a polygonal array appears to involve microtubules. In early electron micrographs of cultured cells there were reports of parallel alignment of the ER with microtubules (F'ranke, 1971). Terasaki et al., (1986) later observed a close correspondencebetween the paths of ER strands and microtubules in the periphery of cells. Using an in vitro system with membranes isolated from chick embryo fibroblasts, it has been possible to generate an extensive reticular array on a microtubule bed using a soluble fibroblast extract containing microtubule motors (Fig. 4). This membrane network can be stained with markers for the ER. The formation of tubular membrane strands is a general property of the focal application of force to a membrane (Hochmuth et al., 1982; Waugh, 1982). As might be expected for a general physical property of membranes, similar networks could be formed by fluid shear (Vale and Hotani, 1988). Alternatively, an active role might be played by actin-based motility (Kachar and Reese, 1988) and by kinesin-driven movements of microtubules to which membranes were statically bound (Vale and Hotani, 1988). Observations of ER movements in live cells have revealed that ER formation in vivo (Lee and Chen, 1988) is remarkably similar to the process
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FIG. 4. Video micrograph of an ER network formed in vitro from a postnuclear supernatant fraction of CEF cells (Dabora and Sheetz, 1988b). Note that the microtubules are lower in contrast than the ER strands and that fingers of ER are seen moving out along the microtubules.
we have reconstituted in vitro (Dabora and Sheetz, 1988b). It would appear that the construction of the ER in vivo requires the movement of ER membrane strands along microtubules as opposed to the microtubule gliding (Lee et al., 1989). According to our current model, the movement of the ER to the cell periphery involves the kinesin-dependent movement of ER tubules on microtubules (see Fig. 4). This is the most obvious part of the process because those strands can be readily visualized using the fluorescent dye, DiOC6(3). It is possible that cytoplasmic dynein bound at other sites on the ER might serve to bring tubules toward the centrosome and the Golgi apparatus (see Fig. 5). After the network is extended, stabilizing proteins might serve to link the ER to the cytoskeleton until other signals are given for redistribution. Indeed, Lee and Chen (1988) found that only 10%of the cells in an unsynchronized culture contain actively spreading ER. Because microtubules are highly dynamic structures in interphase cells, it is likely that the ER would form linkages to more stable elements of the cytoskeleton such as the intermediate filaments. Another important aspect of the ER is the modulation of ER assembly during
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1 Extension
FIG. 5 . Diagram depicting the two major steps in the process of ER extension in vivo. They are depicted here in sequence, but it is expected that there will be considerable overlap of fusion and extension.
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the cell cycle. The transition into mitosis involves the complete randomization of the intracellular membranes into a dispersed population. We might expect that any stabilizing linkages between membranes and cytoskeleton would also be disrupted. There are many candidate proteins (synapsins, microtubule-associated proteins or MAP) that could serve as stable linkages between organelles and the cytoskeleton, and the assembly and disassembly of the nuclear envelope provides a model of how stable attachments would organize the membranes. During mitosis the ER, Golgi, and nuclear membranes vesiculate and are randomly spread between the two daughter cells. For an interphase ER to form, the vesicles must fuse and then be extended on the cellular microtubules. The motility of both plus-end and minus-end-directed organelles appears to be inhibited during mitosis and reactivated in interphase (Tooze and Burke, 1987; Matteoni and Kreis, 1987). It is possible that organelle motility is modulated by regulating the activity of the accessory components required for transport. This is consistent with our finding (see Section 11) that organelle movement in vitro is limited not by the concentration of motors but by the amount of accessory factors (Schroer et al., 1988). According to this model, the organelles contain signals for either plus-end or minus-end-directed movement and the motors are present in excess in cytoplasm, but motile activity is regulated by the amount of the accessory components. An alternative possibility is that the membrane receptors themselves are modified to control motor binding and subsequent motility. The development of quantitative in vitro assays of organelle motility has opened the way to understanding the molecular components needed to create a motile organelle-motor complex. Our understanding of these in vivo processes involving organelle motility, such as ER extension, provide the phenomenological basis to propose testable models that can guide experiments to understand how the processes actually do occur. Further, the purification of additional molecular components involved in organelle motility will make it possible to probe their effects on in vitro motility and to approach gene deletion or other gene modification experiments to understand how these components interact in the in vivo process of motility. ACKNOWLEDGMENTS This work was supported by grants from NIH, the Muscular Dystrophy Association, and the Juvenile Diabetes Foundation. REFERENCES Allen, R. D., Weiss, D . G., Hayden, J. H., Brown, D. T.,Fujiwake, H., and Simpson, M. (1985). Gliding movement of a bidirectional transport along single native microtubules from squid axoplasm: Evidence for an active role of microtubules in axonal transport. J . Cell B i d . 100, 1736-1752. Benfenati, F., Bahler, M . , Jahn, R . , and Greengard, P. (1989). Interactions of synapsin I with small
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synaptic vesicles: Distinct sites in synapsin I bind to vesicle phospholipids and vesicle proteins. J. Cell Biol. 108, 1863-1872. Dabora, S. L., and Sheetz, M. P. (1988a). The microtubule-dependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts. Cell 54, 27-35. Dabora, S. L., and Sheetz, M. P. (1988b). Cultured cell extracts support organelle movement on microtubules in vitro. Cell Motil. Cytoskeleton 10, 482-495. Franke, W. W. (1971). Cytoplasmic microtubules linked to endoplasmic reticulum with crossbridges. Exp. Cell Res. 66, 486-489. Gundersen, G. G., and Bulinski, J. C. (1988). Selective stabilization of microtubules oriented toward the direction of cell migration. Proc. Nutl. Acad. Sci. U.S.A. 85, 5946-5950. Haimo, L. T., Thaler, C. D., and knton, R. D. (1989). Mechanism of bidirectional organelle movements in melanophores. Cell Motil. Cytoskeleton 11, 197. Hochmuth, R. M., Wiles, H. C., Evans, E. A., and McCown, J. T. (1982). Extensional flow of erythrocyte membrane from cell body to elastic tether: 11. Biophys. J. 39, 83-89. Jones, R., and Sheetz, M. P. (1986). Inhibition of motility by inactivated myosin heads. In “The Cytoskeleton” (T. W. Clarkson, P. R. Sager, and T. L. M. Syversen, eds.), pp. 213-220. Plenum, New York. Kachar, B., and Reese, T. S. (1988). The mechanism of cytoplasmic streaming in Characean algal cells: Sliding of endoplasmic reticulum along actin filaments. J. Cell Biol. 106, 1545-1552. Lee, C., and Chen, L. B. (1988). Behavior of endoplasmic reticulum in living cells. Cell 54,36-42. Lee, C., krguson, M., and Bo Chen, L. (1989). Construction of the endoplasmic reticulum. J. Cell Biol. 109, 2045-2055. Lye, R. J., Porter, M. E., Scholey, J. M., and McIntosh, J. R. (1987). Identification of a microtubule-based cytoplasmic motor in the nematode C . elegans. Cell 51, 309-318. Matteoni, R., and Kreis, T. J. (1987). Translocation and clustering of endosomes and lysosomes depends on microtubules. J . Cell B i d . 105, 1253-1266. Meusen, R. L., and Cande, W. Z. (1979). N-Ethylmaleimide-modifiedheavy meromyosin. A probe for actomyosin interactions. J. Cell Biol. 82, 57-65. Paschal, B. M., and Vallee, R. B. (1987). Retrograde transport by the microtubule-associated protein MAP 1C. Nature (London) 330, 181-183. Schnapp, B. J., and Reese, T. S. (1989). Dynein is the motor for retrograde axonal transport of organelles. Proc. Nutl. Acad. Sci. U.S.A. 86, 1548-1552. Schnapp, B. J., Vale, R. D., Sheetz, M. P., and Reese, T. S. (1985). Single microtubules from squid axoplasm support bidirectional movement of organelles. Cell 40,455-462. Schroer, T. A., and Sheetz, M. P. (1989). The role of kinesin and kinesin-associated proteins in organelle transport. In “Cell Movement” (J. R. McIntosh and F. D. Warner, eds.), Vol. 2, pp. 295-306. Alan R. Liss, New York. Schroer, T. A., Schnapp, B. J., Reese, T. S., and Sheetz, M. P. (1988). The role of kinesin and other soluble factors in organelle movement along microtubules. J. Cell Biol. 107, 1785-1792. Schroer, T. A , , Steuer, E. R., Sheetz, M. P. (1989). Cytoplasmic dynein is a minus end-directed motor for membranous organelles. Cell 56, 937-946. Singer, S . J., and Kupfer, A. (1986). Directed migration of eukaryotic cells. Annu. Rev. Cell Biol. 2 , 337-365. Terasaki, M., Chen, L. B . , and Fujiwara, K. (1986). Microtubules and the endoplasmic reticulum are highly interdependent structures. J . Cell B i d . 103, 1557- 1568. Tooze, J., and Burke, B. (1987). Accumulation of adrenocorticotropin secretory granules in the midbody of telophase AtT20 cells: Evidence that secretory granules move anterogradely along microtubules. J . Cell Biol. 104, 1047-1057. Vale, R. D. (1987). Intracellular transport using microtubule-based motors. Annu. Rev. Cell Biol. 3, 347-378.
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Vale, R. D., and Hotani, H. (1988). hnnation of membrane networks in vitro by kinesin-driven microtubule movement. J . Cell Biol. 107, 2233-2230. Vale, R. D., Reese, T. S., and Sheetz, M. P. (1985a). Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 41, 34-4 1. Vale, R. D., Szent-Gyorgyi, A., and Sheetz, M. P. (1985b). Troponin-tropornyosin complex confers C a + + control to myosin bead movement in vitro. Biophys. J . 45, 145a. Waugh, R. E. (1982). Surface viscosity measurements from large bilayer vesicle tether formation: 11. Biophys. J. 38, 29-37.
Part IV
Signaling and Communication
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 36
Chapter I I G Protein-Coupled Receptors: Structure and Function of Signal-Transducing Proteins ERIC M . PARKER AND ELLIOTT M . ROSS Department of Pharmacology University of Texas Southwestern Medical Center Dallas, Texas 75235
I. 11. 111. IV. V. VI .
Introduction Mechanism of G-Protein Activation by Agonist-Liganded Receptors General Structure of G Protein-Coupled Receptors Structure of the Ligand-Binding Domain Structure of the G Protein-Binding Domain Receptor-Mimetic Peptides as Models for the G Protein-Binding Domain References
1.
INTRODUCTION
Every cell must interpret and respond to a wide variety of extracellular signals such as hormones, neurotransmitters, odors, and light. This task is often accomplished by three-component signal transduction systems based on GTP-binding regulatory proteins, or G proteins (see Gilman, 1987; Stryer and Bourne, 1986; Ross, 1989, for reviews). When a receptor’s extracellular binding site is occupied by a hormone, the receptor facilitates the exchange of bound GDP for GTP by a G protein on the inner face of the plasma membrane. The GTP-liganded G protein is thus activated such that it can regulate the activity of an effector protein that generates an intracellular chemical or electrical signal. Numerous receptors act on 10 G proteins to regulate perhaps a dozen effector proteins, including adenylyl cyclase, cyclic GMP phosphodiesterase, phospholipases A, and C, and ion channels. G proteins are heterotrimers of a distinct (Y subunit (39-52 ma),one of two p subunits (35 and 36 m a ) , and a y subunit (8-10 kDa) (See Gilman, 1987, for
-
131 Copynght 0 1990 by Academic R e s s . Inc A11 rights of reproduction in any form reserved.
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review). The 01 subunit is unique to each G protein and is responsible for guanine nucleotide binding and hydrolysis. Several 01 subunits have been identified both biochemically and by molecular cloning, and all have been found to have highly homologous primary structures (Lochrie and Simon, 1988). Each G protein is defined by its unique a subunit andlor by the effector protein that it regulates. Thus, G, stimulates adenylyl cyclase and Gi was identified as mediating the inhibition of the enzyme. At least three closely related Gi forms have now been identified (Jones and Reed, 1987); G i l , Gi2, and G,, share several functions in addition to inhibition of adenylyl cyclase (e.g., regulation of phospholipases and ion channels). Two distinct but highly homologous P subunits (Fong et al., 1988; Gao et al., 1987) and at least three different y subunits have been identified (Sternweis and Robishaw, 1984; Hildebrandt et al., 1985; Hurley et al., 1984). The Py subunits modulate the binding of nucleotides to a subunits, and may also exert independent regulatory functions. It has not been possible to separate the P and y subunits under nondenaturing conditions, and their individual activities are thus unknown. With the exception of the retinal G protein transducin, there is no indication that a particular Py complex associates specifically with a particular 01 subunit; each a subunit copurifies with a heterogeneous mixture of fir cornplexes. GTP-activated 01 subunits regulate effector proteins independently of the Py subunits, at least in most cases. Cell surface receptors that utilize G proteins represent a large and diverse group of proteins. They include many neurotransmitter receptors (e.g., adrenergic, muscarinic cholinergic, serotonergic, and peptidergic receptors), receptors for pituitary protein hormones and eicosanoids, the rhodopsins, and pheromone receptors in yeast and slime molds. All of these receptors are integral membrane glycoproteins that display significant structural and functional homology. The application of molecular cloning techniques to the study of G proteincoupled receptors has led to the elucidation of the primary structure of several of these receptors. This article will review current knowledge of the structure of G proteincoupled receptors and the structural features that are responsible for the various functions ascribed to these receptors. Particular emphasis will be given to the (3adrenergic receptor because it, along with rhodopsin, is the most extensively studied of the G protein-coupled receptors.
II. MECHANISM OF G-PROTEIN ACTIVATION BY AGONIST-LIGANDED RECEPTORS G proteins are activated when they bind GTP and deactivated when the bound GTP is hydrolyzed to GDP. The mechanism of activation and how activation is
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mediated by cell surface receptors have been studied in detail using intact membranes, purified G proteins, and purified G proteins and receptors that have been co-reconstituted into unilamellar phospholipid vesicles (Asano el al., 1984; Asano and Ross, 1984; Brandt and Ross, 1986). The regulatory interactions of receptors, G-protein subunits, and their many ligands are extremely convoluted, but a general pattern of reactions has emerged from these studies (Fig. 1). G proteins exist predominantly in an inactive complex with a single molecule of tightly bound GDP. The dissociation of GDP from the G protein is slow and limits the rate of GTP binding and consequent activation. Agonist-liganded receptor decreases the affinity of the G protein for guanine nucleotitrles, essentially converting the nucleotide-binding site from a “closed” state to an “open” state that freely exchanges nucleotide. Because the GTP/GDP ratio in the cell is large, the net result of agonist binding is an increase in activated, G’I’P-boundG protein. The activated G protein regulates its effector protein until the bound GTP is hydrolyzed to GDP by an intrinsic GTPase activity, the rate of which is slow (k,,, = 4 min- I ) and has not been shown to be regulated. Because the rate of receptor-catalyzed guanine nucleotide exchange exceeds the rate of GTP hydrolysis, a receptor can maintain several G proteins in an active form. A single P-adrenergic receptor can activate >30 molecules of G, (Brandt and Ross, 1986), and a single rhodopsin molecule can activate 1000 molecules of transducin (Liebman et al., 1987). The catalytic ability of receptors to regulate multiple G proteins yields considerable amplification of hormonal signals. It also explains, in part, the pharmacologically defined phenomenon of spare receptors, wherein only a fraction of the total receptor population need bind agonist in order to activate an effector maximally.
FIG. 1. Regulatory GTPase cycle. The activated species is shown as G*-GTP. Hydrolysis of bound GTP (reaction 2) can occur in the presence or absence of effector, E. The receptor-hormone complex (R.H) catalyzes both release of GDP and binding of GTP, increasing both the steady-state GTPase rate and the fraction of G protein in the activated form.
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Ill. GENERAL STRUCTURE OF G PROTEIN-COUPLED RECEPTORS Over the past 3 years, the primary sequences of 20 different G protein-coupled receptors have been determined (see Fig. 2). All are homologous to the visual opsins, which also signal via G proteins and are among the best studied integral membrane proteins. The most striking aspect of the primary structure of the G protein-coupled receptors is the presence of seven stretches of hydrophobic amino acids. In the case of rhodopsins, biophysical, proteolytic, immunocytochemical, and chemical modification experiments have shown that the seven hydrophobic regions are largely helical and span the lipid bilayer (see Findlay and Pappin, 1986, for review). It is presumed that the seven hydrophobic stretches in the other receptors also represent transmembrane a-helices, although direct evidence is lacking. By further analogy to bacteriorhodopsin, the structure of which is known at low resolution from electron-microscopic studies (Henderson and Unwin, 1975), these helices are presumed to form a bundle around the activating ligand. Such a structure is consistent with spectroscopic data on visual rhodopsin (Findlay and Pappin, 1986). Given the analogy with the rhodopsins, the predicted topology of G proteincoupled receptors is illustrated in Fig. 3 using the avian P-adrenergic receptor. The amino termini of the receptors typically have one or more consensus sites for N-linked glycosylation and, hence, are presumed to be oriented extracellularly. This orientation dictates the disposition of the putative connecting loops and the carboxy terminus relative to the membrane. As shown in Fig. 2, homology among the G protein-coupled receptors is concentrated in the putative membrane-spanning helices. Homology is minimal in the amino- and carboxy-terminal domains, in the largest intracellular loop, and in the extracellular connecting loops. Limited deletions or amino acid substitutions in the amino terminus and extracellular connecting loops of the hamster p,-adrenergic receptor do not affect its function and, hence, these regions may only be required for folding and processing of the protein (Dixon et a l . , 1987). The first two cytoplasmic loops are fairly short and generally display some homology among the various receptors, but the third cytoplasmic loop is typically longer and is quite divergent. Its length is also quite variable; it is -150 amino acids longer in the muscarinic cholinergic receptor than in the substance K receptor or rhodopsin. Variability in this cytoplasmic loop may indicate that it confers specificity to the interaction of the receptors with G proteins or other cytoplasmic structural or regulatory molecules. The carboxy terminus is also quite divergent among receptors and is of variable length, being relatively long in the p- and a,-adrenergic receptors and much shorter in most of the others. It may also be involved in a receptor-specific function. In some cases, most notably the P-adrenergic receptors and rhodopsin, the carboxy terminus is rich in serine and threonine residues. In the case of rhodop-
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FIG. 2. Alignment of the amino acid (aa) sequences of several representative G protein-coupled receptors. Nonhomologous regions at the amino and carboxy termini and in the loop between spans V and VI are not shown, although the sizes of the omissions are indicated. The seven putative membrane-spanning domains are overlined and numbered. Dashes indicate gaps placed in the sequence to optimize the alignment. The sequences were taken from the following references: turkey p-adrenergic (quasi+,) receptor (aBET) from Yarden et al., (1986); human PI-adrenergic receptor (BET1) from Frielle er al., (1987); hamster pz-adrenergic receptor (BET2) from Dixon ef al. (1986); hamster a,-adrenergic receptor (ALFI) from Cotecchia et al. (1988); human a,-adrenergic receptor (ALF2) from Kobilka er al. (1987b); human 5HT-la receptor (HTla) from Kobilka et al. (1987a); rat 5HT-lc receptor (HTlc) from Julius er al. (1988); porcine muscannic cholinergic receptor (MAc2) from Peralta er al. (1987); porcine muscarinic cholinergic receptor (MAc1) from Kubo er af. (1986); bovine substance K receptor (SUBK) from Masu et al. (1987); human rhodopsin (RHOD) from Nathans and Hogness (1984).
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EXTRACELLULAR SPACE
FIG. 3. Proposed topography of the avian erythrocyte f3-adrenergic receptor. Putative membranespanning regions are boxed and were assigned on the basis of hydropathy analysis and by analogy to rhodopsin. Those regions that can be proteolytically removed with no loss of function (Rubenstein et al.. 1987) are shown in dashed boxes. Positively charged residues are enclosed in squares and negatively charged residues are enclosed in circles. The single consensus site for N-linked glycosylation in the N-terminal region is shown. The arrangement of putative cytoplasmic and extracellular domains is arbitrary and of no significance.
sin, several of these serine and threonine residues are phosphorylated by a retinaspecific rhodopsin kinase upon bleaching. This light-induced phosphorylation is thought to be involved in light adaptation (Liebman et al., 1987). By analogy, phosphorylation of these residues by a receptor kinase may also be involved in desensitization of other G protein-coupled receptor systems (Sibley et al., 1988). Their removal by in vitro mutagenesis alters desensitization of the receptor (Bouvier et al., 1988). Regions rich in hydroxyl groups are also found in the third cytoplasmic loop of several receptors and may serve similar functions.
IV. STRUCTURE OF THE LIGAND-BINDING DOMAIN The strong homology among the G protein-coupled receptors in the putative membrane-spanning domains suggests that their hydrophobic core of these proteins carries out the same function in every case. One likely function for this region is the binding of ligands specific for the various receptors. In the case of
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rhodopsin, retinal is linked to a lysine residue located in the seventh membrane span and extends into a hydrophobic pocket formed by the remaining membranespanning helices (Findlay and Pappin, 1986). Interestingly, the biogenic amine and muscarinic cholinergic ligands of G protein-coupled receptors are chemically similar to the retinal-lysine conjugate in that they consist of a hydrophobic moiety linked to a cationic side chain. Hence, it is possible that these ligands are also bound within a hydrophobic pocket formed by the membrane-spanning domains. The hypothesis that the ligand-binding domain of G protein-coupled receptors lies in the hydrophobic core of the protein is supported by several lines of evidence. Site-directed mutagenesis studies by Dixon et al. (1987) have shown that large sections of the amino terminus, carboxy terminus, and the intracellular and extracellular hydrophilic loops of the hamster P,-adrenergic receptor are not required for ligand binding. Similarly, Rubenstein et al. (1987) found that proteolytic removal of most of the amino- and carboxy-terminal domains and a large portion of the third cytoplasmic loop left ligand binding intact. The two peptides obtained in a limited digest, which remained noncovalently associated in detergent solution, represented only the hydrophobic core and associated short loops. Wong et al. (1988) used two (3-adrenergic photoaffinity labels, [1251]iodocyanopindolol-diazirineand [ 1251]iodoazidobenzylpindolol, to study the ligandbinding site of the turkey erythrocyte P-adrenergic receptor. Both of the tryptic peptides described by Rubenstein et al. (1987) incorporated label upon photolysis in roughly equal amounts. Similar results have been obtained by Dohlman et al. (1987b, 1988, and personal communication). Further proteolysis followed by peptide sequencing localized one site to Trp330,which is located in the middle of the seventh membrane span. This is very close to the corresponding residue in rhodopsin to which retinal is attached ( L Y S * ~ The ~ ) . labeled site in the larger fragment has not been determined as precisely, but it is localized somewhere between the carboxy-terminal ends of spans I1 and V. The observation that label is incorporated into two regions that are widely separated in the primary sequence suggests that the P-adrenergic receptor folds such that span VII is closely apposed to a region in spans 11-V. A similar orientation of the membranespanning a helices has been proposed for rhodopsin by aligning the rhodopsin structure to the low-resolution crystal structure of bacteriorhodopsin (Findlay and Pappin, 1986). This is consistent with the idea that the membrane-spanning domains fold to form a hydrophobic pocket that comprises the ligand-binding domain. Furthermore, these results suggest that all G protein-coupled receptors are folded in a similar fashion and probably bind ligand, undergo ligand-induced conformational changes, and activate G proteins in a similar, if not identical, manner. By examining the ligand-binding specificity of a series of au,-adrenergicreceptor-P,-adrenergic receptor chimeras, Kobilka ef al., (1988) concluded that the
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seventh membrane-spanning domain was a major determinant of the ligandbinding characteristics of a particular receptor. Relative a-versus P-adrenergic specificity seemed to reflect the relative amount of a-or P-adrenergic receptor sequence in the chimera, but sequence from span VII seemed to have a somewhat larger effect than did sequence from the other spans. Regardless, the observation that these chimeras can bind adrenergic ligands and activate G proteins reinforces the notion that all G protein-coupled receptors have a common structural arrangement and that agonist binding is coupled to G-protein activation by a common mechanism. If ligands bind in a hydrophobic pocket formed by the membrane-spanning domains, the cationic side chain of these ligands must be buried in this hydrophobic environment. This could be made energetically palatable by pairing the cationic side chain with acidic amino acid side chains in the membrane-spanning domains of the receptor. There are several aspartate residues in the second and third membrane-spanning domains that are conserved with only rare exception in all the G protein-coupled receptors sequenced to date (see Fig. 1). These aspartate residues in the P,-adrenergic receptor have been replaced by asparagine by side-directed mutagenesis (Strader et al., 1987b; Fraser et al., 1988). Of particular interest is the observation that replacement of Asp1I3 eliminates the ability of the receptor to bind iodocyanopindolol with high affinity and, based on measurements of adenylate cyclase activity, also decreases the affinity of the receptor for agonists by several orders of magnitude. Hence, this residue is a good candidate for the counterion that participates in an electrostatic interaction with the cationic amine moiety that exists in most ligands of G protein-coupled receptors.
V.
STRUCTURE OF THE G PROTEIN-BINDING DOMAIN
Despite the high degree of homology among their a subunits, each G protein must display selectivity for the receptor that regulates it. Similarly, the homologous G protein-coupled receptors presumably undergo a similar conversion to an activating conformation in response to the binding of agonist. Based on these considerations, it is likely that the structural components of receptors and G proteins that determine their interaction are generally similar, but yet sufficiently (and subtly) different to allow the required selectivity. Because G proteins behave as peripheral membrane proteins, it is reasonable to assume that one or more of the cytoplasmic domains of the receptors are responsible for regulating G proteins. Several recent experiments have yielded evidence supporting this assumption. Strader et al. (1987b) deleted several segments of the third putative cytoplasmic loop of the hamster P,-adrenergic receptor and found that deletion of residues immediately adjacent to the membrane abolished the ability of the receptor to activate adenylate cyclase. Deletion of
11.
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other regions of this loop, either by site-directed mutagenesis or by proteolysis (Rubenstein et al., 1987), did not alter the ability of the receptor to activate adenylate cyclase. The importance of the third cytoplasmic loop is also highlighted by the data of Kubo er al. (1988). These investigators constructed several chimeric Ml-M2 muscarinic cholinergic receptors and showed that MI sequence in the large cytoplasmic loop is sufficient to determine characteristic M1 function when these receptors are expressed in frog oocytes. No data on M2 responses were shown for any of the chimeras. In similar studies by Kobilka et al. (1988), a chimeric a,-P,-adrenergic receptor having p-receptor sequence only in the fifth and sixth transmembrane domains and in the third cytoplasmic loop was able to activate adenylate cyclase in response to a a,-adrenergic agonists. The G,-coupled a2functions were likewise not assayed. Hence, it appears that regions of the third cytoplasmic loop that are proximal to the membrane play a major role in the selectivity of receptors among G proteins. Mutations in the carboxy-terminal cytoplasmic domain of the human P,-adrenergic receptor immediately adjacent to the membrane also hinder the ability of the receptor to activate G, (O’Dowd er al., 1988). As is the case with the third cytoplasmic loop, deletions of the carboxy-terminal domain distal to the membrane have no effect on the ability of the receptor to activate G, (Strader et al., 1987b; Rubenstein et al., 1987). The possible roles of the first and second cytoplasmic loops in coupling to G proteins is still unclear, but it is certainly conceivable that all four putative cytoplasmic domains act in concert to form the G protein-binding domain. As discussed earlier, it is likely that the structural determinants of selectivity are subtle and not absolute. Indeed, data from our laboratory suggest that the ability of receptors to select among G proteins is not absolute. The P-adrenergic receptor acts predominantly via G, in most cells, and the receptor regulates G, with high efficiency when both purified proteins are reconstituted into phospholipid vesicles. In addition, however, when reconstituted in phospholipid vesicles, the avian P-adrenergic receptor can also activate three pertussis toxinsensitive forms of Gi, albeit with less efficiency (Asano et al., 1984; Rubenstein and Ross, unpublished data; Abramson and Molinoff, 1987; Abramson et al., 1988). Some selectivity among the Gi forms was noted, and there was no evidence for the receptors’ regulation of Go or transducin. Abramson et al. (1988) have also detected regulation of the P-adrenergic receptor by Gi in membranes of the cyc- mutant of S49 lymphoma cells. A G,-receptor complex was detected chromatographically, and Gi regulated the receptor’s affinity for agonists. Muscarinic receptors can couple to Gi, Go, and at least one other uncharacterized G protein that regulates phospholipase C and is insensitive to pertussis toxin (Florio and Sternweis, 1985; Masters er al., 1984; Peralta et al., 1988). Cerione et al. (1985) have demonstrated that reconstituted rhodopsin can stimulate not only its physiological target, transducin, but also Gi. In the converse experiment, a,-
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adrenergic receptors, which normally couple to G,, could also activate transducin (Cerione et af.,1985). Careful analysis of the efficiency with which natural and mutant receptors activate various G proteins will probably be required to sort out the mechanisms of selectivity. Our laboratory is one of several that are constructing chimeric receptors that will test the importance of specific structural determinants for the regulation of G proteins and their selectivity. These studies should also clarify the extent to which divergence of a receptor’s signal to different G protein-mediated pathways is a common regulatory motif in animal cells.
VI.
RECEPTOR-MIMETIC PEPTIDES AS MODELS FOR THE G PROTEIN-BINDING DOMAIN
Higashijima et al. (1988) speculated that a group of peptide toxins known as mastoparans may serve as structural models for the G protein-regulatory domain of G protein-coupled receptors. Mastoparans are amphipathic, cationic, tetradecapeptides found in wasp venoms. These authors showed that mastoparans activate G proteins in a manner that resembles agonist-liganded receptors in several characteristic ways. Mastoparans increase the rate of GDP release and GTP binding by several G proteins, particularly Gi and Go. Activation of Go or Gi by mastoparans is completely blocked by pertussis toxin-catalyzed ADP ribosylation, an effect often diagnostic of receptor interaction with a pertussis toxinsensitive G protein. Although mastoparans can regulate nucleotide exchange in free a subunits, the ability of mastoparan to activate G proteins is greatest when the a p y trimer is reconstituted into phospholipid vesicles, a phenomenon that is also seen with receptors. Finally, mastoparans, like receptors, activate G proteins at micromolar concentrations of Mg2 . Thus, it seems possible that the mechanism by which mastoparans activate G proteins may provide clues to the mechanisms by which receptors activate G proteins. Mastoparans display no sequence similarity with the G protein-coupled receptors but can be hypothesized to have conformational similarity. Nuclear magnetic resonance analysis has shown that membrane-bound mastoparans are &-helices that are arranged such that the four positive charges present in the molecule are directed away from the membrane (Higashijima et al., 1983). Interestingly, all of the putative cytoplasmic loops of the G protein-coupled receptors are rich in basic amino acid residues (see Figs. 1 and 2). It is therefore possible that the G protein-binding face of receptors consists of some ordered array of positively charged amino acid residues that are either exposed or redistributed upon agonist binding. However, many integral membrane proteins have an asymmetric distribution of basic amino acids, with these residues being preferred on the cytoplasmic domains of these proteins (von Heijne and Gavel, 1988). It has been postulated that this asymmetric distribution of basic amino acids determines the +
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proper topography of the protein during insertion into the membrane. Further experimentation will be necessary to test the hypothesis that a specific structural arrangement of basic amino acid residues on the cytoplasmic face of G proteincoupled receptors and in mastoparans are critical for activation of G proteins. Synthetic mastoparans are currently being designed that should shed some light on the primary and secondary structural features of these molecules that are required for G-protein activation and that determine selectivity among G proteins. The ability of synthetic mastoparans and chimeric receptors to activate particular G proteins should give us a better understanding of the structure of the G protein-binding face of receptors. ACKNOWLEDGMENTS Studies from the authors’ laboratory have been supported by NIH grant GM30355 and postdoctoral fellowship GMI 1943 (E. M. P.) and by R. A. Welch Foundation grant 1-982. REFERENCES Abramson, S. N., and Molinoff, P. B. (1987). Interactions of P-adrenergic receptors with a membrane protein other than the stimulatory guanine-nucleotide binding protein. Biochem. Pharmacol. 36, 2263-2269. Abramson, S. N., Shorr, R. G. L., and Molinoff, P. B. (1988). Interactions of P-adrenergic receptors with a membrane protein other than the stimulatory guanine nucleotide-binding protein. Biochem. Pharmacol. 36, 2263-2269. Asano, T., and Ross, E. M. (1984). Catecholamine-stimulated guanosine 5‘-@(3-thiotriphosphate) binding to the stimulatory GTP-binding protein of adenylate cyclase. Biochemistry 23, 54675471. Asano, T., Pederson, S. E., Scott, C. W., and Ross, E. M. (1984). Reconstitution of catecholaminestimulated binding of guanosine 5’-0-(3-thiotriphospate)binding to the stimulatory GTP-binding protein of adenylate cyclase. Biochemistry 23, 5460-5467. Bouvier, M . , Hausdorff, W. P., DeBlasi, A., O’Dowd, B. F., Kobilka, B. K . , Caron, M. G., and Lefkowitz, R. J. (I 988). Removal of phosphorylation sites from the Pz-adrenergic receptor delays onset of agonist-promoted desensitization. Nature (London) 333, 370-373. Brandt, D. R., and Ross, E. M. (1986). Catecholamine-stimulated GTPase cycle. Multiple sites of regulation of P-adrenergic receptor and Mg2+ studied in reconstituted receptor-(;, vesicles. J . Biot. Chem. 261, 1656-1664. Cerione, R. A . , Staniszewski, C., Benovic, J. L., Lefkowitz, R. J . , Caron, M. C . , Gierschik, P., Somers, R., Speigel, A. M . , Codina, J . , and Birnbaumer, L. (1985). Specificity of the functional interactions of the P-adrenergic receptor with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles. J . Biol. Chem. 260, 1493- 1500. Cotecchia, S., Schwinn, D. A., Randall, R. A , , Lefkowtiz, R. J., Caron, M. G., and Kobilka, B. K. (1988). Molecular cloning and expression of the cDNA for the hamster a,-adrenergic receptor. Proc. Natl. Acad. Sci. U.S.A. 85, 7159-7163. Dixon, R. A. F., Kobilka, B. K., Strader, D. J., Benovic, J. L., Dohlman, H. K., Frielle, T., Bolanowski, M. A., Bennett, C . D., Rands, E., Diehl, R. E., Mumford, R. A , , Slater, E. E., Sigal, 1. S . , Caron, M. G . , Lefkowitz, R. J., and Strader, C. D. (1986). Cloning of the gene and cDNA for mammilian P-adrenergic receptor and homology with rhodopsin. Nature ( L o d o n ) 321, 75-79. Dixon, R. A. F., Sigal, I . , Candelore, M. R., Register, R. B., Scattergood, W., Rands, E., and
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Strader, C. D. (1987). Structural features required for ligand binding to the P-adrenergic receptor. EMEO J. 6, 3269-3275. Dohlman, H. G., Bouvier, M., Benovic, J. L., Caron, M. G., and Lefkowitz, R. J. (1987). The multiple membrane spanning topography of the P2-adrenergic receptor. Localization of the sites of binding, glycosylation, and regulatory phosporylation by limited proteolysis. J. Biol. Chem. 262, 14282- 14288. Dohlman, H. G., Caron, M. G., Strader, C. D., Amlaiky, N., and Lefkowitz, R. J. (1988). Identification and sequence of a binding site peptide of the P2-adrenergic receptor. Biochemistry 27, 1813-1817. Findlay, J. B. C., and Pappin, D. J. C. (1986). The opsin family of proteins. Biochern. J . 238, 625642. Florio, V. A., and Sternweis, P. C. (1985). Reconstitution of resolved muscarinic cholinergic receptors with purified GTP-binding proteins. J. Eiol. Chern. 260, 3477-3483. Fong, H. W. K., Amatruda, T. T., Birren, B. W., and Simon, M. L. (1987). Distinct forms of the p subunit of GTP-binding regulatory proteins identified by molecular cloning. Proc. h'atl. Acud. Sci. U.S.A. 84, 3792-3796. Fong, H. W. K., Yoshimoto, K. K., Eversole-Cire, P. E., and Simon, M. L. (1988). Identification of a GTP-binding protein o subunit that lacks an apparent ADP-ribosylation site for pertussis toxin. Proc. Nutl. Acad. Sci. U.S.A. 85, 3066-3070. Fraser, C. M., Chung, F., Wang, C., and Venter, J. C. (1988). Site-directed mutagenesis of human padrenergic receptors: Substitution of aspartic acid-130 by asparagine produced a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase. Proc. Nuti. Acad. Sci. U.S.A. 85, 5478-5482. Frielle, T., Collins, S., Daniel, K. W., Caron, M. G., Lefkowitz, R. J., and Kobilka, B. K. (1987). Cloning of the cDNA far the human P1-adrenergic receptor. Proc. Nutl. Acud. Sci. U.S.A. 84, 7920-7924. Gao, B., Gilman, A. G., and Robishaw, J. D. (1987). A second form of the p subunit of signaltransducing G proteins. Proc, Nutl. Acud. Sci. U.S.A. 84, 6122-6125. Gilman, A. G. (1987). G proteins: Transducers of receptor-generated signals. Annu. Rev. Biochem. 56, 615-649. Henderson, R., and Unwin, P. N. (1975). Three-dimensional model of purple membrane obtained by electron microscopy. Nature (London) 257, 28-32. Higashijima, T., Wakamatsu, K., Takemitsu, M., Fujino, M., Nakajima, T., and Miyazawa, T. (1983). Cornformational change of mastoparan from wasp venom on binding with phospholipid membrane. FEBS Lett. 152, 227-230. Higashijima, T., Uzu, S., Nakajima, T., and Ross, E. M. (1988). Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins). J. Biol. Chem. 263, 6491-6494. Hildebrandt, J. D., Codina, J., Rosenthal, W., Birnbaumer, L., Neer, E. J., Yamazaki, A., and Bitensky, M. (1985). Characterization by two-dimensional peptide mapping of the y subunits of N, and Ni, the regulatory proteins of adenylyl cyclase, and of transducin, the guanine nucleotide-binding protein of rod outer segments of the eye. J. Eiol. Chem. 260, 14867-14872. Hurley, J. B., Fong, H. K. W., Teplow, D. B., Dreyer, W. J., and Simon, M. I. (1984). Isolation and characterization of a cDNA clone for the y subunit of bovine retinal transducin. Proc. Nutl. Acad. Sci. U.S.A. 81, 6948-6952. Jones, D. T., and Reed, R. R. (1987). Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J. Eiol. Chem. 262, 14241- 14249. Julius, D., MacDermott, A. B., Axel, R., and Jessel, T. M. (1988). Molecular characterization of a functional cDNA encoding the serotonin l c receptor. Science 241, 558-564. Kobilka, B. K., Frielle, T., Collins, S., Yang-Feng, T., Kobilka, T. S., Francke, U., Lefkowitz, R.
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J., and Caron, M. G. (1987a). An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins. Nature (London) 329, 75-79. Kobilka, B. K., Matsui, H., Kobilka, T. S., Yang-Feng, T. L., Francke, U., Caron, M. G., Lefkowitz, R. J., and Regan, J. W. (1987b). Cloning, sequencing, and expression of the gene coding for the human platelet a*-adrenergic receptor. Science 238, 650-656. Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M. G., and Lefkowitz, R. J. (1 988). Chimeric a2-, P2-adrenergic receptors: Delineation of domains involved in effector coupling and ligand binding specificity. Science 240, 1310- 1316. Kubo, T., Fukuda, K., Mikami, A,, Maeda, A,, Takahashi, H., Mishina, M., Haga, T., Haga, K., Ichiyama, I . , Kangawa, K., Kojima, M., Matsuo, M., Hirose, T., and Numa, S. (1986). Cloning, sequencing, and expression of a complementary DNA encoding the muscarinic acetylcholine receptor. Nurure (London) 323, 41 1-416. Kubo, T., Bujo, H., Akiba, I., Nakai, J., Mishina, M.,and Numa, S. (1988). Location of a region of the muscarinic acetylcholine receptor involved in selective effector coupling. FEBS Lett. 241, 119- 125. Liebman, P. A., Parker, K. R., and Dratz, E. A. (1987). The molecular mechanism of visual excitation and its relation to the structure and composition of the rod outer segment. Annu. Rev. Physiol. 49, 765-791. Lochrie, M. A,, and Simon, M. L. (1988). G protein multiplicity in eukaryotic signal transduction systems. Biochemistry 27, 4958-4965. Masters, S. B., Harden, T. K., and Brown, J. H. (1984). Relationship between phosphoinositide and calcium response to muscarinic agonists in astrocytoma cells. Mol. Phurmucol. 26, 149-155. Masu, Y., Nakayama, K., Tamaki, H., Harada, T., Kuno, M., and Nakanishi, S. (1987). cDNA cloning of bovine substance K receptor through oocyte expression system. Nature (London) 329, 836-838. Nathans, J., and Hogness, D. S. (1984). Isolation and nucleotide sequence of the gene encoding human rhodopsin. Proc. Nutl. Acud. Sci. U.S.A. 81, 4851-4855. O’Dowd, B. F., Hnatowich, M., Regan, J. W., Leader, W. M., Caron, M. G., and Lefkowtiz, R. J. (1988). Site-directed mutagenesis of the cytoplasmic domains of the human P2-adrenergic receptor. Localization of regions involved in G protein receptor coupling. J . Biol. Chem. 263, 15985- 15992. Peralta, E. G., Winslow, J. W., Peterson, G. L., Smith, D. H., Ashkenazi, A,, Ramachandran, J., Schimerlik, M. I., and Capon, D. J. (1987). Primary structure and biochemical properties of an M2 muscarinic receptor. Science 236, 600-605. Peralta, E. G., Ashkenazi, A,, Winslow, J. W., Ramachandran, J., and Capon, D. J. (1988). Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes. Nature (London). 334., 434-437. Ross, E. M. (1989). Signal sorting and amplification through G protein-coupled receptors. Neuron 3, 141-152. Rubenstein, R. C., Wong, S. K.-F., and Ross, E. M. (1987). The hydrophobic tryptic core of the padrenergic receptor retains G, regulatory activity in response to agonists and thiols. J. Biol. Chem. 262, 16655-16662. Sibley, D. R., Benovic, J. L., Caron, M. G., and Lefkowitz, R. J. (1988). Phosphorylation of cell surface receptors: A mechanism for regulating signal transduction pathways. Endocr. Rev. 9, 38-56. Sternweis, P. C . , and Robishaw, J. D. (1984). Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J. Biol. Chem 259, 13806- 13813. Strader, C.D., Sigal, I. S., Register, R. B., Candelore, M. R., Rands, E., and Dixon, R. A. F. (1987a). Identification of residues required for ligand binding to the P-adrenergic receptor. Proc. Nutl. Acud. Sci. U.S.A. 84, 4384-4388.
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Strader, C. D., Dixon, R. A. F., Cheung, A. H., Candelore, M. R., Blake, A . D., and Sigal, I. S . (1987b). Mutations that uncouple the P-adrenergic receptor from G , and increase agonist affinity. J. Biol. Chem. 262, 16439-16443. Stryer, L., and Bourne, H. (1986). G. Proteins: A family of signal transducers. Annu. Rev. Cell B i d . 2, 391-419. von Heijne, G., and Gavel, Y. (1988). Topogenic signals in integral membrane proteins. Eur. J. Biochem. 174, 671-678. Wong, S. K.-F., Slaughter, C., Ruoho, A,, and Ross, E. M. (1988). The catecholamine binding site of the P-adrenergic receptor is formed by juxtaposed membrane-spanning domains. J. B i d . Chem. 263, 7925-7928. Yarden, Y., Rodriguez, H., Wong, S. K.-F., Brandt, D. R., May, D. C., Burnier, J., Harkins, R. N., Chen, E. Y., Ramachandran, J., Ullrich, A,, and Ross, E. M. (1986). The avian P-adrenergic receptor: Primary structure and membrane topology. Proc. Nutl. Acad. Sci. U.S.A. 83, 67956799.
CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 36
Chapter 12 Mechano-Sensitive Ion Channels in Microbes and the Early Evolutionary Origin of Solvent Sensing CHING KUNG, YOSHIRO SAIMI, AND BORIS MARTINAC Laboratory of Molecular Biology and Department of Genetics University of Wisconsin-Madison Madison, Wisconsin 53706
I. Introduction 11. A Stretch-Activated Ion Channel of Escherichiu coli 111. A Stretch-Activated Ion Channel in Yeast IV. Touch Receptors and Channels of Paramecium V. Mechano-Sensitive Channels and the Concept of Solvent Senses References
1.
INTRODUCTION
A room without a door is but a tomb. One could therefore argue on first principles that some kind of portals should emerge with, or soon after, the primordial cell membranes. Speculations aside, we have now shown that protozoans, yeast, and even bacteria all have one class of such portals, the ion channels. It therefore appears that all cellular forms of life have ion channels, and we are forced to conclude that they must have emerged and evolved very early. Ion channels are gated pores. A certain stimulus can increase the probability of a given channel being open. Such a stimulus (gating principle) can be an external ligand (e.g., acetylcholine for the nicotinic acetylcholine receptor or channel); an internal second messenger (e.g., Ca2+ for Ca2+-gated K + channel, cGMP for the cGMP-gated channel in the rod outer segment); or cross-membrane voltage 145 Copynghl Q 1990 by Acadernlc Press. Inc All nghts of reproduction in any form reserved
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(e.g., the voltage-gated Na+ channel and delayed rectifier K + channel of nerves). These classes of channels have been extensively studied and reviewed (Hille, 1984). Other ion channels have subsequently been found to be gated by GTP-binding proteins (Yatani et a l . , 1988) or by arachidonic acid (Kim and Clapham, 1989; Ordway et al., 1989). Last, but not least, is a class of channels that are gated by mechanical forces in the membrane. The activities of the last type of channels, those gated by mechanical forces, have been studied in the hair cells of the inner ear (Howard et al., 1988) and in ciliated protozoans (Machemer and Deitmer, 1985). The activities of individual stretch-activated channels were first demonstrated by Guharay and Sachs (1985) in chick skeletal muscle with a patch clamp. Since then, stretch-activated channels have been found in neurons, endothelial cells, blood cells, eggs, cultured plant cells, and guard cells, through patch-clamp examinations (see Sachs, 1988, for a review). Surprisingly, we encountered stretch-activated ion channels in both the fission yeast Saccharomyces cerevisiae and the bacterium Escherichia coli in patchclamp survey of their membranes. These findings led us to speculate that they might represent ancient devices for the detection of water concentrations through osmotic pressure. Reviews of microbial channels can be found in Saimi et al., (1988a,b) and Martinac et al. (1988).
II. A STRETCH-ACTIVATED ION CHANNEL OF ESCHERlCHlA COLl A typical bacillus, being < 1 pm in diameter, is too small for electrophysiology, even with a patch-clamp electrode. However, there are ways to generate giant cells or giant spheroplasts, some 5-10 pm in diameter, sufficient in size for patch-clamp experiments. Escherichia coli cells can be grown into filaments 50150 pm long by culturing with cephalexin, which prevents septation. Lysozyme, together with EDTA, can then be applied to nick the peptidoglycan wall, thereby converting the filaments into giant spheroplasts (Ruthe and Adler, 1985). The activities of ion channels that we found in these spheroplasts are not artifacts of cephalexin or EDTA-lysozyme, since four other methods gave similar results. Some of these methods exclude the use of one or both of these agents (Buechner et a l . , 1990). There is a stretch-activated channel on the surface of E. coli (Fig. 1). Suction equivalent to a few centimeters of mercury applied on a membrane patch in an on-cell mode recording opens these channels. The applied forces are physiological. The pressure of 5 crn Hg column is equivalent to the pressure generated by