MEMBRANE PROTEIN TRANSPORT A Multi-Volume Treatise Volume 2 • 1995
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MEMBRANE PROTEIN TRANSPORT A Multi-Volume Treatise Volume 2 • 1995
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MEMBRANE PROTEIN TRANSPORT A Multi-Volume Treatise Editor:
STEPHEN S. ROTHMAN University of California San Francisco, California
VOLUME 2 • 1995
JAI PRESS INC. Greenwicli, Connecticut
London, England
Copyright © 7 995 JAI PRESS INC. 55 Old Post Road, No. 2 Greenwich, Connecticut 06836 JAI PRESS LTD. The Courtyard 28 High Street Hampton Hill, Middlesex TW12 IPD England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher. ISBN: U55938-983-4 Manufactured in the United States of America
CONTENTS LIST OF CONTRIBUTORS THE ROLE OF MOLECULAR CHAPERONES IN TRANSPORT OF PROTEINS ACROSS MEMBRANES Elizabeth A. Craig, B. Diane Gambill, Wolfgang Voos, and Nikolaus Pfanner THE NUCLEAR PORE COMPLEX IN YEAST Paola Grandi and Eduard C. Hurt ATP BINDING CASSETTE TRANSPORTERS IN YEAST: FROM MATING TO MULTIDRUG RESISTANCE Ralf Egner, Yannick Make, Rudy Pandjaitan, Veronika Huter, Andrea Lamprecht, and Karl Kuchler
vii
1
29
57
THE APICAL SORTING OF GLYCOSYLPHOSPHATIDYLINOSITOL-LINKED PROTEINS Michael P. Lisanti, ZhaoLan Tang, Philipp E. Scherer, and Massimo Sargiacomo CAVEOLAE: PORTALS FOR TRANSMEMBRANE SIGNALING AND CELLULAR TRANSPORT Michael P. Lisanti, ZhaoLan Tang, and Massimo Sargiacomo NUCLEAR TRANSPORT OF URACIL-RICH SMALL NUCLEAR RIBONUCLEOPROTEIN PARTICLES Elisa Izaurralde, lain W. Mattaj, and David S. Goldfarb
97
111
123
vi
CONTENTS
PHOSPHORYLATION-MEDIATED REGULATION OF SIGNAL-DEPENDENT NUCLEAR PROTEIN TRANSPORT: THE "CcN MOTIF" David A. Jans
161
MEMBRANE PROTEIN TOPOGENESIS IN ESCHERICHIA COLI Gunnar von Heijne
201
MODEL FOR INTEGRATING P-TYPE ATPases INTO ENDOPLASMIC RETICULUM Randolph Addison andjialing Lin
215
NUCLEAR TRANSPORT AS A FUNCTION OF CELLULAR ACTIVITY Carl M. Feldherr and Debra Akin
237
INDEX
261
LIST OF CONTRIBUTORS Randolph Addison
Department of Biochemistry Health Science Center University of Tennessee Memphis, Tennessee
Debra Akin
Department of Anatomy and Cell Biology College of Medicine University of Florida Gainsville, Florida
Elizabeth A. Craig
Department of Biomolecular Chemistry University of Wisconsin Madison, Wisconsin
Carl M. Feldherr
Department of Anatomy and Cell Biology College of Medicine University of Florida Gainsville, Florida
B. Diane Cambill
Department of Biomolecular Chemistry University of Wisconsin Madison, Wisconsin
David S. Goldfarb
Department of Biology University of Rochester Rochester, New York
Paola Grandi
European Molecular Biology Laboratory Heidelberg, Germany
RalfEgner
Department of Molecular Genetics University and Biocenter of Vienna Vienna, Austria
VII
VIM
LIST OF CONTRIBUTORS
Eduard C. Hurt
European Molecular Biology Laboratory Heidelberg, Germany
Veronika Huter
Department of Molecular Genetics LJniversity and Biocenter of Vienna Vienna, Austria
Elisa Izaurralde
European Molecular Biology Laboratory Heidelberg, Germany
David A. Jans
Division for Biochemistry and Molecular Biology John Curtin School of Medical Research Australian National University Canberra, Australia
Karl Kuchler
Department of Molecular Genetics University and Biocenter of Vienna Vienna, Austria
Andrea Lamprecht
Department of Molecular Genetics University and Biocenter of Vienna Vienna, Austria
Jianling Lin
Department of Biochemistry Health Science Center University of Tennessee Memphis, Tennessee
Michael P. Lisanti
The Whitehead Institute for Biomedical Research Cambridge, Massachusetts
Yannick Mah6
Department of Molecular Genetics University and Biocenter of Vienna Vienna, Austria
lain W. Mattaj
European Molecular Biology Laboratory Heidelberg, Germany
Rudy Pandjaitan
Department of Molecular Genetics University and Biocenter of Vienna Vienna, Austria
List of Contributors
IX
Nikolaus Pfanner
Biochemisches Institut Universitat Freiburg Freiburg, Germany
Massimo Sargiacomo
Department of Hematology and Oncology Instituto Superiore di Sanita Rome, Italy
Philipp E. Scherer
The Whitehead Institute for Biomedical Research Cambridge, Massachusetts
ZhaoLan Tang
The Whithehead Institute for Biomedical Research Cambridge, Massachusetts
Gunnar von Heijne
Department of Biochemistry Arrhenius Laboratory Stockholm University Stockholm, Sweden
Wolfgang Voos
Biochemisches Institut Universitat Freiburg Freiburg, Germany
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THE ROLE OF MOLECULAR CHAPERONES IN TRANSPORT OF PROTEINS ACROSS MEMBRANES
Elizabeth A. Craig, B. Diane Gambill, Wolfgang Voos, and Nikolaus Pfanner
I. Introduction 2 A. hsp70 Structure and Biochemical Properties 2 B. ThchsplOs of Saccharomyces cerevisiae 3 C. The Basics of Import into Mitochondria 4 II. A Role for CytosolichspTO in Protein Translocation into Organelles . . 4 III. Role ofMitochondrialhspTO in Protein Translocation 5 A. In vivo Analysis of a Mitochondrial hsp70 Mutant 5 B. Mitochondrial hsp70 Is Required for Translocation into the Matrix 7 C. Degree of Requirement of Ssc 1 p Activity in Transport Is Correlated with the Conformation of the Precursor 14 D. Model of hsp70 Action in Translocation Across Mitochondrial Membranes 18 Membrane Protein Transport Volume 2, pages 1-28 Copyright © 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-983-4
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E. A. CRAIG, B. D. GAMBILL, W. VOOS, and N. PFANNER
E. Requirements for Mitochondrial hsp70 in the Translocation of Proteins into the Matrix Compared to the Intermembrane Space 20 F. Components Other Than Mitochondrial hsp70 Required for Protein Movement across the Outer and Inner Membranes . . 22 IV. Role of ATP in Protein Translocation 23 V. Role of hsp70 of the Endoplasmic Reticulum in Protein Translocation 24 VI. Summary 25 References 25 I.
INTRODUCTION
A. hsp70 Structure and Biochemical Properties The process of transporting proteins from their site of synthesis in the cytosol into organelles such as the mitochondrion or endoplasmic reticulum is facilitated by a group of proteins known as the molecular chaperones. Among the best characterized molecular chaperones are the hsp70 proteins. The hsp70s are highly conserved polypeptides found in all species, with identity ranging from 50% between procaryotic and eucaryotic examples to as high as 99% between members of a HSP70 gene family in yeast (Boorstein et al, 1994). The hspTOs possess two biochemical activities: a weak ATPase activity and peptide binding activity (reviewed in Gething and Sambrook, 1992). The ATPase activity can be stimulated severalfold by binding the hsp70 to peptides or polypeptides. Biochemical and structural studies of mammalian cytosolic hsp70 have shown that an N-terminal 44-kDa proteolytic fragment has ATPase activity that cannot be stimulated by peptide binding. The structure of the N-terminal proteolytic fragment of mammalian HSP70 has been solved by X-ray crystallography (Flaherty et al., 1990). The roughly U-shaped structure consists of two lobes separated by a deep cleft in which ATP binds. The most highly conserved amino acids of the amino-terminal domain are found lining this cleft. The peptide binding activity resides in the carboxyl-terminal portion of the protein. It should be emphasized that there is no "consensus" binding site of hsp70s, inasmuch as they are able to interact with a wide variety of peptides. However, studies of a mammalian hsp70 indicated a preference for binding of peptides rich in aliphatic and hydrophobic amino acids that were at least seven residues in length (Flynn et al., 1991). The cycles of binding and release of peptide require the hydrolysis
Molecular Chaperones and Protein Transport
3
of ATP, implying an interaction between the C-terminal peptide binding domain and the N-terminal ATPase domain. The structure of the C-terminal domain of hsp70 has not been solved; however, two groups have proposed a structure similar to that of the well-characterized major histocompatibility complex class I antigen presenting (HLA) molecules, based on slight similarities in primary sequence and secondary-structure predictions (Rippmann et al., 1991; Flajnik et al., 1991). The HLA structure and the proposed hsp70 peptide binding region consist primarily of P sheets with peptides binding in an extended conformation in the groove bounded by a helices (Fremont et al., 1992). The universal abihty of hsp70s to undergo cycles of binding and release with short regions of peptides determines their role in a great variety of intracellular functions. In this chapter we will discuss how, based on analysis of mutants of the yeast Saccharomyces cerevisiae, hspTOs function in the translocation of proteins from the cytosol into mitochondria and the endoplasmic reticulum has begun to be elucidated. B. The hsp70s of Saccharomyces cerevisiae
Like other eucaryotes, the budding yeast Saccharomyces cerevisiae has multiple hsp70 proteins. This large gene family is divided into subfamilies that correlate with the subcellular localization of the gene products (reviewed in Craig et al., 1993). The SSA and SSB subfamilies of proteins are found in the cytosol. The SSB subfamily has two members that are not absolutely essential for growth. This subfamily of hspTOs is found in association with translating ribosomes, perhaps bound to the nascent polypeptide chain. The four SSA gene products are found dissolved in the cytosol. The SSA subfamily is essential; cells require at least one of the SSA proteins in substantial amounts for growth at any temperature. SSA proteins function to regulate the transcription of heat shock responsive genes (Stone and Craig, 1990) and as discussed below are important for efficient translocation of at least some proteins from the cytosol into the ER and mitochondria. The other two yeast hsp70s are organellar. Kar2p is an essential protein found in the lumen of the endoplasmic reticulum (Rose et al., 1989; Normington et al., 1989). The mitochondrial hsp70, Ssclp, is a soluble protein of the matrix and is essential for growth (Craig et al., 1987, 1989). As discussed below both organellar hsp70s are required for transport of proteins into their respective organelles. Here, we focus on the role of mitochondrial hsp70 in translocation since it is the subject of work from our laboratories.
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E. A. CRAIG, B. D. GAMBILL, W. VOOS, and N. PFANNER
C. The Basics of Import into Mitochondria The biogenesis of mitochondrial proteins is a multistep process (reviewed in Pfanner et al., 1991; Click and Schatz, 1991). Precursor proteins synthesized in the cytosol with an N-terminal presequence and probably bound to cytosolic hsp70s associate with receptors on the mitochondrial surface. The presequence is then inserted into the outer membrane and then across the inner membrane triggered by the membrane potential, A\|/, across the inner membrane. The presequence is proteolytically removed by the processing protease of the mitochondrial matrix. Then the bulk of the polypeptide moves vectorially across the outer and inner membranes. Experimentally the translocation of precursor proteins across the inner membrane can be broken down into two steps: (1) the insertion of the presequence and (2) the translocation of the remainder of the polypeptide. The first step is dependent on Ay; the second is not. As described below Ssclp mainly functions in the second step, the movement of the bulk of the polypeptide into the matrix. A genetic approach has proved to be a very useful companion to biochemical analysis in understanding a number of physiological processes in both eucaryotes and procaryotes. The translocation of proteins from the cytosol into mitochondria has been no exception. Temperaturesensitive mutants have allowed the assessment of the effect of the absence of a single functional protein in vivo. When cells are grown under permissive conditions, the protein of interest is functional. Defects that are detected within a very short time after a shift to the nonpermissive temperature are likely a direct result of the inactivation of the protein. In this chapter we discuss the results of our combined genetic and biochemical analysis of the role of mitochondrial hsp70 in the translocation of proteins into mitochondria using temperature-sensitive hsp70 mutants.
IL A ROLE FOR CYTOSOLIC hsp70 IN PROTEIN TRANSLOCATION INTO ORGANELLES Several years ago it was suggested that cytosolic hspTOs of 5. cerevisiae were important for efficient translocation of at least some proteins from the cytosol to the lumen of the ER or matrix of the mitochondria. In vivo evidence came from the observation that strains depleted of the SSA proteins accumulated precursors of two proteins, the (3 subunit of the FJFQ ATPase, a mitochondrial protein, and a factor, a secreted protein, as the amount of SSA proteins dropped below the level found in wild-type
Molecular Chaperones and Protein Transport
5
(wt) cells (Deshaies et al., 1988). This initial experiment suffered from the fact that several hours elapsed after new expression of SSA proteins was stopped before a decrease in protein translocation was observed. Therefore it was possible that the effect of SSA depletion on translocation was an indirect one. However, recent experiments with temperature-sensitive SSA mutants have shown a defect in translocation of the same proteins within 10 minutes after a shift to the nonpermissive temperature, suggesting a direct effect (Becker and Craig, unpublished results). In vitro experiments supported the notion that SSA proteins are important for translocation. Translocation of radiolabeled proteins into microsomal vesicles and mitochondria was facilitated by the addition of SSA proteins (Chirico et al., 1988). Incubation of the precursor with urea substituted for the addition of SSA proteins in facilitating translocation, suggesting that the SSA proteins may aid in maintaining the precursor in a partially unfolded, translocation-competent conformation. This is an appealing idea because of the known peptide-binding activity of hsp70s and because of their propensity to bind to regions rich in hydrophobic residues as would be exposed in unfolded proteins. However, at this point there is no demonstration in vivo of a direct interaction of SSA proteins with precursors, although such interactions have been observed in reticulocyte lysates (Chirico, 1992).
III. ROLE OF MITOCHONDRIAL hsp70 IN PROTEIN TRANSLOCATION A. In vivo Analysis of a Mitochondrial hsp70 Mutant The first indication that mitochondrial hsp70 is required for the translocation of proteins from the cytosol into the matrix of mitochondria came from analysis of a temperature-sensitive mutant of S. cerevisiae (Kang et al., 1990). This mutant, called sscl-2, was obtained by directly mutagenizing SSCl DNA in vitro. Because of the suggestion that the cytosolic hsp70s, the SSA proteins, are involved in the translocation of at least some proteins from the cytosol into the endoplasmic reticulum and mitochondria, the sscl-l mutant was tested for its ability to carry out translocation at the nonpermissive temperature of 37°C. Specifically, the effect of a shift to 37°C on the conversion of the precursor form of mitochondrial proteins to the mature form was tested. The proteins in extracts made from cells 30 minutes after a shift from 23°C to 37°C were separated by electrophoresis and reacted with antibodies specific for
E. A. CRAIG, B. D. GAMBILL, W. VOOS, and N. PFANNER
*^
4- 4- ^ /
••••NliM
/ HSP60
f^f^
JL 23* C
37* C
Figure 1. Accumulation of precursor proteins in SSC1 mutants in vivo. Cultures of WT, ssc1'2 and ssc1-3 cells growing at 23°C were divided, and half of each culture was shifted to 37°C for 30 minutes prior to harvest. Protein extracts were fractionated by SDS-PAGE, electrotransferred to nitrocellulose filters, and probed with hsp60- and F^p-specific antiserum, p, precursor, m, mature.
mitochondrial proteins. Accumulation of the precursor form of hsp60 (Fig. 1) as well as a number of other proteins was observed. Since the protease that catalyzes the cleavage of matrix-destined mitochondrial proteins is located in the matrix, the lack of efficient cleavage in the sscl-2 mutant suggested a defect in the translocation process. Since initially cells were shifted to the nonpermissive temperature for 30 minutes prior to harvest of the cells, the effect on processing could easily have been due to a secondary rather than a direct effect of a mutation. To determine how rapidly the processing defect occurred after temperature shift, cells were labeled between 5 and 10 minutes after the shift and the
Molecular Chaperones and Protein Transport
7
P subunit of the FJFQ ATPase (FjP) was precipitated using specific antibodies. Whereas in wild-type (wt) cells all of the p subunit synthesized in this period was converted to the mature form, all of the detectable P subunit in the mutant was found in the precursor form, indicating a rapid decrease in precursor processing after the shift to the nonpermissive temperature. To ascertain the location of the precursor in the sscl-l cells, pulselabeled cells were fractionated prior to immunoprecipitation with FjPspecific antibody (Kang et al., 1990). After a 5-minute pulse, nearly all of the labeled subunit was associated with the mitochondria. However, it remained sensitive to added protease compared to matrix-localized proteins, indicating that it was associated with the mitochondrial surface. B. Mitochondrial hsp70 Is Required for Translocation into the Matrix Functional Ssclp Is Required for Translocation into Isolated Mitochondria
In vivo experiments are limited in allowing the determination of a specific defect in the translocation process; therefore we attempted to analyze translocation in sscl'2 mitochondria in a cell-free system (Kang et al., 1990). Mitochondria were isolated from wt and sscl-2 cells grown at 23°C. Prior to use in in vitro translocation assays the mitochondria were incubated at 37^C for 15 minutes to inactivate SSCl protein. Radiolabeled precursor proteins synthesized in the presence of [^^S] methionine in reticulocyte lysates were added to these isolated, energized mitochondria. Although efficient processing of the precursor proteins occurred, the proteins remained sensitive to exogenously added protease, indicating that the bulk of the proteins was not imported. In initial experiments similar results were obtained with precursors of Fe/S protein, the p subunit of the FJFQ ATPase, and cytochrome b2, suggesting that a decrease in SSCl function causes a general defect in translocation. The in vitro and in vivo results are in apparent contradiction. The in vivo experiments suggested a defect in translocation resulting in failure of insertion of the N-terminal targeting sequence across the inner membrane. However, the in vitro experiments suggested that the membrane potential-dependent insertion of the targeting sequence is not inhibited, whereas translocation of the remainder of the protein is defective. We think that the in vitro experiments provide a better indication of the role
8
E. A. CRAIG, B. D. GAMBILL, W. VOOS, and N. PFANNER
of mitochondrial hsp70 in translocation. An explanation of the apparent discrepancy may be the differences in the conditions. In an in vitro assay, radiochemical amounts of precursor are being imported; sites of import are in vast excess over the number of precursor proteins. However, in the in vivo experiment cells are labeled over a period of 5 minutes, 5 minutes after the shift to the nonpermissive temperature. The amount of precursor synthesized during the labeling time exceeds the number of import sites available. Therefore if translocation were blocked as indicated by the in vitro experiments with precursors "stuck" in an import site, the precursors synthesized after this point would be expected to accumulate in the unprocessed form. The observed association of precursors with mitochondria is probably due to interaction with receptors or other interactions with the outer surface of the outer membrane. Precursors labeled at later times after the temperature shift were found in the cytosol, suggesting that binding sites on the mitochondrial surface were saturated. Ssclp Interacts Directly with Precursor Proteins Since hsp70s were known to bind to unfolded proteins it was not unreasonable to propose that Ssclp's role in translocation might be due to its direct association with the precursor as it enters the mitochondria. An indication of a direct association came from co-immunoprecipitation experiments (Ostermann et al., 1990). Labeled proteins that were trapped during translocation into sscl'2 mitochondria at the nonpermissive temperature were precipitated by Ssclp-specific antibody. In agreement with these findings, cross-linking experiments identified Ssclp as a protein in extremely close proximity to translocating polypeptides in wild-type mitochondria (Scherer et al., 1990). To test the effect of the conformation of the precursor on its ability to be translocated into mutant mitochondria, the fusion protein Su9-DHFR was denatured by incubation in 8 M urea. The denatured preprotein was diluted directly into import reactions. Unlike partially folded precursor added directly from reticulocyte lysates, the import rates for the unfolded precursors into wt and sscl-2 mitochondria were very similar. These results suggested that Ssclp is involved in the unfolding of preproteins during translocation into mitochondria. In summary: initial studies indicated that mitochondrial hsp70 was required for translocation of a number of proteins into mitochondria. Initial insertion of the targeting sequence across the outer and inner membrane is not dependent upon hsp70 function. However, the translo-
Molecular Chaperones and Protein Transport
9
cation of the bulk of the protein across the membranes requires functional hsp70 and involves a direct interaction between the precursor protein and mitochondrial hsp70. Isolation and Analysis of a Second SSC1 Allele^ ssc1-3
In order to more fully understand the role of mitochondrial hsp70 in translocation, additional temperature-sensitive SSCl mutants were sought. One additional mutant, called sscl-3, was isolated by the same procedure used in the isolation of ssc 1-2 (Gambill et al., 1993) and was found to be defective in processing precursor proteins at the nonpermissive temperature of 37°C (Fig. 1). Surprisingly, sscl-2 and sscl-3 mitochondria had different properties with regard to the translocation of the fusion protein Su9-DHFR (dihydrofolate reductase) (Fig. 2). Su9-DHFR contains the presequence plus three amino acids of the mature portion of the Neurospora crassa F^-ATPase subunit 9 and the entire protein coding region of murine DHFR (Pfanner et al., 1987). Unlike most presequences the Su9 presequence contains two cleavage sites for the matrix-localized protease, after amino acids 35 and 66. Wild-type mitochondria efficiently cleaved Su9-DHFR to the mature form and translocated it to a proteaseresistant location. Consistent with the initial results described above, addition of Su9-DHFR to heat-treated sscl-l mitochondria resulted in cleavage to the mature form, but it did not result in translocation to a protease-resistant location. However, most of the Su9-DHFR added to sscl-3 mitochondria was only cleaved at the first cleavage site at amino acid 35 and not translocated to a protease-resistant location. This difference in precursor translocation into mitochondria suggested that the sscl-3 mitochondria might be more defective in translocation than sscl-2 mitochondria. To further address this difference, we compared the ability of the two types of mitochondria to import Su9-DHFR that was artificially denatured by incubation in 8 M urea (Gambill et al., 1993). Both sscl-2 and sscl-3 mitochondria efficiently translocated denatured precursor to a protease-protected location (Fig. 2). However, while the precursor was translocated across both the inner and outer membranes in sscl-2 mitochondria, it was translocated across only the outer membrane of the sscl-3 mitochondria. This was shown by subjecting the mitochondria to a hypotonic solution after import, causing rupture of the outer, but not the inner membrane (Fig. 3). After swelling, most of the protein imported into sscl-2 mitochondria was still resistant to exogenously added pro-
+ATP mm
^
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+PK Figure 2. Undenatured Su9-DHFR precursor accumulates in ssc^-2 and sscl-3 mitochondria in a protease-sensitive form, but after denaturation is translocated to a protease-resistant location. ^^S-labeled precursor of Su9DHFR synthesized in reticulocyte lysate was incubated with isolated mitochondria (25 ^g/lane) from WT, sscl-2, or ssc1-3 that had been preincubated for 15 minutes at 37°C to inactivate mutant Ssdp. Where indicated the mitochondria were treated with proteinase K (PK) after the import reaction. The reisolated mitochondria were analyzed by SDS-PAGE and fluorography. (A) The lysate was added directly to the mitochondria. (B) The reticulocyte lysate containing the labeled precursor was precipitated with ammonium sulfate and dissolved in 8 M urea to denature it (Kang et al., 1990). The precursor was then added directly to mitochondria; the final import mix had a concentration of 200 m M urea. Reprinted (Gambill et al., 1993) with permission. 10
Molecular Chaperones and Protein Transport
WT
Swelling PK
-
+ +
11
sscl-2
+
-
+ +
sscl-3
+
-
Figure 3. Urea-denatured Su9-DHFR is transported into the matrix of sscl-2 mitochondria but remains in the intermembrane space in sscl-3 mitochondria. Import was performed as described in Figure 2B. After import mitochondria were reisolated, a portion was diluted into a hypotonic solution (+ swelling), and a portion was diluted into an isotonic buffer containing 0.6 M sorbitol (-swelling). Where indicated (+ PK), proteinase K was added for 20 minutes after the dilution. The mitochondria were reisolated and analyzed by SDS-PAGE and fluorography. The treatment in a hypotonic solution results in the rupture of the outer but not the inner membrane, as confirmed by the localization of the intermembrane space protein cytochrome 62/ the inner membrane protein ADR/ATP carrier, and the matrix protein Ssd p. Taken from Gambill et al. (1993) with permission.
tease, indicating that the protein had been completely translocated into the matrix. But a large portion of the protein imported into sscl-3 mitochondria became susceptible. Therefore in sscl-3 mitochondria most of the Su9-DHFR protein accumulated as an intermediate that was cleaved once and spanned from the matrix, across the inner membrane into the intermembrane space. This type of intermediate has been termed the "intermembrane space intermediate" (Hwang et al., 1989; Rassow and Pfanner, 1991; Jascur et al., 1992; Pfanner et al., 1992). Even the small amount of mature form accumulated was exposed to the intermembrane space. It is possible that the failure of the sscl-3 mitochondria to translocate Su9-DHFR across the inner membrane in these experiments was due to the refolding of the precursor prior to crossing the inner membrane into a conformation that prevented its passage. To test this possibility translocation was carried out with mitochondria whose outer membranes had been disrupted (Gambill et al., 1993). Previously Schatz and co-workers
12
E. A. CRAIG, B. D. GAMBILL, W. VOOS, and N. PFANNER
(Hwang et al., 1989) had shown that such mitochondria, referred to as mitoplasts, were capable of translocating some preproteins. While denatured Su9-DHFR could be transported across the inner membrane of sscl-2 mitoplasts, almost no translocation into sscl-3 mitoplasts occurred. We concluded that unfolding of the polypeptide chain is not sufficient to allow translocation across the inner membrane of sscl-3 mitochondria. Since hsp70 function is required for translocation of proteins even when denatured, we concluded that mitochondrial hsp70 is a bonafide component of the mitochondrial translocation machinery. Comparison of the sscl-l and sscl-S Alleles In the in vitro translocation assays described above the sscl-S mutant displayed a much stronger phenotype than the sscl-2 mutant (Fig. 4). In the sscl-3 mutant only the first site of cleavage of Su9-DHFR was cleaved, whereas in the sscl-2 mutant both were cleaved. In addition, after denaturation Su9-DHFR only traversed the outer membrane, whereas complete translocation into the matrix was observed in the sscl-2 mutant. These in vitro results were somewhat surprising to us since our initial observations of the growth phenotypes of the two strains suggested that sscl-3 was a "weaker" allele than sscl-2. Upon the shift to 37°C sscl-3 is able to form small, barely visible colonies before growth ceases, whereas sscl-2 forms no visible colonies, sscl-3 cells can resume growth and form colonies at 23°C after being at 37°C for several days. While less than 1 day at 37°C prevents colony formation of sscl-2 at any temperature. Therefore the sscl-3 mutation is merely cytostatic at the nonpermissive temperature, whereas the sscl -2 mutation is cytocidal. This phenotypic difference is likely due to differences in the effect of the two mutations on the function of Ssclp. We tested the ability of the mutant hsp70s to interact with translocating polypeptides. Urea-denatured and labeled Su9-DHFR was incubated with wt, sscl-2, and sscl-3 mitochondria. The capacity of the mature protein to be co-immunoprecipitated by Ssclp antibody was tested. At short incubation times, Su9-DHFR was co-immunoprecipitated in both wt and sscl-2 mitochondria. The immunoprecipitation was about 70% efficient as compared to precipitation with DHFR antibody in wt mitochondria and about 80% as efficient in sscl-2 mitochondria. Interestingly, after longer times of incubation the DHFR could no longer be precipitated by Ssclp antibody in wt mitochondria, presumably because the DHFR protein had become properly folded. However, efficient precipitation was still observed in
.OM > IM
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SSC1-3 (-urea)
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Figure 4. Depiction of the location of the Su9-DHFR in ssc1-2 (A, B) and sscl-3 (C, D) mitochondria. As described in the text and shown in Figure 3. (A) When the precursor is not denatured by incubation in 8 M urea, both N-terminal cleavage sites of Su9-DHFR enter the matrix, but the bulk of the protein remains outside the outer membrane of ssc1-2 mitochondria. (B). After denaturation with urea the entire protein is imported into the matrix of ssc1-2 mitochondria. (C) In ssc1-3 mitochondria the N-terminus of Su9-DHFR is imported to a point that allows cleavage only at the first site. (D). Upon denaturation with urea, the protein enters the intermembrane space, but no further import into the matrix is detected. Asterisk (*) indicates the sites of cleavage of the matrix-localized processing protease. For diagrammatic purposes, cleavage is not shown.
13
14
E. A. CRAIG, B. D. GAMBILL, W. VOOS, and N. PFANNER
sscl-2 mitochondria, suggesting that the Sscl-2p remained bound to imported protein for a much longer time. Analysis of the interaction of Sscl-3p with precursor was more difficult to evaluate. Only a small amount of mature protein was formed in the sscl-3 mitochondria and only about 20% of that imported was able to be co-immunoprecipitated with Ssclp antibody. Thus it appears that Sscl-3p is strongly impaired in binding Su9-DHFR, whereas Sscl-2p fails to release from Su9DHFR. Although the exact effect of the two mutations on substrate protein binding and release will not be determined until purified protein is analyzed, the results from the analysis of the interaction of Su9-DHFR with the different SSCl proteins in isolated mitochondria suggests an explanation for the seemingly contradictory effects of the mutant proteins on translocation in the in vitro system and the effects on cell growth. The translocation assays suggest that sscl-3 mutant protein is less effective than sscl-2 in protein translocation into mitochondria. Our working model proposes that Sscl-2p still has substantial affinity for substrate proteins but is also defective in releasing the substrates that are bound. This residual binding activity would be sufficient for translocation under certain conditions, such as when the precursor protein is denatured. However, the defect in release indicated by prolonged association with imported proteins could be problematic in vivo. After a shift to the nonpermissive temperature, failure to release from proteins after binding might well interfere with mitochondrial processes more than a simple failure to bind, causing a lethal effect. Because it could not be efficiently immunoprecipitated with precursor proteins, Sscl-3p likely has a greatly decreased affinity for substrate proteins. C. Degree of Requirement of Sscl p Activity in Transport Is Correlated with the Conformation of the Precursor The results described above, concerning the failure of Su9-DHFR precursor to be imported across the inner membrane into sscl-3 mitochondria even when denatured by treatment with urea, indicate that Ssclp is necessary for translocation across the inner membrane. However, the ability of urea-denatured Su9-DHFR to cross the inner membrane of sscl-2 mitochondria implies an effect of the structure of the precursor on its ability to be translocated. In order to better understand the role of Ssc Ip on protein translocation we tested the ability of a variety of different precursors to be imported into isolated wt, sscl-2, and sscl-3
Molecular Chaperones and Protein Transport
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Time (min) Figure 5. 62(167)-DHFR precursor is imported into sscl-2 and sscl-3 mitochondria as well as into wt mitochondria. Import experiments using )b2(167)-DHFR precursor were carried out as described in Figure 2. After the import reaction was carried out for various times, the mitochondria were treated with proteinase K to degrade any protein on the mitochondrial surface and were reisolated and analyzed by SDS-PAGE and fluorography. Taken with permission from Voos et al. (1993).
mitochondria (Voos et al., 1993). Surprisingly, we found a precursor that was imported into the three types of mitochondria with nearly identical efficiency (Fig. 5). The precursor, Z72(167)-DHFR, consisted of 167 N-terminal residues of the yeast cytochrome ^2 protein and the entirety of murine DHFR. The 167 residues of the cytochrome b^ protein included 80 residues of the presequence followed by 87 residues of the mature protein. Cytochrome ^2 is located in the intermembrane space; the 80 residues of the presequence contain the information for targeting to this compartment. In the process of import the precursor is cleaved once by the matrix processing protease to give an intermediate-sized form and then a second time on the outer side of the inner membrane by inner membrane protease I to yield the mature form. The processing of fe2(167)-DHFR into the intermediate and mature forms occurred at the same rate in wt, sscl-2, and sscl-3 mitochondria that had been incubated at 37°C for 15 minutes prior to import to inactivate the mutant hsp70s.
16
E. A. CRAIG, B. D. GAMBILL, W. VOOS, and N. PFANNER
I
rsi
ro
(N
ro
u
u
U
U
CO (/)
5 min
CO CO
(/) U5 1
U.
CO CO
15 min
Figure 6. Urea-denatured fa2(220)-DHFR is imported with similar efficiency into WT, sscl-2 and ssclS mitochondria. Reticulocyte lysate containing ^^S-labeled b2(220)-DHFR was precipitated with ammonium sulfate and dissolved in 8 M urea. The denatured precursor was diluted 40-fold into the import reaction containing mitochondria. Import into mitochondria was performed for 5 or 15 minutes, as described in Figure 2. Taken from Voos et al. (1993) with permission.
In all cases the mature form was protected against digestion with exogenous protease; however, it was susceptible to digestion after incubation with hypotonic buffer, indicating that the imported protein was appropriately localized to the intermembrane space. We had previously observed that the import of authentic cytochrome ^2 into sscl'2 mitochondria was partially inhibited (Kang et al., 1990). We repeated this result and determined that import of the full-length cytochrome 63 protein into sscl-3 mitochondria was almost completely blocked. To track down the reason for the difference in the translocation of authentic cytochrome 63 and the b2167-DHFR fusion, we tested other fusions that contained larger portions of cytochrome 62- It was possible that the DHFR moiety conferred the SSCl independence. However, when we tested two other fusions that contained larger portions (220 and 330 amino acids) of cytochrome ^2 linked to DHFR we found that their
Molecular Chaperones and Protein Transport
17
100en W
^^ffl
(IH
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^
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«20 nm) were imported only when associated
Nuclear Transport of U snRNPs
131
with high numbers of NLSs. The impUcation from these studies is that NLS copy number influences the efficiency of translocation through the pore complex. Thus NLS copy number effects may act during both targeting and translocation stages of import. U snRNPs are relatively large assemblies and may, therefore, require multiple NLSs for efficient import. The import of RNA and DNA viruses is vaguely similar to the import of U snRNPs because the import of the nucleic acid component is mediated by associated proteins. During the import of adenovirus (Greber et al, 1993) and HIV-1 (Bukrinsky et al., 1993b), capsid proteins must be removed before the particle can be imported. Like U snRNPs, therefore, these viruses have latent periods in the cytoplasm before they become karyophilic. However, the mechanisms of viral and U snRNP latency are probably dissimilar. Being extremely large, most karyophilic viruses appear to display multiple NLSs in the form of associated NLS-containing polypeptides. A recent example is the import of HIV-1 (Bukrinsky et al., 1993a). Import of one HIV-1 strain is mediated by a NLS-containing gag-matrix protein. The mutation of the gag-matrix NLS inhibited the nuclear transport of the 160S HIV-1 preintegration complex. Perhaps as many as 100-200 copies of the gag-matrix protein are normally associated with the preintegration complex. Among all the different polynucleic acids that are imported into nuclei, both in nature and in the laboratory, including viruses, retrotransposible elements, plasmids, and transforming DNAs, the role of the m^'^'^GpppN cap of U snRNAs is unique in its apparent role as a factor in signaling import (see below). Although the fundamental mechanics of nuclear transport are probably conserved among all eukaryotes, quantitative differences in import rates are commonly observed among different cell types. As will be described below, the particulars of U snRNP import in somatic cells can differ significantly from their transport in oocytes. Furthermore, the capacity of the transport apparatus can vary according to the physiological state of the cell. ProUferating cells import larger colloidal gold particles more efficiently than cells that have been maintained in a nonprolifcrating state for many days (Feldherr and Akin, 1990). This effect is a property of the nuclear envelope and not the cytoplasm. Studies using heterokaryons between proliferating and nonproliferating cells showed that the nonproliferating nucleus retained its poor import capacity even when bathed in the cytoplasm of a proliferating cell; the proliferating nucleus continued to import efficiently (Feldherr and Akin,
1 32
ELISA IZAURRALDE, IAIN W. MATTAJ, and DAVID S. COLDFARB
1993). The role of cell activity in nuclear transport is the topic of another article in this series (Feldherr, vol3). We hope that the preceding paragraphs will provide a general context for the specific discussion of U snRNP nuclear trafficking. In the next section we will summarize some aspects of U snRNP composition, assembly, and function. These topics have been reviewed recently (Liihrmann et al., 1990; Andersen and Zieve, 1991; Mattaj et al, 1993). For more extensive background information, interested readers are referred to these articles.
IV. COMPOSITION AND FUNCTION OF SPLICEOSOMAL U snRNPs Spliceosomal U snRNPs are composed of one (Ul, U2, and U5) or two (U4/U6) short uracil rich RNAs (U snRNAs) in association with a set of proteins. Some of the proteins are common to the various snRNPs, and others are specific for individual snRNPs. The particles are named according to the RNA molecules that they contain, either Ul, U2, U5, or U4 and U6 snRNA. In the U4AJ6 snRNP particle, U4 snRN A extensively base-pairs with U6 snRNA. A tri-snRNP complex composed of U4/U6 and U5 snRNPs has also been described. Formation of the tri-snRNP is functionally essential for splicing activity, but this form of the snRNPs seems to be more labile to isolation conditions than are the individual U4/U6 or U5 snRNPs. In fact, all spliceosomal snRNPs interact with each other, and with additional factors, in a sequential order to form a functional splicing complex, the spliceosome, on the precursor mRNA. Most of these interactions are transient and change during the dynamic process of pre-mRNA splicing (see Moore et al., 1993, for a recent comprehensive review).
V. THE RNA CONSTITUENTS OF SPLICEOSOMAL snRNPs With the exception of U6, spliceosomal U snRNAs are transcribed by RNA polymerase II. Polymerase Il-transcribed U snRNAs are synthesized as precursors with extended y ends, which are removed during the assembly of U snRNPs (Elicieri, 1974; Madore et al., 1984). Our knowledge of the activities involved in this process is still limited and will be discussed below in more detail. According to the polymerase involved in their transcription, U snRNAs differ at their 5' ends. Polymerase Il-transcribed U snRNAs, in
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common with other pol II transcripts, have a monomethyl guanosine cap (m^GpppN) added cotranscriptionally (Reddy and Bush, 1988, and references therein). This cap structure will be subsequently hypermethylated during the transient cytoplasmic phase of U snRNP assembly (see below). U6, the only spliceosomal snRNA transcribed by polymerase III, carries a y-methyltriphosphate at its 5' terminus (Singh and Reddy, 1989). In addition to the modifications of their 5' and 3' termini, U snRNAs possess multiple modified internal nucleotides (Reddy and Bush, 1988). It is not yet clear in which cellular compartment these post-transcriptional modifications occur and whether association with snRNP proteins is required for intemal modification. The functional significance of these modifications has also not been studied. VI. THE PROTEIN COMPONENTS A. The Common Proteins The four spliceosomal U snRNPs, Ul, U2, U5, and U4/U6, have at least nine polypeptides in common, with molecular masses ranging from 9 to 69 kDa (Table 1; see Wolin and Walter, 1991; Will et al., 1993, and references therein). The latest member of this group, a polypeptide with an apparent molecular mass of 69 kDa, was only recently described (Hackl et al., 1993), probably because its interaction with snRNP particles appears to be more labile than that of the other common proteins. The common proteins are also named core proteins or, because of their interaction with sera of the anti-Sm serotype, Sm proteins (Lemer et al., 1979). The particles formed by association of the core proteins with individual U snRNAs are called core particles to distinguish them from the mature particles, which contain, in addition, specific proteins. The core proteins are made and stored in the cytoplasm in the form of protein complexes that associate with the U snRNAs after their export from the nucleus (Eliceiri, 1974; De Robertis et al., 1982). Apparently, these proteins do not contain an exposed nuclear localization signal of the type found on karyophilic proteins, and their nuclear migration takes place only upon their binding to the U snRNAs (Zeller et al., 1983; Mattaj and De Robertis, 1985). In Xenopus oocytes, the Sm proteins are stored in very large amounts in the cytoplasm in the absence of snRNAs for use in early embryogenesis (Zeller et al., 1983). Although there is little apparent storage of common snRNP proteins in the cytoplasm of somatic cells, the pathway of snRNP assembly is the same as in oocytes. Zieve
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Table 1. The Common Proteins of Spliceosomal U snRNPs
Name
Apparent molecular (kDa)
G F E Dl D2
9 11 12 16 16.5
D3 B B'
18 28 29
mass
69 Note: The core proteins are named according to their electrophoretic mobility. The molecular masses are according to Will et al. (1993).
and co-workers have described three different core protein complexes in murine cells: a complex with a sedimentation coefficient of 6S, composed of D, E, F, and G core proteins with a suggested stoichiometry of D2EFG (note that these results were obtained before it became clear that there are three distinct D-sized proteins, Dl, D2, and D3; see Table 1). This particle seems to be the first protein complex that assembles with snRNA (Fischer et al., 1985; Zieve and Sauterer, 1990). However, since additional core proteins have been identified subsequent to these initial studies, the snRNP assembly pathway should be reinvestigated in more detail. A 20S protein complex was also found, composed of the D core protein in association with a 70-kDa protein. Whether this 70-kDa protein corresponds to the recently described 69-kDa core polypeptide is not yet known. The B/B' core proteins sedimented at around 5S-6S, at l i s , and at 20S; however, it is not clear whether this heterogeneity reflects self-association or interaction with other proteins. The core proteins interact in a sequential order with a partially conserved RNA structural motif that is designated the Sm binding site or domain A and is present in U1, U2, U4, and U5, but not in U6 snRNAs (Branlant et al., 1982). The core of the Sm binding site consists of a single-stranded region containing the consensus sequence PuA(U)^GPu, with n > 3. This core is usually flanked by hairpin loop structures (Jacob et al., 1984). In spite of this apparent conservation, different Sm binding
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sites are not equivalent, and the contribution of neighboring RNA sequences and structural elements to the binding of Sm proteins varies in different snRNPs (Jarmolowski and Mattaj, 1993). Nevertheless, it appears at present that exactly the same set of core proteins interact with each of the individual RNAs. Indeed, electron microscopic studies have revealed that U1, U2, U4, and U5 core particles are morphologically very similar and form a spherical body about 8 nm in diameter (Kastner et al., 1990). This basic structural element of the snRNPs has been called the Sm core domain. Interestingly, the Sm binding site of U7 snRNAs from various organisms does not correspond well to the consensus sequence listed above. This results in inefficient assembly with the core proteins (Grimm et al., 1993). This inefficient interaction, paradoxically, has been shown to be essential for the assembly of functional U7 snRNPs (Grimm etal., 1993). Core proteins play an important role in snRNP assembly and subcellular localization. In particular, hypermethylation of the cap structure of U snRNAs, transport of the assembled particles into the nucleus, and possibly some post-transcriptional modifications of the RNA strictly depend on binding of the core proteins to the Sm domain (Mattaj and De Robertis, 1985; Mattaj, 1986). No evidence for a direct functional role of these proteins in splicing has been provided. Indeed, it has been shown in vitro, in an extract of human cell nuclei, that a U4AJ6 snRNP lacking stably bound core proteins is functionally active in splicing complementation (Wersig and Bindereif, 1992). B. The Specific Proteins
Apart from the common proteins, each snRNP contains unique proteins (see Table 2). Ul- and U2-specific proteins were initially identified by the use of autoimmune antibodies. For example, antibodies against the U1 -specific proteins 70K, A and C, are found in the serum of patients suffering from systemic lupus erythematosus and mixed connective tissue diseases. Autoantibodies to the U2-specific proteins A' and B'^ can also be detected in patients suffering from autoimmune diseases. Specific proteins associated with U5 and the triple U4/U6.U5 snRNP, as well as additional U2-associated proteins, were identified by biochemical fractionation (Will et al., 1993, and references therein). The protein composition of purified snRNPs varies according to the ionic conditions used during the isolation procedure. At high salt, U2 snRNP sediments with a coefficient of 12S and is composed of the core proteins
136
ELISA IZAURRALDE, IAIN W. MATTAJ, and DAVID S. GOLDFARB Table 2. The Specific Proteins of Spliceosomal U snRNPs Ul (12S) C(22) A (34) 70K (70)
U2 (17S) B " (28.5) A'(31)
35 53 60 66 92 110 120 150 160
U5 (20S)
15 40 52 100 102 116 200 205
U4/U6.U5 (25S)
15 40 52 100 102 116 200 205 + 15.5 20 27 60 60 60 90
in association with the two U2-specific proteins A' and B''. At low ionic strength U2 sediments at 17S. This shift in the sedimentation coefficient is due to the presence of nine additional proteins (Table 2). Similarly, the triple snRNP, which has a sedimentation coefficient of 25S, is observed at low salt concentration and contains, in addition to the proteins already associated with the individual U5 and U4/U6 snRNPs, five additional polypeptides specific for the tri-snRNP complex. It is likely that other proteins transiently associate with the snRNPs during assembly of the particles (chaperones), their transport across the nuclear pore (transport receptors), or splicing (non-snRNP splicing factors). In contrast with the common proteins, whose nuclear accumulation is strictly dependent on their recruitment into U snRNP core particles, some of the specific proteins move to the nucleus separately from the rest of the snRNP (Feeney et al., 1989; Jantsch and Gall, 1992; Kambach and Mattaj, 1992,1994). Examples of this are the two Ul snRNP-specific proteins Ul A and UlC and the two U2-specific proteins U2A' and U2B'^ Nuclear targeting of these proteins appears to be mediated by the mechanism common to karyophilic proteins. However, the nuclear localization signals of Ul A and U2B'' proteins, which were subject to a more detailed analysis, are unusual in primary structure and do not correspond to either of the
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"canonical" types of NLS previously defined for nuclear proteins (see below). VIL ASSEMBLY PATHWAY OF SPLICEOSOMAL U snRNPs Assembly of U snRNPs takes place in the cytoplasm where, as discussed above, pools of core proteins exist in the form of various complexes (see Figure 1). Newly synthesized pol II U snRNAs are exported into the cytoplasm and associate with the common proteins before migrating back to the nucleus (EUcieri, 1974; Mattaj and De Robertis, 1985; Zieve et al., 1988). The major exception to this pathway is the U6 snRNA, which lacks a binding site for the core proteins and appears not to leave the nucleus after transcription (Vankan et al., 1990). The interaction of U6 with U4 snRNA to form the double U4AJ6 snRNP occurs in the nucleus, upon re-entry of U4 core particles (Vankan et al., 1990; Wersig et al., 1992). In most cell types examined, there is an excess of U6 RNA over U4 snRNA, perhaps to ensure efficient U4/U6 assembly. During the transient cytoplasmic phase, the monomethyl cap structure of U snRNAs is converted into a trimethyl cap (m^'^'^GpppN) by the addition of two methyl groups at position 2 of the guanosine. The identity of the methylase is not known, but the observation that binding of the Sm proteins is a prerequisite for its activity suggests that the enzyme must recognize, in addition to the cap structure, some determinants on the snRNP core particle (Mattaj, 1986). It is also during assembly that trimming of the 3' ends and modification of the internal nucleotides take place. Processing of the extended 3' termini is initiated in the nucleus, since on the appearance of Ul, U2, and U4 snRNAs in the cytoplasm, most of the 3' additional nucleotides have been trimmed (Zieve and Sauterer, 1990). However, the remaining few additional 3' nucleotides seem to be processed after binding of the core proteins, which is dependent on the RNA cap structure and on the occurrence of transport (Neuman de Vegvar and Dahlberg, 1990; Yang et al., 1992). Thus, accurate 3' end formation appears to be coupled to, and might affect the efficiency of, transport of U snRNPs through the nuclear pore complex and seems to involve interaction with both termini of the U snRNAs (Neuman de Vegvar and Dahlberg, 1990; Yang et al., 1992). Precise 3' end formation, on the other hand, is not essential for reaccumulation of U snRNAs in the nucleus after snRNP assembly (Konings and Mattaj, 1987; Jarmolowski and Mattaj, 1993).
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In the next sections we focus our attention on the steps of the pathway that involve the transport of U snRNPs back and forth through the nuclear pore complex. It should be noted that, although the assembly pathway described above is well supported by experiments carried out in various vertebrate cell types, the same pathway has not yet been demonstrated to be universal among eukaryotes. Furthermore, the more detailed knowledge of signals and mechanisms described below is largely based on experiments carried out in a single, highly specialized cell type, the Xenopus laevis oocyte, because of the ease with which RNA transport can be examined and manipulated in these cells.
VIII. NUCLEAR EXPORT OF U snRNPs Many of the data on U snRNA export have been obtained using a Ul snRNA mutant, UlASm (previously called UlAD), which carries a substitution of six nucleotides in its Sm binding site (Hammet al., 1987). Unlike the wild-type RNA, whose cytoplasmic residence is transient, the mutant RNA accumulates in the cytoplasm after export, since it is no longer able to interact with the core proteins. This uncoupling of export from re-import makes UlASm RNA suitable for export studies. If export takes place, the RNA is in the cytoplasm, whereas if export is blocked (i.e., by inhibitors, modification of the RNA, or the use of antibodies) the RNAremains in the nucleus. UlASm snRNA export, in common with rRNA, tRNA, and mRNA export, exhibits the characteristics of a receptor-mediated process, such as temperature dependence and saturability (Jarmolowski et al., 1994). One of the first insights into the U snRNA export mechanism was provided by the observation that after transcription U6 snRNA is not exported into the cytoplasm, unlike pol II U snRNAs (Vankan et al., 1990). Since U6 snRNA is the only spliceosomal RNA transcribed by RNA polymerase III, it was important to determine whether it was the polymerase responsible for the transcription of a particular RNA, which determines whether this RNA will be exported or retained into the nucleus. This question was investigated by Hamm and Mattaj (1990), who compared the export of UlASm RNA synthesized by pol II with an artificial UlASm RNA whose transcription was driven by the RNA polymerase III pro motor derived from the U6 gene. The pol III UlASm RNA was retained in the nucleus, whereas the pol II transcript, as expected, accumulated in the cytoplasm. Since the only difference between these two RNAs was the presence of the cap structure at the 5' end
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of the pol II transcript as opposed to the triphosphate 5' end present on the pol III transcript, the role of the cap structure in Ul snRNA export was investigated in more detail. First, it was shown that UlASm snRNA export could be competitively inhibited by a cap analog, the dinucleotide m^GpppG. Second, the export of an in vitro synthesized RNA, carrying a trimethyl cap structure, was found to be slower than that of the monomethyl-capped RNA. This study provided the first indication that the cap structure played a role in RNA export. The observation that the export of a capped RNA could be inhibited by a large excess of the cap analog strongly suggested that the cap structure was recognized directly by one or more factors that participate in Ul snRNA export. In the competition experiment, this factor was titrated by the excess of cap analog, resulting in inhibition of transport. The observation that a trimethyl capped RNA was still exported, but with slower kinetics, indicated that the physiological cap structure (m'^GpppN) was not absolutely required for RNA export, but that it would accelerate the export rate of the RNA. There are two plausible explanations for this observation. One is that an RNA with a trimethyl cap can still be recognized by the cap binding factor(s), but with a lower affinity, resulting in slower kinetics of export. The second is that interaction with the cap binding factor is not essential for export, but affects export rate when present on the RNP export substrate (see below). More recent studies on RNA export in Xenopus oocytes have confirmed the conclusions of this work and further showed that the role of the cap structure is not restricted to U snRNA export, but can also be extended to mRNA export (Dargemont and Kiihn, 1992; Jarmolowski et al., 1994). It appears, however, that although factors that recognize the cap structure are essential for the export of U snRNAs from oocyte nuclei, their effect on mRNA is less important, being confined to affecting the rate of export (Jarmolowski et al., 1994).
IX. THE ROLE OF THE CAP STRUCTURE IN U snRNA EXPORT The role of the cap structure in U snRNA export was further investigated by Jarmolowski et al. (1994). By microinjection of increasing quantities of m vitro synthesized, unlabeled Ul ASm snRNA competitor into oocyte nuclei these authors could progressively inhibit export of labeled Ul ASm RNA transcripts. The inhibition was observed when the competitor RNA had a m^GpppG cap but not an ApppG. These experiments show not only
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ELISA IZAURRALDE, IAIN W. MATTAJ, and DAVID S. GOLDFARB
that Ul snRNA export is a saturable process but furthermore, the export factor that is first saturated by increasing quantities of UlASm RNA substrate requires the cap structure in order to recognize RNA. The essential role of the cap structure was confirmed by the observation that a capped RNA, not related in sequence to Ul snRNA, was also able to inhibit Ul snRNA export in a concentration-dependent manner. Similar results were obtained when the export of ASm mutant versions of U2 and U5 snRNAs were analyzed. Increasing amounts of capped Ul snRNA progressively inhibited export of these two U snRNAs, whereas an ApppG-capped Ul snRNA was not inhibitory at the maximal concentration tested. Export of U2 and U5 snRNAs could also be competed with by increasing amounts of the unrelated RNA mentioned above when an m^GpppG cap structure was present at its 5' end. Together, these results strongly suggest that a cap recognition factor(s) is generally involved in U snRNA export. As previously observed for the RNA with a trimethyl cap (Hamm and Mattaj, 1990), an RNA with the nonphysiological ApppG cap structure could still be exported from the nucleus but with slower kinetics, when injected in the absence of a competitor. This result can best be reconciled with the competition results in terms of relative affinities of the cap binding factor involved in U snRNA export. The prediction would be that the cap binding factor should be able to interact with the RNAs with nonphysiological cap structures, but with a reduced affinity, resulting in slower export kinetics. This prediction was strongly supported by the observation that export of RNAs with nonphysiological cap structures can also be competitively inhibited by m^GpppG capped RNAs (Jarmolowski et al., 1994, and unpublished data). It is not yet clear, however, whether the cap binding factor is able to recognize nonphysiological caps directly, although with a lower affinity, or is recruited onto the RNP export substrate via protein-protein interactions. In conclusion, these results suggest that the physiological cap is not required for export per se, but it has a kinetic effect on this process. In contrast, the cap recognition factor appears to be essential.
X. CHARACTERIZATION OF THE NUCLEAR CAP BINDING ACTIVITY INVOLVED IN U snRNA EXPORT Having established the role of the cap structure in U snRNA export and the involvement of a cap recognition factor in this process, the next step was the identification of such a factor. Several nuclear and cytoplasmic
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cap binding proteins (CBPs) had been described in extracts of mammalian, plant, and yeast cells (Sonenberg, 1981; Patzelt et al., 1983; Rozen and Sonenberg, 1987; Ohno et al, 1990), the best studied being the eukaryotic translation initiation factor 4E (eIF-4E; for reviews see Shatkin, 1985; Rhoads, 1988; Sonenberg, 1988). It was important to determine whether the cap binding activity (CBA) involved in nuclear transport was related to any of these CBPs and in particular to the translation initiation factor eIF-4E, which, in spite of its cytoplasmic function, is also found in low concentration in the nucleus (Lejbkowicz et al., 1992). To investigate this question a series of cap analogs, whose affinity for human eIF-4E had previously been determined, were tested for their ability to inhibit nuclear export of Ul ASm snRNA transcripts in Xenopus oocytes (Izaurralde et al., 1992). Five dinucleotide cap analogs, m^GpppG, Et^GpppG, m^'^GpppG, m^'^'^GpppG, and GpppG, and the mononucleotide m^'^GMP were tested by microinjection into oocytes transcribing the Ul ASm gene. In oocytes injected with either m^GpppG or Et^GpppG, UlASm transcripts were retained in the nucleus. Injection of GpppG, m^'^GpppG, m^'^'^GpppG or m^'^GMP did not affect the cellular distribution of UlASm transcripts, which accumulated exclusively in the cytoplasm. Subsequently, similar results were obtained when instead of using dinucleotide competitors, the various cap analogs were incorporated into an unrelated RNA, which was then used as a competitor (Jarmolowski and Izaurralde, unpublished data). These competition experiments showed that, in addition to 7methyl diguanosine triphosphate, the only N-7 substituted cap analog able to inhibit nuclear export of Ul snRNA transcripts was 7-ethyl diguanosine triphosphate. The dinucleotide m^'^GpppG was not able to inhibit Ul snRNA export, whereas it has been shown to be more efficient than Et^GpppG in binding to eIF-4E and inhibiting protein synthesis (Darzynkiewicz et al., 1989; Carberry et al., 1990). In addition, neither m^'^GMP nor other mononucleotides that bind tightly to eIF-4E in vitro inhibited nuclear export of UlASm transcripts (unpublished data). These results demonstrated that the CBA involved in RNA export does not exhibit the same binding specificity as eIF-4E. In contrast, an 80-kDa nuclear cap binding protein (CBP80) from human cell nuclei initially identified and purified by Ohno et al. (1990) did exhibit the same cap analog recognition specificity as the CBA involved in Ul snRNA nuclear export (Izaurralde et al., 1992). The only dinucleotides shown to efficiently inhibit binding of CBP80 to a capped RNA probe in vitro were m^GpppG and Et^GpppG. All other dinucleotides and mononucleotides
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ELISA IZAURRALDE, IAIN W. MATTAJ, and DAVID S. GOLDFARB
tested were inefficient competitors. Whether CBP80, or other nuclear CBPs, plays a role in U snRNA export should soon be clarified through isolation and characterization of the proteins and their cognate genes.
XL NUCLEAR IMPORT OF U snRNPs The presence of large pools of snRNP proteins in the cytoplasm of Xenopus oocytes has been of enormous advantage for the study of U snRNP assembly and transport in vivo. In vitro synthesized U snRNAs injected into the oocyte cytoplasm are assembled into snRNP core particles by association with the Sm proteins and subsequently transported into the oocyte nuclei. Therefore, the Xenopus oocyte offers the possibility of uncoupling U snRNA import from export and directly studying the requirements for U snRNP nuclear uptake. Recently, a major breakthrough in the study of U snRNP import has been achieved with the development of an in vitro reconstitution system for snRNP particles (Sumpter et al., 1992) and the adaptation of preexisting in vitro and in vivo systems for protein nuclear import to the study of U snRNP import (Fischer et al., 1993, 1994; Marshallsay and Luhrmann, 1994). The availability of these two systems made possible the study of U snRNP assembly and transport in mammalian cells. Comparison and synthesis of the results emerging from the amphibian and mammalian systems will be essential to unraveling the basic mechanisms involved in this process. U snRNP nuclear import, in common with import of karyophilic proteins, exhibits the characteristics of a receptor-mediated process, such as temperature dependence and saturability (Fischer et al, 1993,1994). Earlier studies of U snRNA import had shown that mutation of the Sm binding site, but not other regions of U2 snRNA, including those required for stable binding of U2-specific proteins, affected the nuclear accumulation of U2 snRNA (Mattaj and De Robertis, 1985). Binding of Sm proteins was also found to be required for cap hypermethylation (Hernandez and Weiner, 1986; Mattaj, 1986). Because binding of the Sm proteins was essential for both nuclear migration and cap trimethylation, it was not clear whether the trimethyl guanosine cap structure, the Sm proteins, or both elements were necessary for nuclear migration. Subsequently, it has been shown that, in Xenopus oocytes, hypermethylation of the cap structure and binding of Sm proteins are both required to direct the assembled Ul and U2 snRNPs back to the nucleus. Evidence for the involvement of the trimethyl cap (m^'^'^GpppN) in signaling the import of Ul snRNP came from various experiments
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(Fischer and Liihrmann, 1990; Hamm et al., 1990). First, after injection into the cytoplasm, Ul snRNAs with ApppG 5' termini were not transported to the nucleus, unUke Ul snRNAs with an intact trimethyl cap. All of these RNAs were, however, apparently able to assemble normally with both the core and the specific Ul snRNP proteins. Second, Ul snRNA transport was specifically inhibited after removal of its 5' terminus via RNase H cleavage mediated by a DNA oligonucleotide complementary to the Ul 5' end. Third, Ul snRNP nuclear import was competitively inhibited by coinjection into Xenopus oocytes of a large excess of the dinucleotide m^'^'^GpppG but not of the m^GpppG dinucleotide. Furthermore, antibodies directed against the trimethyl cap, or oxidation of the ribose ring of the trimethyl cap by treatment with periodate, severely inhibited nuclear transport of Ul snRNPs. These data clearly established that a factor(s) recognizing the trimethyl cap of Ul snRNA is required to ensure movement of the assembled snRNP back to the nucleus. This factor can be specifically saturated by the trimethyl cap analog but not by the monomethyl dinucleotide. Interestingly, in contrast to the inability of mononucleotides to inhibit nuclear export, the mononucleotide m^'^'^GTP was able to inhibit Ul snRNA import with the same efficiency as the trimethyl dinucleotide m^'^'^GpppG (unpublished data), suggesting a differential mode of recognition of monomethyl and trimethyl cap structures by their respective "receptors." The same authors also showed that the trimethyl cap is not the only feature of the snRNPs recognized by the cellular import machinery. In vitro synthesized Ul snRNA mutants unable to bind to the core proteins but carrying a trimethyl cap structure were not transported into the nucleus after injection into the oocyte cytoplasm. These experiments clearly demonstrated that the Sm proteins not only ensure hypermethylation; they also participate directly in the nuclear targeting of Ul snRNA. Confirmation, and even stronger evidence, of the role of Sm proteins as part of the U snRNP nuclear localization signal came from a series of elegant experiments described by Fischer et al. (1993) using in vitro assembled snRNP core particles. These particles were shown to accumulate in oocyte nuclei with the same kinetics and requirements similar to those of core particles assembled in vivo by injecting U snRNAs into the oocyte cytoplasm. By taking advantage of the availability of these two alternative in vivo and in vitro assembly systems, it was possible to petform competition studies. Oocytes were pre-injected with increasing amounts of unlabeled U snRNAs with different cap structures. These RNAs associate, as expected, with the large pool of Sm proteins present
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in the oocyte cytoplasm to form competitor core particles. Subsequently, radioactivelly labeled, in vitro assembled particles containing trimethyl capped RNAs were injected into the cytoplasm and their nuclear accumulation monitored. Inhibition of import of labeled particles was observed even when the competitor particles lacked the trimethyl cap structure. Therefore, like the dinucleotide m^'^'^GpppG, the core particles are able to saturate a limiting component required for U snRNP nuclear accumulation. This putative U snRNP transport receptor recognizes the nuclear localization signal in the core particles independently of the cap structure. In summary, the nuclear localization signal of Ul snRNP in Xenopus oocytes results from the contribution of two elements, the trimethyl cap structure on the RNA and the bound core proteins. The nature of the receptor(s) interacting with the two elements of this bipartite nuclear localization signal is not yet known. It is also not known which features of the core particles are recognized in this process (see below).
XII. THE REQUIREMENT OF THE TRIMETHYL CAP STRUCTURE FOR U snRNA NUCLEAR IMPORT IS NOT GENERAL The requirement of the trimethyl cap structure for Ul snRNP import is not general for all spliceosomal U snRNPs or for all cell types. In Xenopus oocytes, U2 snRNP behaves like Ul and its transport can be inhibited by the dinucleotide m^'^'^GpppG, but U4 and U5 are not as sensitive as Ul or U2 snRNPs to the presence of the trimethylated dinucleotide (Fischer et al, 1991). Furthermore, unlike Ul and U2, whose nuclear import strictly depends on their having a trimethyl cap, U4 and U5 have a much less stringent requirement and are transported into the nucleus, although with slower kinetics, even when the nonphysiological ApppG cap structure is present at their 5' termini. Thus, although the trimethyl cap structure is not essential for nuclear uptake in the case of U4 and U5 snRNPs, its presence does affect the rate of import (Fischer etal., 1991). This observation suggests that the cap functions as an accessory signal to increase the efficiency of nuclear import. In the absence of the cap, the nuclear localization signal present on the Sm core domain would be sufficient to allow nuclear accumulation of the U5 snRNP, but not of the Ul or U2 snRNPs. The possibility that the different core domains are not functionally equivalent can be excluded by the observation that U5
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snRNP transport can be inhibited by an excess of Ul snRNP core particles and vice versa, suggesting that their transport is mediated by a common receptor that recognizes similar features on the Sm core domain of both snRNPs (Fischer et al., 1993). The reason for the differential requirement for the cap structure is not yet completely understood. Possible clues were provided by the finding that a minimal Ul snRNP particle reconstituted in vitro on an RNA lacking the 5'-terminal stem loops of Ul RNA (stem loops I to III) was targeted to the nucleus, in spite of the absence of a trimethyl cap (Fischer et al., 1993), and that precise deletion of stem loops I and II from Ul snRNA resulted in an snRNP whose nuclear transport was trimethyl cap independent (Jarmolowski and Mattaj, 1993). Thus, in some ill-defined way, the structure of the 5^ region of Ul snRNA is responsible for the trimethyl cap requirement of U1 snRNA transport. It is not obvious how to interpret these observations in mechanistic terms. The absolute requirement for a trimethyl cap structure for Ul and U2 in the oocyte system is not maintained in somatic cells. This observation was made by microinjection of either Ul snRNAs or in vitro assembled Ul snRNP particles into the cytoplasm of Vero cells (African green monkey kidney cells) and mouse fibroblast 3T3 cells (Fischer et al., 1994). In these somatic cells a Ul particle with an ApppG cap instead of the trimethyl cap was imported into the nucleus. The ApppG-capped snRNP was accumulated in the nucleus at a reduced rate, however, indicating that the trimethyl cap, although dispensable, retained a signaling role for nuclear targeting of Ul snRNP. The finding that Ul snRNP particles were transported in a very similar way in the two somatic cell types tested suggested that this behavior may apply generally for all somatic cells, irrespective of their origin. It will be necessary, however, to test Xenopus somatic cells before accepting the conclusion that the difference is between somatic and germ cells rather than between species. Thus, in somatic cells the cap appears to function as an accessory signal to increase the efficiency of Ul snRNP nuclear import, as was the case for U4 and U5 snRNPs in the oocyte system. Perhaps the most remarkable result obtained so far in this context is the observation that, in vitro, somatic cell extracts confer trimethyl cap independence on Ul snRNP accumulation in the nucleus, whereas oocyte extracts confer cap dependence (Marshallsay and Liihrmann, 1994). Thus, the difference between the cell types reflects functional differences in cytoplasmic factors required for snRNP nuclear transport (see below).
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XIII. A UNIFYING MODEL FOR THE ROLE OF THE TRIMETHYL CAP AND THE CORE DOMAIN IN SIGNALING U snRNP NUCLEAR IMPORT Since the Sm core domain is both necessary and, for some U snRNPs or in certain cell types, sufficient to direct U snRNP particles into the nucleus, it is likely that an absolutely essential snRNP NLS is located on the core domain and is created by the interaction of the Sm proteins with the U snRNA. Several possibilities can be envisaged; for example, a nuclear localization signal may preexist in the core proteins and become exposed upon binding to the RNA. It is also possible that binding of the core proteins may induce a conformational change in the RNA and that some features on the RNA rather than the proteins are recognized by factors that mediate nuclear import. These two possibilities are not mutually exclusive, and the nuclear localization signal may be generated by the contribution of the two constituents of the core snRNP, the core proteins, and the U snRNA. The trimethyl cap may act as an accessory signal to increase the efficiency of nuclear transport. In oocytes, transport of Ul snRNP is much less rapid than in somatic cells, and therefore the role of the cap may become essential because of a simple difference in transport efficiency. In somatic cells, the role of the cap would be limited to an accessory function since transport is more efficient than in oocytes and therefore, even a suboptimal NLS may be recognized by the transport receptor. The higher efficiency of the transport apparatus in somatic cells versus oocytes could be due to a stronger interaction of the receptor with the Sm core domain or, alternatively, be a consequence of higher receptor concentration in these cells or the presence of different forms of, or perhaps even entirely different, receptors in the two cell types. The observation that the concentration of Ul snRNP required to saturate its own transport in somatic cells is 5-10-fold higher than in the oocytes supports the possibility that effective receptor concentration is higher in somatic cells (Fischer et al., 1994). Note that it is not yet clear for either cell type whether there are two distinct snRNP receptors or one unique factor that recognizes simultaneously both parts of the bipartite snRNP NLS. In mechanistic terms the trimethyl cap may increase the affinity of the receptor recognizing the Sm domain by interacting directly with this receptor, with an additional factor, or directly with one or several of the Sm proteins bound to the U snRNA to influence the formation of the core NLS. Clearly, a major goal for future studies will be the identification of
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the receptor system that recognizes the bipartite nuclear targeting signal and investigation of the nature of this recognition.
XIV. NUCLEAR IMPORT OF U6 snRNP AFTER MITOSIS The U snRNP import mechanism described above is Ukely to be valid for the reimport of the particles into the nucleus after mitosis as well as during interphase. In the case of U6 snRNP, which does not have a transient cytoplasmic phase, a nuclear import signal was nevertheless shown to exist, presumably to ensure the reaccumulation of the free U6 snRNP in the nucleus after mitosis. The import of U6 snRNP particles has been investigated by microinjection into the oocyte cytoplasm of in vitro transcribed U6 RNA (Hamm and Mattaj, 1989). A region of U6 snRNA (nucleotides 20-24) required for nuclear migration has been proposed to be the binding site of a U6 protein. This protein has not yet been identified. However, the observation that the nuclear targeting signal of U6 particles appears to be similar in nature to the NLSs of karyophilic proteins leads to the suggestion that U6 RNA may be directed into the nucleus via a NLS carried by this protein. The role of the 5' terminus of U6 RNA in signaling nuclear import has also been addressed. Nuclear accumulation of U6 transcripts does not require the presence of a y-methyl triphosphate at the 5' end of the U6 RNA. U6 RNAs having several different cap structures accumulate in the nucleus, the single exception being U6 transcripts with a monomethyl cap structure (m'^GpppG), which are retained in the cytoplasm. Cytoplasmic retention of these monomethyl-capped U6 RNAs could be overcome by coinjection of the dinucleotide m^GpppG, suggesting that a cap binding activity was responsible for anchorage of the RNA in the cytoplasm (Fischer et al., 1991).
XV. THE ROLE OF CAP STRUCTURES IN SIGNALING RNA SUBCELLULAR LOCALIZATION The observation that a U6 snRNA with a m^GpppG cap structure is retained in the cytoplasm, and that this retention can be relieved by coinjection of m^GpppG dinucleotide (Fischer et al., 1991), points to the existence of several different roles for cap structures in targeting RNAs to different subcellular compartments. The m^GpppN cap structure seems to be part of the RNA export signal in the nucleus, whereas in the cytoplasm, it promotes cytoplasmic retention. To allow U snRNPs to
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migrate efficiently back to the nucleus this structure has to be modified by the hypermethylation described for the pol II U snRNPs. Indeed, cytoplasmically microinjected U6 RNA with a trimethyl cap is imported into the nucleus. In this case the trimethyl cap is not a positive signal for import, since this process is insensitive to the presence of a large excess of the dinucleotide m^'^'''GpppG. This finding demonstrates that hypermethylation reverses the anchorage in the cytoplasm caused by the monomethyl cap. Therefore hypermethylated caps can act in some cases as an essential part of the nuclear import signal (e.g., in Ul and U2 import in oocytes) and influence the rate of import in other snRNPs and cell types, but more generally, hypermethylation may prevent interaction with cytoplasmic cap binding proteins to overcome the resulting anchorage of the RNA in the cytoplasm.
XVI. INHIBITORS OF NUCLEAR TRANSPORT In this section we will focus on the uses of transport inhibitors to elucidate aspects of nuclear transport. Generally, inhibitors that reduce the function of the nuclear pore complex inhibit the rate of all mediated import and export processes. The passive diffusion of molecules through the 10-nm aqueous channels is affected by only a single inhibitory antibody (see below). In contrast, reagents that act on targeting steps are usually more selective and may only inhibit the transport of one kind of substrate. We will deal first with general inhibitors of nuclear transport. The depletion of a cell's energy charge with metabolic inhibitors such as azide and 6-deoxyglucose arrests protein import at a stage after targeting. Thus, fluorescence-labeled substrates accumulate at the external surface of the nuclear envelope of cells depleted of ATP. Where studied, the export of macromolecules, such as ribosomal subunits or mRNA, is inhibited by ATP depletion (Battaille et al., 1990; Dargemont and Kiihn, 1992). Studies such as these have led researchers to conclude that bidirectional translocation through the nuclear pore complex requires phosphate bond energy (Forbes, 1992). The energy-requiring step(s) in translocation have not, however, been identified. Translocation, but not targeting, is also inhibited by chilling (Richardson et al., 1988). Sensitivity to chilling does not necessarily indicate that transport is blocked at an ATP-requiring step. Most enzyme-catalyzed reactions are slowed when incubated on ice, and the nuclear pore complex can certainly be viewed as an enzyme catalyst. Rather, chilling sensitivity indicates that the translocation reactions have a significant positive
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energy of activation. Targeting stages, because they are insensitive to both ATP depletion and chilling, apparently involve only simple diffusion and binding reactions. The role of phosphate bond energy in nuclear transport has recently become complicated by observations in cell-free nuclear transport assays. The addition of nonhydrolyzable GTP analogs inhibited targeting and GDP analogs inhibited translocation (Moore and Blobel, 1993; Melchior et al., 1993). These effects have been attributed to the RanyTC4 GTPase (Goldfarb, 1994, and references therein) and raise the possibility that GTP hydrolysis instead of, or in addition to, ATP hydrolysis is required for transport. Competitive inhibition of transport can be achieved by injecting high concentrations of import or export substrates that titrate or saturate the transport apparatus. A distinguishing feature of competition kinetics is that under conditions of saturation, the transport apparatus runs at maximum velocity. Under saturating conditions of maximum transport rate, the mole percentage of available substrate that is transported per unit time is decreased, a condition that provides for competition among substrates. Saturation kinetics has been shown for tRNA (Zasloff, 1983), U snRNA (Jarmolowski et al., 1994), mRNA (Dargemont and Kiihn, 1992; Jarmolowski et al., 1994) and ribosome export (Bataille et al, 1990), and protein and U snRNP import (Goldfarb et al., 1986; Fischer et al., 1993). Saturation kinetics is a classical characteristic of receptormediated processes. For protein and U snRNP import, saturation appears to occur during an early targeting step, presumably at the level of NLS recognition in the cytoplasm. The use of competition kinetics to explore U snRNP targeting is described below. Two related classes of inhibitors, lectins and anti-nucleoporin antibodies, bind directly to pore complex nucleoporins. Wheat germ agglutinin (WGA) and related lectins bind to a large number nucleoporins that are modified in the cytoplasm with O-linked N-acetylglucosamine (OGlcNAc) (Forbes, 1992). Immunogold labeling studies showed that WGA, which inhibits both import and export, binds to both sides of the nuclear pore complex (Starr and Hanover, 1992). The specificity of WGA action is uncertain because many other intracellular proteins in addition to the GlcNAc-containing nucleoporins are also modified with GlcNAc residues and their binding by WGA could cause pleiotropic effects. This problem is partially abrogated by the fact that WGA, a dimer, has significantly higher avidity for clustered GlcNAc residues such as those found at the pore complex than for the isolated GlcNAc residues found on most other proteins.
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The inhibitory anti-nucleoporin monoclonal antibodies bind to groups of nucleoporins that contain common epitopes (Featherstone et al., 1988; Forbes, 1992; Starr and Hanover, 1992). These nucleoporin subgroups represent subsets of those bound by WGA, which is more general, so it is likely that the two inhibitors work by the same mechanism. However, not all anti-nucleoporin antibodies inhibit nuclear transport. The mechanism of action of WGA and anti-nucleoporin antibodies may be due to steric effects. Steme-Marr et al. (1992) demonstrated that WGA interferes with the binding of uncharacterized transport factors to the pore complex. Also, because WGA and anti-nucleoporin antibodies are bivalent, it was suggested that they inhibit transport by cross-linking adjacent pore complex subunits and mechanically restricting their activity (Akey and Goldfarb, 1989). Neither WGA nor anti-nucleoporin antibodies inhibit the passive diffusion of small microinjected proteins or dextrans. Thus these reagents do not occlude the 10-nm diffusion channels. Second-generation anti-nucleoporin antibodies, with specificities for single nucleoporins, have been developed and should serve as more specific probes into pore complex function (Cordes et al, 1993; Wilken et al., 1993). Interestingly, some karyophiles are significantly less sensitive to WGA inhibition than others. The import of U6 snRNA, for example, is several times more sensitive to WGA than is Ul snRNAimport (Michaud and Goldfarb, 1992). Variation in sensitivity to WGA might reflect the use of different transport factors or disparate pore complex docking sites. Alternatively, the differences may be due to steric factors and/or biochemical characteristics of the karyophiles such as ionic charge or hydrophobicity. A unique inhibitor of nuclear transport is a monoclonal IgG directed against a linear peptide epitope of gp210, an integral membrane protein that is thought to function as a membrane anchor for the annulus of the pore complex (Greber et al., 1990). When mRNA encoding anti-gp210 IgG was microinjected into tissue culture cells, the mRNA was translated and the nascent IgG chains secreted into the lumen of the ER, where they bound gp210 and inhibited transport (Greber and Gerace, 1992). The fact that an antibody could inhibit nuclear transport by binding to the lumenal portion of a protein, situated across the nuclear membrane and at the extreme periphery of the pore complex, suggests that the entire 125-MDa pore complex structure is conformationally active during transport. The anti-gp210 monoclonal is unique in that it inhibits both NLS-mediated transport and passive diffusion through the 10-nm diffusion channels.
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XVII. KINETIC EVIDENCE FOR MULTIPLE NUCLEAR TARGETING PATHWAYS IN U snRNP IMPORT Most nuclear proteins contain amino acid sequences that are at least vaguely related to the archetypal SV40 large T-antigen NLS (PKKKRKV) or the nucleoplasmin NLS, which comprises two domains of basic amino acids separated by a spacer of 10 amino acids (KRPAATKKAGQAKKKK)(Garcia-Bustosetal., 1991;Forbes, 1992). The single basic domain T-antigen NLS will target proteins to nuclei of yeast (Silver et al., 1989) and Tetrahymena (White et al., 1989), demonstrating that fundamental aspects of nuclear targeting are conserved among eukaryotes. Robbins et al. (1991) suggested that the T-antigen NLS may be an extreme single-ended variant of the bipartite nucleoplasmin NLS, and it is likely that the double basic domain NLS motif is most common. The import of synthetic peptide NLS-serum albumin conjugates, such as P(Lys)-BSA, which is based on the SV40 T-antigen NLS, was shown to be kinetically saturable in Xenopus oocytes (Goldfarb et al., 1986). The microinjection of saturating concentrations of P(Lys)-BSA was later shown to compete with the import of most nuclear proteins, indicating that each of these proteins is imported by the same targeting apparatus, regardless of whether they carry single or double basic domain NLSs (Michaud and Goldfarb, 1993). Thus, even though the amino acid sequences of basic domain NLSs vary considerably, sequence differences probably reflect only quantitative variation in NLS "strength" rather than differences in the targeting apparatuses that mediate their import. Although these studies are consistent with a single predominant targeting apparatus, they could not rule out the existence of multiple NLS receptors having relatively broad overlapping sequence specificities for different NLS subfamilies. Early competition kinetic experiments between basic domain NLScontaining proteins and U snRNPs indicated that these two classes of karyophiles use distinct targeting apparatuses (Michaud and Goldfarb, 1991). Here, the import of U2 snRNP was unaffected by saturating concentrations of P(Lys)-BSA. The converse competition experiment, using free m^'^'^GpppG cap dinucleotide, showed that the saturation of cap-dependent import had no effect on basic domain NLS import (Fischer et al., 1991; Michaud and Goldfarb, 1992). Together, these studies provided the kinetic basis for the idea that the m^'^'^GpppN cap directed U snRNPs along a distinct targeting pathway. One important interpreta-
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tion of these data is the conclusion that the saturable steps in import lay upstream of the nuclear pore within the targeting pathways (Goldfarb and Michaud, 1991). The idea that the m^'^'^GpppN cap was a novel signaUng moiety was directly supported by experiments demonstrating that the import of Ul snRNP into oocyte nuclei depended on the m^'^'^GpppN cap, as discussed in preceding sections. More recent results, also described above, indicated that the m^'^'^GpppN cap is not always essential for import in somatic cells or in oocytes. However, whereas the nuclear import of Ul-type snRNPs in somatic cells is less stringently dependent on the m^'^'^GpppN cap than in oocytes, it still appears to follow a pathway that is independent of the basic domain NLS targeting pathway. This conclusion came from studying import in tissue culture cells that expressed a cytoplasmically anchored mutant SV40 T-antigen (FS T antigen) (van Zee et al., 1993). FS T antigen is membrane-bound and appears to inhibit the nuclear import of basic domain NLS-containing karyophiles by sequestering targeting factors that bind to its NLS. High levels of cytoplasmic FS T antigen interfered with the nuclear import of basic domain NLS-containing proteins, but not with the import of Ul snRNP. It will be interesting to learn if the import of a microinjected ApppG-capped Ul snRNA, which is imported slowly in tissue culture cells, is sensitive to a FS T antigen import block. A key to resolving the role of 5' cap structures in U snRNP import will be the biochemical identification and functional characterization of the m^'^'^GpppN cap binding protein.
XVIII. MULTIPLE U snRNP IMPORT PATHWAYS Complexity in U snRNP targeting pathways was discovered when competition analyses were extended to U snRNPs that differed from the Ul-type U snRNPs in their biochemical composition and cap structure. In oocytes, the import of Ul, U2, U4, and U5 snRNPs (Ul-type snRNPs) was distinguished from the import of basic domain-containing proteins by the competitive effects of free m^'^'^GpppG cap dinucleotide and P(Lys)-BSA. U6 snRNP showed the converse behavior, being saturable by P(Lys)-BSA but not by m^'^'^GpppG, much like NLS-containing proteins. These results suggest that U6 snRNA import is mediated by a NLS-containing U6 snRNA-binding protein. Because U6 snRNP is assembled in the nucleus and is present in the cytoplasm only during mitosis (or following microinjection), the cell's predominant protein import pathway appears to serve the trafficking needs of U6 snRNP.
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U3 snoRNP contains a 3mGpppG cap but is not complexed by the Sm proteins. In oocytes U3 snoRNP is imported by a cap-independent pathway (Baserga et al., 1992), and the import of SmGpppG-capped U3 snRNA is not competed with by cap dinucleotide. The cap independence of U3 snRNA import may be related to the fact that U3 snoRNA does not bind Sm proteins, which are essential for the import of Ul-type U snRNPs. Thus the unique import properties of U3 snoRNA reinforce the conclusion that both the cap and Sm proteins play physiological roles in Ul-type snRNP import. The import of U3 snoRNP is also not competed with by saturating concentrations of P(Lys)-BSA, suggesting that it is not imported by the basic domain NLS pathway used by U6 snRNP (Michaud and Goldfarb, 1992). These results suggest that U3 snoRNP may be imported by a specialized targeting apparatus, although it is unclear why this would be necessary. In conclusion, kinetic competition studies suggest that U snRNPs may be imported along three distinct import pathways, depending on the cap structure of the snRNA and the proteins that comprise the karyophilic U snRNP. These studies, and those of van Zee et al. (1993), predict that the import of Ul-type snRNPs will be mediated by transport factors that are distinct from those used to import NLS-containing proteins. The test of this hypothesis will probably come from in vitro fractionation and reconstitution studies of U snRNP import (Marshallsay and Liihrmann, 1994). This powerful approach will allow the identification of cytoplasmic components that direct the nuclear transport of U snRNPs. Cell extract-dependent in vitro transport systems should also lead to the elucidation of the exact roles of the 3mGpppG cap structure and Sm proteins in Ul-type snRNP import. REFERENCES Adam, S.A. & Gerace, L. (1991). Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell 66, 837-847. Adam, S.A., Lobl, T.L., Mitchell, M.A., & Gerace, L. (1989). Identification of specific binding proteins for a nuclear location sequence. Nature 337, 276-279. Adam, S.A., Steme-Marr, R.E., & Gerace, L. (1990). Nuclear protein import in permeabihzed mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. I l l , 807-816. Akey, C. & Goldfarb, D.S. (1989). Nuclear import through the nuclear pore complex is a multi-step process. J. Cell Biol. 109, 971-982. Akey, C.W. & Radermacher, M. (1993). Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy. J. Cell Biol. 122, 1-19.
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Andersen, J. & Zieve, G.W. (1991). Assembly and intracellular transport of snRNP particles. Bioessays 13, 57-64. Baserga S.J., Gilmore-Hebert M., & Yang E.W. (1992). Distinct molecular signals for nuclear import of the nucleolar snRNA, U3. Genes Dev. 6,1120-1130. Bataille, N., Helser, T, & Fried, H.M. (1990). Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus laevis oocyte nucleus: a generalized, facilitated process. J. Cell Biol. HI, 1571-1582. Borer, R.A., Lehner, C.F., Eppenberger, H.M., & Nigg, E.A. (1989). Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell 56, 379-390. Branlant, C , Krol, A., Ebel, J.R, Lazar, E., Haendler, B., & Jacob, M. (1982). U2 RNA shares a structural domain with Ul, U4 and U5 RNAs. EMBO J. 1, 1259-1265. Breeuwer, M. & Goldfarb, D.S. (1990). Facilitated nuclear transport of histone HI and other nucleophilic proteins. Cell 60, 999-1008. Bukrinsky, M., Haggerty, S., Dempsey, M., Sharova, N., Adzhubei, A., Spitz, L., Lewis, P., Goldfarb, D., Emerman, M., & Stevenson, M. (1993a). A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 365,666-669. Bukrinsky, M.I., Sharova, N., McDonald, T.L. Pushkaraskaya, T. Tarpley, W.G., and Stevenson, M. (1993b) Association of HIV-1 integrase, matrix, and reverse transcriptase antigens with viral nucleic acids following acute infection. Proc. Natl. Acad. Sci. USA 90, 6125-6129. Carberry, S.E., Darzynkiewicz, E., Stepinski, J., Tahara, S.M., Rhoads, R.E., & Goss, D.J. (1990). A spectroscopic study of the binding of N-7-substituted cap analogs to human protein synthesis initiation factor 4E. Biochemistry 29, 3337-3341. Cordes, V.C, Reidenbach, S., Kohler, A., Sniurman, N., van Driel, R., & Franke, W.W. (1993). Intranuclear filaments containing a nuclear pore complex protein. J. Cell Biol. 123,1333-1344. Dargemont, C. & Kuhn, L.C. (1992). Export of mRNA from microinjected nuclei of Xenopus leavis oocytes. J. Cell Biol. 118, 1-9. Darzynkiewicz, E., Stepinski, J., Ekiel, I., Goyer, C , Sonenberg, N., Temeriusz, A., Jin, Y, Sijuwade, T., Haber, D., & Tahara, S. M. (1989). Inhibition of eukaryotic translation by nucleoside 5'-monophosphate analogues of mRNA 5'-Cap: changes in N7 substituent affect analogue activity. Biochemistry 28, 4771-4778. De Robertis, E.M., Lienhard, S., & Parisot, R.F (1982). Intracellular transport of microinjected 5S and small nuclear RNAs. Nature 295, 572-577. Dingwall, C , Shamick, S.V., & Lasky, R.A. (1982). A polypeptide domain that specifies migration of nucleoplasmin into the nucleus. Cell 30,449-453. Dworetzky, S.I., Lanford, R.E., & Feldherr, CM. (1988). The effect of variations in the number and sequence of targeting signals on nuclear uptake. J. Cell Biol. 107, 1279-1288. Eliceiri, G.L. (1974). Short-lived, small RNAs in the cytoplasm of HeLa cells. Cell 3, 11-14. Featherstone, C, Darby, M.K., & Gerace, L. (1988). A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo. J. Cell Biol. 107, 1289-1297.
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Feeney, R.J., Sauterer, R.A., Feeney, J.L., & Zieve, G.W. (1989). Cytoplasmic assembly and nuclear accumulation of mature small nuclear ribonucleo-protein particles. J. Biol. Chem. 264, 5776-5783. Feldherr, CM. & Akin, D. (1990). The permeability of the nuclear envelope in dividing and nondividing cell cultures. J. Cell Biol. I l l , 1-8. Feldherr, CM. & Akin, D. (1991). Signal-mediated nuclear transport in proliferating and growth-arrested BALB/c 3T3 cells. J. Cell Biol. 115, 933-939. Feldherr, CM. & Akin, D. (1993). Regulation of nuclear transport in proliferating and quiescent cells. Exp. Cell Res. 205, 179-186. Fischer, U. & Ltihrmann, R. (1990). An essential signaling role for the m3G cap in the transport of Ul snRNPto the nucleus. Science 249, 786-790. Fisher, D.E., Conner, G.E., Reeves, W.H., Wisniewolski, R., & Blobel, G. (1985). Small nuclear ribonucleoprotein particle assembly in vivo: demonstration of a 6S RNAfree core precursor and posttranslational modification. Cell 42, 751-758. Fischer, U., Darzynkiewicz, E., Tahara, S.M., Dathan, N.A., Liihrmann, R., & Mattaj, I.W. (1991). Diversity in the signals required for nuclear accumulation of U snRNPs and variety in the pathways of nuclear transport. J. Cell Biol. 113, 705-714. Fischer, U., Sumpter, V., Sekine, M., Satoh, T, & Luhrmann, R. (1993). Nucleo-cytoplasmic transport of U snRNPs: definition of a nuclear location signal in the Sm core domain that binds a transport receptor independently of the m3G cap. EMBO J. 12, 573-583. Fischer, U., Heinrich, J., van Zee, K., Fanning, E., & Luhrmann, R. (1994). Nuclear transport of Ul snRNP in somatic cells: differences in signal requirement compared with Xenopus laevis oocytes. J. Cell Biol. 125, 971-980. Forbes, D.J. (1992). Structure and function of the nuclear pore. Annu. Rev. Cell Biol. 8, 495-528. Garcia-Bustos, J., Heitman, J., & Hall, M.N. (1991). Nuclear protein localization. Biochim. Biophys. Acta 1071, 83-101. Goldfarb, D.S. (1991). Shuttling proteins go both ways. Curr. Opin. Biol. 1, 212-214. Goldfarb, D.S. (1992). Are the cytosolic components of the nuclear, ER, and mitochondrial import apparatus functionally related? Cell 70, 185-188. Goldfarb, D. (1994). A GTPase cycle for nuclear transport. Curr. Biol. 4,57-60. Goldfarb, D. & Michaud, N. (1991). Pathways for the nuclear transport of proteins and RNAs. Trends Cell Biol. 1,20-24. Goldfarb, D.S., Gariepi, J., Schoohiik, G., & Romberg, R.D. (1986). Synthetic peptides as nuclear localization signals. Nature 322, 641-644. Greber, U.F. & Gerace, L. (1992). Nuclear protein import is inhibited by an antibody to a lumenal epitope of a nuclear pore complex glycoprotein. J. Cell Biol. 116,15-30. Greber, U.F., Senior, A., & Gerace, L. (1990). A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide with a large lumenal domain and a small cytoplasmic tail. EMBO J. 9, 1495-1502. Greber, U.F, Willetts, M., Webster, R, & Helenius, A. (1993). Stepwise dismantling of adenovirus during entry into cells. Cell 75,477-486. Grimm, C , Stefanovic, B., & Schumperli, D. (1993). The low abundance of U7 snRNA is partly determined by its Sm binding site. EMBO J. 12, 1229-1238.
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Hackl, W., Fischer, U., & Liihrmann, R. (1994). A69-kD protein that associates reversibly with the Sm core domain of several spliceosomal snRNP species. J. Cell Biol. 124, 261-272. Hamm, J. & Mattaj, I.W. (1989). An abundant U6 snRNPfound in germ cells and embryos of Xenopus laevis. EMBO J. 8,4179-4187. Hamm, J. & Mattaj, I.W. (1990). Monomethylated cap structures facilitate RNA export from the nucleus. Cell 63,109-118. Hamm, J., Kazmaier, M., & Mattaj, I.W. (1987). In vitro assembly of Ul snRNPs. EMBO J. 6, 3479-3485. Hamm, J., Darzynkiewicz, E., Tahara, S.M., & Mattaj, I.W. (1990). The trimethylguanosine cap structure of Ul snRNA is a component of a bipartite nuclear targeting signal. Cell 62, 569-577. Hernandez, N. & Weiner, A.M. (1986). Formation of the 3'-end of Ul snRNA requires compatible snRNA promoter elements. Cell 47, 249-258. Hinshaw, J.E., Carragher, B.O., & Milligan, R.A. (1992). Architecture and design of the nuclear pore complex. Cell 69, 1133-1141. Imamoto, N., Matsuoka, Y, Kurihara, T, Kohno, K., Mihagi, M., Sakiyama, F, Okada, Y, Tsunasawa, S., & Yoneda, Y (1992). Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins. J. Cell Biol. 119,1047-1062. Izaurralde, E., Stepinski, J., Darzynkiewicz, E., & Mattaj, I.W. (1992). A cap binding protein that may mediate nuclear export of RNA polymerase Il-transcribed RNAs. J.CellBiol. 118,1287-1295. Jacob, M., Lazar, E., Haendler, B., Gallinaro, H., Krol, A., & Branlant, C. (1984). A family of small nucleoplasmic RNAs with common structural features. Biol. Cell 51,1-10. Jantsch, M.F & Gall, J.G. (1992). Assembly and localization of the Ul-specific snRNP C protein in the amphibian oocyte. J. Cell Biol. 119,1037-1046. Jarmolowski, A. & Mattaj, I.W. (1993). The determinants for Sm protein binding to Xenopus Ul and U5 snRNAs are complex and non-identical. EMBO J. 12, 223-232. Jarmolowski, A., Boelens, W C , Izaurralde, E., & Mattaj, I.W (1994). Nuclear export of different classes of RNA is mediated by specific factors. J. Cell Biol. 124, 627-635. Kambach, C. & Mattaj, I.W (1992). Intracellular distribution of the Ul Aprotein depends on active transport and nuclear binding to Ul snRNA. J. Cell Biol. 118,11-21. Kambach, C. & Mattaj, I.W. (1994). Nuclear transport of the U2 snRNP-specific U2B" protein is mediated by both direct and indirect signalling mechanisms. J. Cell Sci. 107,1807-1816. Kastner, B., Bach, M., & Liihrmann R. (1990). Electron microscopy of small nuclear ribonucleoprotein (snRNP) particles U2 and U5: evidence for a common structure-determining principle in the major U snRNP family. Proc. Natl. Acad. Sci. USA 87, 1710-1714. Konings, D. A.M. & Mattaj, I.W (1987). Mutant U2 snRNAs of Xenopus which can form an altered higher order RNA structure are unable to enter the nucleus. Exp. Cell Res. 172,329-339.
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Lejbkowicz, R, Goyer, C , Darveau, A., Neron, S., Lemieux, R., & Sonenberg, N. (1992). A fraction of the mRNA 5' cap-binding protein, eukaryotic initiation factor 4E, localizes in the nucleus. Proc. Natl. Acad. Sci. USA 89, 9612-9616. Lemer, E.A., Lemer, M.R., Janeway, C.A., Jr., & Steitz, J.A. (1979). Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease. Proc. Natl. Acad. Sci. USA 78,2737-2741. Liihrmann, R., Kastner, B., & Bach, M. (1990). Structure of spliceosomal snRNPs and their role in pre-mRNA splicing. Biochim. Biophys. Acta 1087,265-292. Madore, S.L., Wieben, D.E., & Pederson, T. (1984). Intracellular site of Ul small nuclear RNA processing and ribonucleoprotein assembly. J. Cell Biol. 98, 188-192. Marshallsay, C. & Liihrmann, R. (1994). In vitro nuclear import of snRNPs: cytosolic factors mediate mjG-cap dependence of Ul and U2 snRNP transport. EMBO J. 13,222-231. Mattaj, I.W. (1986). Cap trimethylation of U snRNA is cytoplasmic and dependent in U snRNP protein binding. Cell, 46,905-911. Mattaj, I.W. & De Robertis, E.M. (1985). Nuclear segregation of U2 snRNA requires binding of specific snRNP proteins. Cell 40, 111-118. Mattaj, I.W., Boelens, W., Izaurralde, E., Jarmolowski, A., & Kambach, C. (1993). Nucleocytoplasmic transport and snRNP assembly. Mol. Biol. Rep. 18, 79-83. Melchior, P., Paschal, B., Evans, J., & Gerace, L. (1993). Inhibition of nuclear protein import by hydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. J. Cell Biol. 123,1649-1660. Michaud, N. & Goldfarb, D.S. (1991). Multiple pathways in nuclear transport: the import of U2 snRNP occurs by a novel kinetic pathway. J. Cell Biol. 112, 215-224. Michaud, N. & Goldfarb, D.S. (1992). Microinjected U snRNAs are imported to oocyte nuclei via the nuclear pore complex by three distinguishable targeting pathways. J. Cell Biol. 116,851-862. Michaud, N. & Goldfarb, D.S. (1993). Most nuclear proteins are imported by the same pathway. Exp. Cell Res. 208,128-136. Moore, M.S. & Blobel, G. (1993). The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors. Cell 69,939-950. Moore, M.S. & Blobel, G. (1992). The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature 365, 661-663. Moore, M.J., Query, C.C, & Sharp, PA. (1993). Splicing of precursors to messenger RNAs by the spliceosome. In: The RNA World (Gesteland, R., and Atkins, J.F., eds.), pp. 303-357. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Neuman de Vegvar, H.E. & Dahlberg, J.E. (1990). Nucleocytoplasmic transport and processing of small nuclear RNA precursors. Mol. Cell Biol. 10, 3365-3375. Newmeyer, D.D. & Wilson, K.L. (1991). Egg extracts for nuclear import and nuclear assembly reactions. Methods Cell Biol. 36, 599-626. Patzelt, E., Blaas, D., & Kuechler, E. (1983). CAP binding proteins associated with the nucleus. Nucleic Acids Res. 11,5821-5835. Pinol-Roma, S. & Dreyfuss, G. (1992). Shuttling of pre-mRN A binding proteins between the nucleus and cytoplasm. Nature 355, 730-732. Ohno, M., Kataoka, N., & Shimura, Y. (1990). A nuclear cap binding protein from HeLa cells. Nucleic Acids Res. 18,6989-6995.
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PHOSPHORYLATION-MEDIATED REGULATION OF SIGNAL-DEPENDENT NUCLEAR PROTEIN TRANSPORT: THE ''CcN MOTIF^^
David A. Jans
I. Introduction II. Nuclear-Cytoplasmic Processes in the Eukaryotic Cell A. The Nuclear Pore Complex as a Molecular Sieve B. Nuclear Localization Signals III. Methodological Considerations in Analyzing Nuclear Transport rV. Regulated (Conditional) Nuclear Entry V. SV40 Large Tumor Antigen and the CcN Motif VI. Mechanisms of Regulation of Signal-Dependent Nuclear Protein Transport A. Cytoplasmic Retention Factors
Membrane Protein IVansport Volume 2, pages 161-199 Copyright © 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-983-4
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B. NLS Masking C. The CcN Motif: Positive and Negative Regulation by Dual Phosphorylation VII. Variants of the CcN Motif: Other Kinases Able to Regulate NLS-Dependent Nuclear Protein Import A. Signal Transduction-Responsive NLSs B. Cell-Cycle-Dependent NLSs VIII. Conclusion and Future Prospects Acknowledgments References
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I. INTRODUCTION Since the sequence responsible for subcellular localization of the SV40 large tumor antigen (T-ag) was defined, research in the field of nuclear protein transport has largely revolved around the idea of a nuclear localization signal (NLS) being exclusively responsible for nuclear localization. NLSs have been defined for a multitude of nuclear proteins, and specific NLS-binding proteins or receptors have been identified. It has become increasingly clear, however, that NLS function can be regulated, with phosphorylation as the main mechanism controlling NLS-dependent nuclear localization of a number of proteins, including transcription factors (TFs) such as N F - K B , c-rel, v-jun, the a-interferonregulated factor ISGF-3, the yeast TF SWI5, and other nuclear proteins. Recent analysis indicates that even the archetypal NLS-containing T-ag is subject to regulated (conditional) nuclear localization. The regulatory module for T-ag nuclear transport (the "CcN motif) has been identified, in which the NLS ("N") determines ultimate subcellular destination, a casein kinase II site ("C") 15 amino acids N-terminal to the NLS determines the rate of nuclear import, and a cdk (cyclin-dependent kinase) site ("c") immediately adjacent to the NLS regulates the absolute amount of T-ag maximally accumulated in the nucleus. Since C and c sites can be identified in the vicinity of the NLS of a variety of other physiologically important proteins, it is likely that the CcN motif represents a conserved structural domain conferring conditional NLS-dependent transport on many TFs. Variants of the CcN motif include "SRNs" (signal transduction-responsive NLSs) and "CDNs" (cell-cycle-dependent NLSs), where kinases other than those acting at the CcN motif specifically regulate nuclear import of particular proteins through phosphorylation at site(s) close to their respective NLSs. Cytoplasmic reten-
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tion factors, intra- and intermolecular NLS masking, and direct NLS masking by phosphorylation are some of the better understood mechanisms by which phosphorylation specifically regulates nuclear transport. The regulation of nuclear transport through such mechanisms appears to be common to eukaryotic cells from yeast and plants to higher mammals. II. NUCLEAR-CYTOPLASMIC PROCESSES IN THE EUKARYOTIC CELL A. The Nuclear Pore Complex as a Molecular Sieve The fact that eukaryotic cells possess a nucleus has important implications for cellular function, since the existence of a nuclear compartment means that the genetic information, the DNA, is separated from the site of protein synthesis, the cytoplasm, by a double membrane structure, the nuclear envelope (Dingwall and Laskey, 1992). Gene transcription and translation accordingly take place in different subcellular compartments, meaning that specific transport events between the two are necessary for a eukaryotic cell to be functional: mRNA must make its way from the nucleus to the cytoplasm in order to be translated into protein; and proteins that are needed in the nucleus (structural nuclear proteins such as histones and lamins, the enzymes required for DNA replication, gene transcription, activation of gene expression, etc.) have to be transported from their site of synthesis in the cytoplasm into the nucleus. All transport goes through the nuclear envelope-localized nuclear pore complexes, very large (about 10^ kDa) almost organelle-like structures composed of at least 30 distinct protein components. Between 100 and 5 x 10^ pore complexes are present per nucleus, depending on the metabolic and differentiation state of the cell, with more complexes present in active or differentiating cells. As its name suggests, the nuclear pore complex has a pore or molecular sieve function whereby molecules smaller than 40-45 kDa can diffuse freely between cytoplasm and nucleus (Miller et al., 1991; Silver, 1991). B. Nuclear Localization Signals Proteins larger than 45 kDa require a nuclear localization signal (NLS) in order to be targeted to the nucleus.^ Through mutation/deletion analysis and/or their abihty to target a normally cytoplasmically localized protein to the nucleus, NLSs have been defined as the sequences neces-
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sary and sufficient for nuclear localization. They function through recognition/ligand-receptor-like interactions rather than through binding to chromatin or nuclear structures, etc. (see Miller et al., 1991; Silver, 1991). Proteins larger than 45 kDa microinjected into the nucleus remain nuclear whether they possess an NLS or not, demonstrating that NLSs do not function as nuclear retention signals (see Miller et al., 1991; Silver, 1991). The archetypal NLS is that of the SV40 large tumor antigen (T-ag) (Pro-Lys-Lys-Lys-Arg-Lys-Val^^^). It is both sufficient and necessary for nuclear localization of T-ag (Kalderon et al., 1984a,b; Lanford and Butel, 1984) and functional in targeting normally cytoplasmic carrier proteins to the nucleus whether present within the coding sequence of the carrier protein, or as a peptide covalently coupled to the carrier. The mutation of a single amino acid residue within this sequence (Lys^^^ to either Thr or Asn) abolishes its nuclear targeting function. The T-ag NLS, with its concentration of basic amino acids preceded by a P-tum, has served as the basis of the search via homology for NLSs in other nuclear proteins (Garcia-Bustos et al., 1991; Miller et al., 1991). Another class of NLS— the "bipartite NLS" with two clusters of basic amino acid residues separated by an intervening 10-12-amino acid spacer whose length appears to be critical—^has more recently been established as a variant of the T-ag NLS archetype (Dingwall and Laskey, 1991, 1992; Robbins et al., 1991); examples are those for the Saccharomyces cerevisiae transcription factor (TF) SWIS and the Xenopus laevis nuclear phosphoproteins nucleoplasmin and N1N2 (see Tables 1 and 2). A further class of NLS is based on the yeast MAT a2 NLS (Asn-Lys-Ile-Pro-Ile-LysAsp^ see Garcia-Bustos et al., 1991; Miller et al., 1991). NLSs have been hypothesized to be recognized by receptor/transporter proteins called NLS-binding proteins (NLSBPs), which may play a direct role in active nuclear transport. Although their precise specificity and subcellular location are still unresolved, NLSBPs have been identified by a number of research groups (Yoneda et al., 1988; Adam et al., 1989;Bendittetal., 1989; Li and Thomas, 1989; Meier and Blobel, 1990; Lee at al., 1991; Stochaj et al., 1991). Direct evidence has been provided that the molecular chaperonin and heat shock-related HSP70/HSC70 protein is both necessary for NLS-dependent nuclear transport in reconstituted systems and capable of specifically binding NLSs (Imamoto et al., 1992; Shi and Thomas, 1992).
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III. METHODOLOGICAL CONSIDERATIONS IN ANALYZING NUCLEAR TRANSPORT A drawback of most nuclear transport studies to date relating to the identification of NLSs is the nature of the experiments and techniques used, whereby eukaryotic cells are generally transfected with a proteinexpressing plasmid construct and then scored qualitatively for subcellular localization 24-^8 hours later using indirect immunofluorescence in fixed cells. Apart from difficulties in interpreting the results through the fact that fixation procedures may artifactually affect subcellular localization, such an approach provides minimal quantitative information with respect to how much protein is in the nucleus. It also provides no information with respect to the rate of nuclear import, since analysis is performed on cells at steady state, and the subcellular localization is clearly a function not only of intrinsic nuclear transport properties, but also of the transfection efficiency and the level of expression of the transfected DNA. In addition, the high levels of protein expression in the cells analyzed are often not physiological, especially in the case of TFs (e.g., N F - K B ) , which are normally present in very low amounts. Some of the above drawbacks can be avoided by examining the transport of carrier proteins such as bovine serum albumin carrying covalently attached NLS peptide microinjected into living cells, where information may be obtained on the initial rate of nuclear transport. However, the carrier protein constructs used to date have generally involved multiple NLS peptides, thus being far from physiologically relevant in terms of the ratio of NLS to molecular weight/size. This has been shown to be of significance in several studies (Nelson and Silver, 1989; Yoneda et al., 1992). Alternative methods such as subcellular fractionation can provide quantitative data if performed in the correct manner, but suffer from the caveat that redistribution of proteins may occur during the fractionation process. Technical advances in the application of confocal laser scanning microscopy (CLSM) have made the quantitative estimation of fluorescence possible, meaning that relative protein concentrations in different subcellular compartments can be measured in vivo (Peters, 1986; Jans, 1992). The use of microinjection of fluorescently labeled protein in conjunction with CLSM enables the quantitative analysis of nuclear import in single living cells (see Figure 1 A), and it has recently proved possible to resolve temporally the nuclear transport process and deter-
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NUCLEAR TRANSPORT IN VIVO IN MICROINJECTED HTC CELLS I Cells fused with PEG i to yield polykaryons
mm Cells grown in standard Wm medium/petri dish without antibiotics
&^ii»i&»@
CKII-SITE-DEPENDENT £ik2-K-INHIBITABLE NLS-DEPENDENT
CLSM
NUCLEAR T R A N S P O R T IN V I T R O IN M E C H A N I C A L L Y P E R F O R A T E D H T C CELLS Cells grown on washed Dincoverslips intracellular buffer
CKII-SITE-DEPENDENT £d£2-K-INHIBITABLE NLS-DEPENDENT
WMMMm
1m .4V. ^'^^ ~ Cytosol lAF-labelled Protein
^ Coverslip removed to leave behind apical cell membrane
Figure 1. Systems to measure the kinetics of signal-dependent nuclear protein transport in vivo (A) and in vitro (B), using quantitative confocal laser scanning microscopy.
mine transport kinetics (Rihs and Peters, 1989; Rihs et al., 1991; Jans et al, 1991; Jans and Jans, 1994; see Section V below). The application of in vitro or reconstituted nuclear import assay systems has enabled an initial assault on understanding the steps of
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Figure 1. (continued) (C) Signal dependence of nuclear transport in the respective systems for microinjected HTC cell polykaryons (37 °C, 15 and 40 minutes, respectively, for a T-ag(amino acids 111-135)-P-galactosidase(amino acids 9-1028) fusion protein (left) and a fluorescein-labeled 70-kDa dextran (right), respectively) (/), and for mechanically perforated HTC cells (23°C, 75 minutes for T-ag (amino acids 111-135)-|i-galactosidase (amino acids 9-1028) fusion proteins without (left) and with (right) a Thr^^^ mutation in the NLS rendering the NLS inactive) (2). Proteins were labeled with 5-iodo-acetamido-fluorescein (lAF), and HTC cells were fused with polyethylene glycol 1 hour before microinjection as described previously (Rihs and Peters, 1989). Microinjection was performed with a Narshige IM200 microinjectorand Zeiss micromanipulator, and images in the case of both assay systems were obtained with an MCR 600 Bio-Rad CLSM.
nuclear protein import, and defining them mechanistically with respect to NLS-binding proteins, including HSP70/HSC70 (Adam et al., 1989; Stochaj et al., 1991; Shi and Thomas, 1992).^ In conjunction with CLSM, in vitro nuclear transport assays can be applied in a quantitative approach to define the components and kinetics of the steps involved in regulating NLS-dependent nuclear transport. We have established an in vitro system based on cells of the HTC rat hepatoma cell line that have been mechanically perforated (see Figure IB) (Jans et al., 1991), using a modification of a procedure first used to investigate intracellular vesicle trafficking
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DAVID A. JANS
(Simons and Virta, 1987). Consistent with the findings from other mv/rro assay systems based on differential permeabilization of the plasma membrane and the nuclear envelope by the detergent digitonin (Adam et al., 1989; Stochaj et al., 1991; Shi and Thomas, 1992; Moore and Blobel, 1993; Adam and Adam, 1994), signal-dependent nuclear transport in mechanically perforated HTC cells can be shown to involve at least two stages (Jans et al., 1991), as first reported by Newmeyer and Forbes (1988). Both stages are dependent on cytosolic factors (unpublished results; Jans et al, 1991; see Figure IB): an ATP-independent docking or binding stage at the nuclear envelope/nuclear pore complex (see Figure 4), and an ATP-dependent active transport step. Signal (NLS) dependence of nuclear transport in vivo and in vitro is illustrated in Figure IC. Analysis of the rates and maximal extent of nuclear transport (see below) indicate quite clearly that nuclear transport is not an all-or-none phenomenon exclusively determined by the possession or absence of an NLS—^rather, as soon as kinetic measurements are quantitatively performed, it becomes clear that other sequences function in a regulatory fashion to modulate NLS-dependent nuclear transport in a very precise way.
IV. REGULATED (CONDITIONAL) NUCLEAR ENTRY Whereas some proteins such as histones and nucleolin appear to be constitutively targeted to the nucleus, others only seem to be targeted to the nucleus under specific conditions, often being mostly in the cytoplasm (see Miller et al., 1991; Schmitz et al., 1991). TFs regulating nuclear gene expression are not different from other proteins in terms of their being synthesized in the cytoplasm and therefore being subject to specific mechanisms regulating nuclear protein import. The advantages of a conditionally cytoplasmic location for a TF include the potential to control its nuclear activity through the regulation of its nuclear uptake, and its direct accessibility to cytoplasmic signal-transducing systems. The nuclear translocation of various TFs (Lenardo and Baltimore, 1989; Shirakawa and Mizel, 1989; Govind and Steward, 1991; Moll et al., 1991; Schmitz et al., 1991) and oncogene products (Gusse et al., 1989; Van Etten et al., 1989; Capobianco et al, 1990; Roux et al., 1990) has been shown to accompany changes in the differentiation or metabolic state of eukaryotic cells precisely, indicating that nuclear protein import can be a key control point in regulating gene expression and signal transduction.
Table I . Examples of Reaulated Nuclear Protein Transport Protein
Kinase/stimulus
Effect on Nuclear Transport (Sequences ~nvolved)~
A. Signal transduction-related T-ag CKll c-re1 PK-A NF-KB
Lamin B2 GR
c-jos Cofilin
rNFIL-6 ISGF-3
NF-AT
CKll site increases the rate of nuclear transport by -40-fold (a) (s"'s"~DDE-10 a.a. spacer-PKKKRKV). PK-A site enhances nuclear localization (b), whereby mutation of SZMabolishes nuclear localization ( R R P S ' ~ ~a.a. -~~ spacer-KAKROR). Phosphorylation (of 1KB or NF-KB subunits) results in unmasking of ~ 5 0 1 ~ NLSs 6 5 and NF-KB nuclear localization (c) IL-1cONFa E - ~spacer-ORKROK ~ human p65 R R P S ~ ~ ~ D R E L S a.a. E - I ~spacer(human p50/p110 R R K S ~ ~ ~ - D L E T Sa.a. PK-C (PK-A/elF2K) EEKRKR). PK-C-mediated phosphorylation inhibits nuclear transport (d) ( R S ~ ~ ~ S ~ " R G K R R R I E ) . PK-C Hormone binding by GR releases it from a cytoplasmic complex with HSP90 and results in its nuclear localization (e). GH Nuclear retention of GR is impaired during the G2 stage of the cell cycle, concomitant with altered phosphorylation PP-2A? of GR. The inhibition of phosphatase PP-2A by okadaic acid similarly leads to inefficient nuclear retention of GH-occupied GR, as does transformation by the v-mos oncogenic kinase, implying that site-specific (cell-cycledependent) dephosphorylationof GR is involved in hormonedependent nuclear translocation (f). (AconstitutiveNLS RKCLQAGMNLEARKTKK (amino acids 479-495 of the rat sequence) is repressed by a second ligand bindingdependent NLS function, which includes amino acids 600-626 and 696-777.) (g) Phosphorylation of c-jos reverses its binding to a putative inhibitor protein and retention in the cytoplasm (h) PK-Nsemm (KRRIRRIRNKMAAAKCRNRRRL-200 a.a. S ~ ~ C ~ ~ - R K G S ~ ~ ~ S S " ) . Heat shock induces unmasking of the cofilin NLS via dephosphorylation at a consensus multifunctional calmodulindeMultifunctional pendent protein kinase site, resulting in its nuclear localization (i) (RKSS~~TPEEKKRKKA). calmodulin dept. protein kinasemeat shock The adenylate c clase activator forskolin stimulatesphosphorylation of rNF1L-6 and its translocation to the nucleus cAMPIPK-A? Z CKPSKKPS' ??I. Interferon-induced tyrosine phosphorylation of the three ISGF3a subunits is required to effect their association and Interferon a/$ translocation to the nucleus (k).' tyrosine kinase ca2+-dependentactivation of calcineurin results in dephosphorylation of cytoplasmicNF-AT, and its nuclear localization ca2+ (1). Through different mechanisms, the immunosuppressants FKS06lcyclosporin inhibit the phosphatase and thereby FK506/cyclosporin induce nuclear translocation of NF-AT. (continued)
Table 1. Continued Prorein
d
U 0
Kinase/stimulus
Effect on Nuclear Transport (sequences ~nvolved)~
B. Cell cycledependent/developmental T-ag cdWcdc2-K cdc2-K-mediated phosphorylation reduces the maximal level of nuclear accumulation, probably through increasing the affinity of binding to a cytoplasmic retention factor (m) ( S T ~ ~ P K K K R K V ) . SWIS cdWCDC28 Phosphorylation-mediatednuclear exclusion through NLS masking--mutation of the CDC28-serines to alanine results 109 - a.a. s p a c e r - ~ ~ ~ ~ in corlstitutive nuclear localization (n) ( s ~ ~ ~ P s K RKDGTSSVSSS~PIK). lodestar CKII? Nuclear exclusion until prophase (DESS~%DS~~~DS%DKNKKRK) (0) v-jun cdk? Cell-cycledependent determination of the rate of nuclear localization conferred by Sa8 (faster during G2); c-jun possesses cZ4' and is constitutively nuclear ( A S ~ ~ ~ R K R(p). KL) dorsal toll Relocalization from the cytoplasm to the nucleus during developmentin a ventral-todorsal gradient, involving multiple PK-A/c~~+? components of the roll si al transduction pathway, including the cytoplasmic retention factor cacrus, and "releasing factors" pelle and rube.8" Phosphorylation of dorsal leads to release from cactus and nuclear localization, whereby activated roll receptor and increased cytoplasmicca2+ appear to be involved (q) ( R R P S ~ ' * a.a. - ~ ~spacer -RRKROR). AdenovirusS ?? Nuclear targeting up to the early neumla stage of Xenopus embryonic development through a "developmentally Ela protein regulated NLS (drNLS) (r) (~~NLs':amino acids 142-182:m- 20 a.a. spacer - MCSLCYMRTCGM!!, distinct from the constitutive C-terminal NLS: a.a. 285-289: KRPRP). RB-1 cdWcdc2-K Cellcyclede ndent hype~hospho~lation reduces nuclear association ("nuclear tethering") (s) (Putative CcN motif: human ADMYLS %v&' where P1 is a putative CKll site, and serines 608 and 612 cdk sites).'
STIR.
Notes:
a.a.. amino acids; U - 1 4 interleukin l a ; TNFa, tumor necrosis factor a ; eIF2K. heme regulated initiation factor 2 kinase; GR, glucocorticoid receptor; GH. glucocorticoid hormone; PP-2A, protein phosphatase type 2A; ISGF-3, interferon-stimulated gene factor; rNFlL-6, rat nuclear factor induced by IL-6;NF-AT,nuclear factor of activated T cells; RB-I. pl loRb,the product of the "retinoblastoma-susceptibility factor'' tumor suppressor gene. single letter amino acid code is used. NLSs are underlined, and phosphorylatedresidues are numbered accordingto their residue number in the respective protein. References are: (a) Ribs et al. (19911; Jans et al. (1991); Jans and Jans (1994); (b)Mosialos et al. (1991); (c) Shirakawa and Mizel(1989); Lenardo and Baltimore (1989); Ghosh and Baltimore (1990); Henkel et al. (1992); Zabel et al. (1993); Beg et al. (1992); (d) Hennekes et al. (1993); (e) Picard et al. (1988); (f) De Franco et al. (1991); Hsu et al. (1992); Qi et al. (1989); (g) Cadepond et al. (1992); (h) Roux et a]. (1990); Auwerx et al. (1990); Tratner et al. (1992); (i) Nishida et al. (1987); Ohta et al. (1989); (i)Metz and Ziff (1991); (k) Pellegrini and Schindler (1993); Schindleret al. (1992); (1) Liu (1993); (m)Jans et al. (1991); (n) Moll et al. (1991); (0) Girdham and Glover (1991); (p) Chida and Vogt (1992); (q) Govind and Steward (1991); Whalen and Steward (1993); Kubota et al. (1993); (r) Standiford and Richter (1992); (s) Templeton et al. (1991); Templeton (1992).
~
~
1
~
b~egulationof nuclear translocation of rNFIL-6 may be cell type specific, since CAMP-mediatedstimulation is required for its nuclear localization in pheochromocytomacells, whereas rNFIL-6 is constitutively nuclear in HeLa cells (Metz and Ziff, 1991). 'The 91-kDa ISGF-3a subunit is also a componentof the interferon y-induced GAF (gamma-activated factor) TF (Shuai eta]., 1992; see Pellegrini and Schindler, 1993).Tyrosine phosphorylation induces nuclear localization of the 91-kDa ISGF-3a subunit in this signaling pathway as well. %he C-terminus (LQISNISIST~'~) of dorsal is implicated in cytoplasmic retention, since its deletion results in nuclear localization of the dorsal gene product. Proteolysis of dorsal or cactus may also be involved. e~omologoussequences have been identified in the rat (amino acids 594-6231. mouse, and human GH binding domains of GR (see Standiford and Richter. 1992). which are known to include a part of the ligand binding-dependent NLS (Picard et a]., 1988; Cadepond et al., 1992). A ' 'bipartite" NLS purported to participate in nuclear localization of mouse RB has been identified: KRSAEFFNPPKPL='~~-~~ ,.a. S ~ ~ C ~ ~ - Swhere ~ ~ ~s~~~ I GisEa , putative CKII site) (Zacksenhaus et al., 1993).
1 72
DAVID A. JANS
TFs able to undergo inducible nuclear import include the glucocorticoid receptor (GR) (Picard et al., 1988), the a-interferon-regulated factor ISGF-3 (interferon-stimulated gene factor 3) (Schindler et al., 1992), the nuclear w-jun oncogenic counterpart of the AP-1 transcription complex member c-jun (Chida and Vogt, 1992), the yeast mating switch/HO endonuclease promoter regulator SWI5 (Moll et al., 1991), the Drosophila melanogaster morphogQu dorsal (Govind and Steward, 1991), and the nuclear factor N F - K B (Lenardo and Baltimore, 1989; Shirakawa and Mizel, 1989; Schmitzetal., 1991). A number of physiological examples of regulated NLS-dependent nuclear protein localization, where the signal and regulatory sequences responsible have been identified to some extent, are listed in Table 1. Examples of signal transduction pathways from extracellular hormonal signal to the nucleus ultimately leading to changes in the regulation of gene expression include the GR, where, upon glucocorticoid hormone (GH) binding, cytoplasmic receptor translocates to the nucleus to modulate gene transcription by direct binding to specific DNA sequences called GH response elements (GREs) (Picard et al., 1988); another example is the hormone-stimulated response to elevated cAMP levels. The latter results in translocation of the cAMP-dependent protein kinase (PK-A) catalytic (C-) subunit from the cytoplasm to the nucleus (Nigg et al., 1985; Meinkoth et al., 1990; Pearson et al., 1991), where it phosphorylates and thereby modulates the activities of nuclear TFs such as the cAMP response element (CRE) binding protein (CREB). In a fashion similar to that of PK-A, the MAP- (ERK) and RSK-kinases also enter the nucleus upon mitogenic stimulation and activation (Chen at al., 1992), whereas the 82-kDa a isoform of the Ca^Vphospholipid-dependent kinase (PK-C) relocalizes to the nuclear envelope upon activation (Leach et al., 1989).^ Hormonally induced nuclear transport of activated kinases is a commonly exploited means of communicating signals to the nucleus in many signal transduction and developmental pathways.
V. SV40 LARGE TUMOR ANTIGEN AND THE CcN MOTIF As stated in Section II.B, nuclear localization of T-ag is dependent on amino acids 126-132. Mutations within the T-ag NLS abolish nuclear targeting (Kalderon et al., 1984a,b; Lanford and Butel, 1984; see Figure IC). Using quantitative CLSM and the systems shown in Figure 1, A and B, to measure nuclear transport kinetics at the single cell level both in vivo and in vitro, we have demonstrated that NLS-dependent nuclear
Phosphorylation-Mediated Nuclear Transport
173
transport of T-ag is regulated by phosphorylation. Sites for the physiologically important casein kinase II (CKII) and the cdk (cyclin-dependent kinase) cdc2-kinase (crfc2-K), which are close to the T-ag NLS, regulate the kinetics of nuclear transport of T-ag, probably through the modulation of specific protein-protein binding affinities and recognition events (Rihs and Peters, 1989; Jans et al., 1991; Rihs et al., 1991; Jans and Jans, 1994). The CKII site (the serine at position 112) increases the rate of NLS-dependent nuclear import, so that maximal accumulation within the nucleus occurs within 20 minutes, contrasting with the 10 hours taken when CKII phosphorylation is prevented through deletion or mutation of the CKII site (Rihs et al., 1991; Jans and Jans, 1994) (see Figure 2A). In contrast to the enhancing effect of the CKII site, phosphorylation by cdc2'K (at threonine 124) inhibits nuclear transport, drastically reducing the level of maximal nuclear accumulation (Jans et al., 1991) (see Figure 2B). We have named this regulatory module for T-ag nuclear transport the "CcN motif," comprising CKII ("C") and cdk/cdc2K ("c") sites, and the NLS ("N") (Jans et al., 1991) (see Figure 2). Aspartic acid (see Figure 3) can substitute functionally for phospho-serine/threonine in the case of both phosphorylation sites of the T-ag CcN motif (Jans etal, 1991; Jans and Jans, 1994), implying that the phosphorylated sites probably represent recognition signals in themselves that are recognized by components of the cellular nuclear transport machinery. The existence of a complex regulatory system for S V40 T-ag nuclear localization, namely the CcN motif involving two different phosphorylation sites, demonstrates the existence of specific mechanisms regulating nuclear entry. Clearly, the NLS is not the sole determinant conferring nuclear localization in an all-or-none phenomenon; rather, the kinetics of NLS-dependent nuclear localization are quantitatively regulated by phosphorylation sites in the vicinity of the NLS. Through phosphorylation at the signal transduction-responsive CKE"^ site positively regulating the rate of nuclear transport, and phosphorylation at the cell cycle-dependent cdk/cc/c2-K site negatively regulating the maximal extent of accumulation, the level of T-ag present in the nucleus can be precisely regulated, presumably exactly as required with respect to the eukaryotic cell cycle and stages of the viral lytic cycle. Significantly, a variety of nuclear proteins in addition to T-ag possess putative CcN motifs (Jans et al., 1991), implying a general role for the CcN motif in regulating nuclear protein transport. Some of these are shown in Table 2; others include the Drosophila "Notch" group of genes.
The CKII Site Enhances the Rate of Nuclear Transport
Fn/c
CKII site intact
C cdc2-K-phosphorylation Inhibits Nuclear Transport
Unphovphorylated
cdc2-pho«phorylated
i^ -J
0
5
I
I
I
i
I
I
10
15
20
25
30
35
40
Time (min)
Nuclear Transport is completely dependent on the NLS
N 174
Phosphorylation-Mediated Nuclear Transport
175
including Notch itself and Enhancer of Split and human homologs (EUisen et al., 1991; Stifani et al., 1992; Fortini et al., 1993); the yeast TF SNF2 (Laurent et al, 1993) and human and Drosophila homologs (Chiba et al., 1994); the family of interferon a- or y-induced TFs, including IFi204 and IFil6 (Choubey and Lengyel, 1992; Trapani et al, 1992); the Swi4 family of mismatch repair enzymes (Fleck et al., 1992), bovine poly-A polymerase (Wahle et al., 1991), yeast DNA topoisomerase II (Shiozaki and Yanagida, 1992), the DNA repair helicase ERCC6 (Troelstra et al., 1992), and the protein tyrosine phosphatase PEP (Matthews et al., 1992). Putative CcN motifs can also be found in the sequences of proteins localizing in the plant cell nucleus (Klimczak et al., 1992; Raikhel, 1992; Tinland et al., 1992; Hicks and Raikhel, 1993) (see Table 2), underlining the universal nature of NLSs and nuclear protein transport across the animal/plant kingdom, as well as of the latter's regulation by phosphorylation. This universality is further supported by the functionality of the T-ag NLS in Drosophila embryos (Stein and Jans, unpublished observations, yeast, and plant cells, etc. (see Raikhel, 1992; Shiozaki and Yanagida, 1992); of the plant cell NLS of Agrobacterium tumefaciens VirD2 protein in yeast cells (Tinland et al., 1992); and of the cdk/CDC28kinase-regulated yeast NLS of SWI5 in rat and monkey cells (Jans et al., 1994). In the latter case, we have been able to show not only that the SWI5-NLS is functional in nuclear targeting, but also that the cdk
Figure 2. Regulation of nuclear protein transport by the CcN motif. Nuclear transport kinetics of fluorescently labeled T-ag (amino acids 111-135)-[J-galactosidase (amino acids 9-1028) fusion protein derivatives in microinjected HTC cells (see Figure 1 A) were measured by quantitative CLSM. Fn/c represents fluorescence quantitated in the nucleus relative to that in the cytoplasm (i.e., fold accumulation in the nucleus). Regulation/dependence of nuclear protein transport by/on the CcN motif component CKII site (A), cdcl-Y. site (B), and NLS (C) is shown. The CKII site mutant (substitution of Ser^^^^^^^ by Ala, and replacement of the CKII recognition site Asp-Asp-Glu by Asn-Asn-GIn) and deletion (of amino acids 111-119, including the CKII site) derivatives shown have been described previously (Jans and Jans, 1994; Rihsetal., 1991). /n wfro phosphorylation with purified HeLa cdc2-kinase(stoichiometry of phosphorylation atThr^^"* of 1.4 mol P/mol tetramer) was performed as described in Jans et al. (1991). Results identical to those shown are obtained in mechanically perforated HTC cells (not shown, Jans et al., 1991).
Negative charge at the CKII site increases the nuclear transport rate Fn/c
Negative charge at the cdc2-site reduces maximal nuclear accumulation 20 r
Fn/c WT {Thrl24)
12 h
10
15
20
25
40
Time (min)
Figure 3. Negative charge at the CcN motif phosphorylation sites is mechanistically important for the regulation of nuclear transport. Nuclear transport kinetics of fluorescently labeled T-ag (amino acids 111-135)-^galactosidase(amino acids 9-1028) fusion protein derivatives in microinjected HTC cells were measured as in Figure 2. Asp at position 112 (Gly at position 111) (above) increases the rate of nuclear transport (compare to transport in the absence of a CKII site, with Gly at position 112 (and Ala at position 111), similar to CKII phosphorylation at the site (Ser^^^) (Jans and Jans, 1994). Asp at position 124 (below) reduces the extent of maximal accumulation, thus simulating cdc2-k phosphorylation of Thr^^^ (Jans et al., 1991); compare to Figure 2B. Comparable results to those shown are obtained in mechanically perforated HTC cells (not shown; Jans et al., 1991). 176
Table 2. Potential and Confirmed CKll and cdk-Kinase Phosphorylation Sites in the Vicinity of the NLS of Various Nuclear Proteins proteina SV40 T-ag Polyoma T-ag
CKII Site($) (Signal Transduction)
cdk Sire($) (Cell Cycle)
S'"S"~DDE (a) SStS6l"TD ES~~'ENE
PPK (b) P R T ' PVS ~~ ATz7' PPK SS~"PQP (e) PST "'RKP TS~PRS SPS'~~PTS(i) APDT'~~PEL (k) P A V S ~ ~ ~ P(k) LL T4 7 4 ~ ~ ~ T4 7 9 ~ ~ ~ T4 4 0 ~ ~ ~ TS~~PVR
Human p53 Human c-myc Lamin N C Mouse c-abl N
D G S ~ ~ ~ L(m) ND Human B-myb Human c-myb
ax^ poly A polymerase (bovine) ERCC6 (human)
S4" FLD s5'7m
S~DNDDIEVES~~DEE (n) DS I4OSS1 4 2 ~ ~ 1 (n) 4 4 DS~~'SLD DDS~~~EESD
~ ~ ~ ~
T'~~s'~%EE S~~EDELEE~ S ' 7 ' ~ ~ ~ ~ S~~TKE E T ~ ~ K S E E T ' ~ ~(0) LDE S~~~K (0)G E
~
NU PKKKRKV'" (c) VSRKRPRP'" P P K K A R E D ~(d) ~~ PQPKKKP 319 (f) PAAKRVKLD"~(g) RQRRNELKRS'~~(~) S V T K K R K L E ~(j) '~ SALIKKKKKMAP~~' (1)
LKRQRKRR~'~ LKK~KQ~~~ KRAHHNALERKRR~~ Q S R K K L R M ' ~(n) ~ KRK-12 a.a. spacer-^^^^^^^^ RRWNKLRLQDKEKRLK~~' KRR- I I a.a. spacer-^^^^^'^^^^ RRRRRR~ KRKSMREEETGVKKSKAAK'94f KRKKSTKEKAGPKGSK'~~~ SKKKKNAK~~ (continued)
Table 2. (Continued) CKII Site(s) (Signal Tramduction)
cdk Site(s) (Cell Cycle)
S'~~PAD S ~ ~ ~ D Q D DSS~~~HYDS~"~DGD DVS~~~NED S~'~~PVD S'~~PAK
RTRS~~~PL
REKYRTR-I0 a.a. s p a c e r - ~ K R R KCsh~ ~ ~
PSS~~~PR
RGTDKRR-9 a.a.
proteina Drosophila gro TLE- I TAN-1' Plectrin (rat)
S'~~PTKK S'~~PPK S"~PLK
Yeast SW15 (S. cerevisiae)
s1362s 1
K R S ~ ~ ~ P(s) RK S S S ~ ~ P (s) IK 3
6
3
~
~
DPS 1 4 5 5 1458 ~ ~ ~ MDEPS'463~~~~
~
spacer-^^^^^^^'^'^
s ~ ~ ~ ~ P H KRAKDLKARRKK~'~~ S~'GSE SPAKKPKV~* (see p)
X. laevis Nucleoplasmin
DNA topoisomerase Il (S. pombe)
NLS
~
~
ST~~'~PK S'~"PIR
KRPAATKKAGQAKKKKL'~~ (q)
R K K R K - ~ ~ ~ . ~ . ~ ~ ~ C(r)~ ~ - K K S K Q E ~ ~ KKYENVVIKRSPRKRGRPRK~~~~ (s) KSRKR-12a.a. spacer-^^^^^^'^^^ RKRPTRR'~~'
Plant Opaque-2 (maize) G B F l (broccoli)
ES'~~NRE S ' ~ G N D A S ' ~ ~ H S(u) DE S'~~SDENDE
Agrobacterium rurnefaciens VirE2 protein VirD2 protein
S%TE DES~'QS'~DDD
(octopine)
EQDT"~~RDD
RAIKTKYGSDTEIKLKSK"~~ (v)
EYLSRKGKLEL~~ (w) S~PKR
K R P R D R H D G E L G G R K R A R ~ ~(x) ~~
Notes: a ~ h single e letter amino acid code is used. The consensus site for C m comprisesan acidic amino acid three residues C-terminal to the phosphorylatable SA', with an elevated number of acidic residues in the vicinity of the site increasing its affinity for CKlI (Krebs et al., 1988); cdks phosphorylate SA' residues N-terminal to proline residues, with a basic amino acid one or two residues C-terminal to the proline. References apply as indicated to confirmed NLS, and CKJI- and cdc2-K sites, with the phosphorylated/phosphorylatableSA' residues numbered: (a) Gfisser et al. (1988); (b)McVey at al. (1989); (c) Kalderon et at. (1984a,b);Lanford and Butel (1984); (d) Richardson et al. (1986); (e) Bischoff et al. (1990); (f) Chelsky et al. (1989); Addison et al. (1990); (g) Dang and Lee (1988); (h) Liischer et al. (1989); (i) Ward and Kirschner (1990); (i)Loewinger and McKeon (1988); (k) Kipreos and Wang (1990); (1) Van Etten et al. (1989); Sawyers et al. (1994); (m) Liischer et al. (1990); (n) F'rendergast et al. (1992); (0) Jin and Burakoff (1993); (p) Chelsky et al. (1989); Wiche et al. (1993); (q) Robbins et al. (1991); (r) Kleinschmidt and Seiter (1988); (s) Moll et al. (1991); (t) Varagona et al. (1992); (u) Klimczak et al. (1992); (v) Citovsky et al. (1992); (w) linland et al. (1992); (x) Howard et at. (1992).
b~denticalregions are found in the mouse Max correlate Myn (see F'rendergast et al., 1992). 'The NLSs of SWIS, nucleoplasmin, and the C-terminus of VirD2, as well as of the proteins above containinga "spacer" of 10-12 amino acids (a.a.), constitute '*bipartite-type"NLSs (see Section 1I.B) (Dingwall and Laskey, 1991, 1992; Robbins et at., 1991). 'Ihe alternatively spliced p70s6kvariant significantly lacks the putative N-terminal NLS and is not nuclear localized (Reinhard et at., 1994). %his putative CKJI site may not be necessary for nuclear targeting-see Reinhard et al. (1994). r~omologousregions are found in the related protein IFi202 (Choubey and Lengyel, 1992). g~omologousregions are found in the bovine FKBP25 (the FK506 binding protein) sequence. h~imilarsequences are found in TLE-2 and -3, the human homologues of the enhancer of split complex genes (Groucho) (Stifani et al., 1992; Ellisen et al., 1991). i Other members of the Notch family of proteins including hN also retain CcN motifs (Stifani et al., 1992; Fo~tiniet at., 1993).
180
DAVID A. JANS
site-mediated regulation of its function (see The relldorsal Family, below) operates in mammalian cells. This argues strongly for phosphorylation and the CcN motif and related sequences being universal in terms of regulating nuclear protein import. Notably in the case of the TF SWI5, we have been able to show that the cdk sites regulate the extent of maximal nuclear accumulation (Jans et al., 1995), exactly as crfc2-k phosphorylation does in the case of T-ag, although the mechanism of the phosphorylation-mediated effect appears to be different (compare SV40 Large T-Antigen and NLS Masking by Phosphorylation, below). Whereas Table 2 shows that an array of proteins other than T-ag possess CcN motifs, only a few have as yet been experimentally examined in terms of the regulation of their nuclear import kinetics. Several examples of cell cycle-dependent (cdk) phosphorylation inhibiting nuclear transport are given in Table 1, but the demonstration of CKII-mediated effects on the transport rate has thus far been restricted to T-ag. This is almost certainly due to the technically demanding nature of the quantitative analysis at the single, cell level required (see Section III). VI. MECHANISMS OF REGULATION OF SIGNAL-DEPENDENT NUCLEAR PROTEIN TRANSPORT A. Cytoplasmic Retention Factors The rel/dorsal Family One mechanism of regulating nuclear protein import is that of cytoplasmic retention, whereby a cytoplasmically localized "anchor" protein or retention factor specifically binds an NLS-containing protein and prevents it from migrating to the nucleus (Hunt, 1989) (see Figure 4). A cytoplasmic retention function has been described for the NF-KB-binding inhibitor protein IKB (also known as MAD-3), where phorbol ester (or other) treatment induces release of the NLS-carrying NF-KB p65 subunit from an inactive cytoplasmic complex with IKB in order to combine with the mature NF-KB p50 subunit and migrate to the nucleus (Lenardo and Baltimore, 1989; Shirakawa and Mizel, 1989; Schmitz et al., 1991). Phosphorylation of IKB by PK-C, PK-A, or the heme-regulated eIF2K (initiation factor 2 kinase) prevents its association with N F - K B in vitro (Ghosh and Balumore, 1990). Roles similar to that of IKB have been proposed for cactus (which shows sequence homology to the N F - K B inhibitor protein IKB) in negatively regulating nuclear localiza-
Reguiation of SV40 Large T-antigen Nuclear cdk (cdc2-K)
Cytoplasm
Accelerated Docking (?)
Nucleus
Figure 4. Regulation of SV40 T-ag nuclear transport by phosphorylation and the CcN nnotif. A simpi istic representation of some of the steps involved in the regulation of NLS-dependent nuclear transport of T-ag by cdk (cdc2-k) and CKII phosphorylation sites is shown. Phosphorylation by cdc2-k appears to result in cytoplasmic retention of T-ag, probably by increasing the affinity for a cytoplasmic anchor protein (see SV40 Large T Antigen, below; and Jans et al., 1991); this effect appears to be titratable. The CKII site is necessary for an accelerated rate of import into the nucleus (steady state within 15-20 minutes as opposed to approximately 10 hours in its absence), probably through increasing the affinity and kinetics of docking at the nuclear envelope/nuclear pore complex. The NLSBP (possibly HSC70/HSP70)-mediated interactions shown are speculative—CKII phosphorylation may conceivably regulate interaction with NLSBPs directly (see Section VI.C). Negative charge is mechanistically important for both cdk and CKII site phosphorylations in the nuclear transport process (Jans et al., 1991; Jans and Jans, 1994), implying that the phosphorylated sites constitute targeting signals themselves, recognized alone or in concert with the NLS, and demonstrating that dephosphorylation at either site is not mechanistically involved in the transport process.
181
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tion of the Drosophila morphogen dorsal (Govind and Steward, 1991) and for the p4(y^^ protein (avian homologue of IKB), which may retain the c-rel proto-oncogene in the cytoplasm in analogous fashion. In the case of the former, phosphorylation of dorsal has been shown to effect its release from cactus and nuclear translocation (Whalen and Steward, 1993); dorsal is constitutively nuclear in the absence of cactus (Roth et al., 1989; Steward, 1989). Other IKB family members include lKBY(a discrete gene product identical to the 70-kDa C-terminus of the NF-KB p50 precursor pi 10 (Inoue et al., 1992) and the proto-oncogene bcl-3 (Wulczyn et al., 1992), both of which can bind the NF-KB p50 subunit in vitro and may function to retain it in the cytoplasm in vivo. All of the lytRlcactus family members contain five to seven ankyrin repeats, structural elements involved in protein-protein interactions. Some of these appear to be directly involved in cytoplasmic retention since deletion of ankyrin repeat 7 together with part of 6 inactivates bcl'3 binding of NF-KB p50 (Wulczyn et al., 1992); and deletion analysis of pi 10 indicates that a short sequence including ankyrin repeat 6 and an adjacent acidic sequence are responsible for cytoplasmic retention of the protein (Blank et al., 1991). The mechanism of cytoplasmic retention appears to operate through masking of the NLS in the case of the IKBS (see Nuclear Factor NF-KB and Intra- and Intermolecular Masking, below). Glucocorticoid Receptor and HSP90 The heat shock protein HSP90 plays a cytoplasmic anchor role in retaining the GR in the cytoplasm in the absence of GH (Picard et al., 1988). Hormone binding by GR directly results in dissociation of the complex with HSP90, and NLS-dependent nuclear translocation of the receptor, which is then active for DNA binding and transcription regulation (Picard et al., 1988). More recent results suggest that cell-cycle-dependent dephosphorylation of the GR, probably by phosphatase PP-2A, may also be involved in this nuclear translocation (Hsu et al., 1992; see Table 1). cAMP'Dependent Protein Kinase Catalytic and Regulatory Subunits As alluded to in Section IV, the PK-A C-subunit translocates to the nucleus upon dissociation from the PK-A holoenzyme complex, which
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occurs subsequent to hormonal stimulation and binding of cAMP by the regulatory (R-) subunit (Nigg et al., 1985; Meinkoth et al., 1990; Pearson et al., 1991). The PK-A R-subunit can be regarded as playing the role of a cytoplasmic anchor, since it functions to retain the C-subunit in the cytoplasm (in the vicinity of the perinuclear Golgi in the case of the type II R-subunit and type II PK-A holoenzyme) (Pearson et al., 1991) in the absence of cAMP-mediated stimulation. c-fos Proto-oncogene
Whereas y-fos is constitutively nuclear, nuclear localization of its c-fos proto-oncogenic counterpart appears to be dependent on cAMPmediated or serum stimulation (Roux et al., 1990). Phosphorylation of c-fos, possibly by PK-A, appears to effect its nuclear translocation by reversing its retention in the cytoplasm, where it is bound to a putative labile inhibitor protein (Roux et al., 1990). SV40 Large T Antigen
Cytoplasmic anchoring appears to be the mechanistic basis of the crfc2-K-mediated inhibition of T-ag nuclear import described in Section V. Maximal inhibition of transport of tetrameric T-ag-p-galactosidase fusion proteins (containing four copies each of NLS and the cdk/cdc2-K site, respectively) is effected by a stoichiometry of phosphorylation of one (rather than four) at the cdc2-K site (Jans et al., 1991), which indicates that one phosphorylated cdc2-K site is sufficient to retain the protein in the cytoplasm, even in the presence of three non-cdc2-K phosphorylated CcN motifs. Phosphorylation presumably increases the affinity of the specific interaction between T-ag and its cytoplasmic anchor. It appears that the inhibitory effect of cdc2-K phosphorylation (or aspartic acid substitution) of Thr^^'* can be oyercome by increasing the concentration of cytosolic T-ag fusion protein (unpublished results), implying that there may be a finite (titratable) cellular level of the cytoplasmic factor capable of retaining Thr^^'^-phosphorylated T-ag in the cytoplasm. B. NLS Masking
A number of proteins possessing apparently functional NLSs when assayed out of context (e.g., as a peptide coupled to a carrier protein) are predominantly cytoplasmic, which is proposed to be due to inaccessibil-
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ity or masking of their NLSs. Masking may take place through interaction with another protein (e.g., a factor binding to the NLS itself) or conformational effects whereby the NLS is masked by other parts of the molecule. Phosphorylation is an efficient and potentially rapidly responsive means of regulating/effecting NLS accessibility. Nuclear Factor NF-KB and Intra- and Intermolecular Masking The active (nuclear) form of NF-KB is composed of the p50 and p65 (relA) protein components that contact specific DNA sequences as a homo- or heterodimer (Lenardo and Baltimore, 1989; Schmitz et al., 1991). Both subunits are homologous within a 300 amino acid NLS-containing sequence required for DNA binding and dimerization, which is shared with members of the rel oncogene family and the D. melanogaster developmental control gene dorsal Initial models (see The vd/dorsal Family, above) for the regulation of nuclear localization of the NF-KB TF revolved around the observation that phorbol ester (or other)^ treatment effected release of the NF-KB p65 subunit from an inactive cytoplasmic complex with the cytoplasmic retention factor IKB ( M A D - 3 ) , in order to be transported in NLS-dependent fashion to the nucleus to activate K light chain gene transcription (Lenardo and Baltimore, 1989; Shirakawa and Mizel, 1989; Ghosh and Baltimore, 1990; Schmitz et al., 1991). A dual mechanism involving NLS masking of both NF-KB components has more recently been favored (Beg et al, 1992; Henkel et al, 1992; Zabel et al, 1993), whereby the C-terminus of the pi 10 precursor of N F - K B p50 (which also exists in several cell types as a discrete gene product called iKBy, purported to be functionally comparable to MAD-3; Inoue et al, 1992) has been shown to function to retain the NF-KB p50 subunit in the cytoplasm through intramolecular masking of its NLS. Antibodies specific to the NLS recognize p50 but not pi 10, implying that the NLS is masked in the larger precursor (Henkel et al, 1992). As discussed above (The rd/dorsalFamily), the C-terminus of pi 10, which is absent from p50, appears to mask the NLS directly through specific ankyrin repeat sequences (Blank et al, 1991), one mechanism of unmasking of the NLS being through proteolysis of the pi 10 C-terminus (Blank et al, 1991; Fan and Maniatis, 1991; Riviere et al, 1991). Intermolecular masking of its NLS by IKB (MAD-3) appears to be the mechanism of cytoplasmic retention of NF-KB p65 (Zabel et al, 1993), whereby deletion or mutation of the p65 NLS eliminates binding to IKB
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(Beg et al., 1992). The mechanism of release from cytoplasmic retention appears to be through IKB degradation (see Beg et al., 1993) induced by PK-C phosphorylation (probably by the PK-C^ isotype, known to be activated by tumor necrosis factor a, one of the physiological stimuli for N F - K B ; Diaz-Meco et al., 1994). Nuclear localization of the NF-KB T F is thus dually regulated by specific intra- and intermolecular masking of the respective NLSs of the two N F - K B subunits. Signal transduction-triggered phosphorylation regulates the masking events precisely to enable rapid response in terms of TF nuclear translocation and gene induction. NLS Masking by Phosphorylation
NLS masking can also be directly effected by phosphorylation, whereby phosphorylation close to or within an NLS masks or inactivates it either through charge or conformational effects. SWI5. Cell-cycle-dependent nuclear exclusion of the 5. cerevisiae TF SWI5 involved in mating type switching is effected by phosphorylation by the cdk/CDC28-kinase (Moll et al., 1991), the yeast homologue of C(ic2-K. Three cdk/CDC28-kinase sites, one of which is within the spacer of the SWI5 bipartite NLS (see Tables 1 and 2), are proposed to inhibit nuclear localization by inactivating or masking the function of the SWI5 NLS through charge or conformational effects (Moll et al., 1991). At anaphase, CDC28 kinase activity falls and SWI5 is dephosphorylated to effect nuclear entry and activation of transcription of the HO mating switch endonuclease; removal by mutation of the CDC28 kinase sites results in constitutive nuclear localization. Lamin B2. NLS-dependent nuclear transport of lamin B2 is inhibited by phosphorylation at two PK-C sites N-terminally adjacent to the NLS (see Table 1) (Hennekes et al, 1993), both in vivo in response to phorbol ester stimulation and in vitro. Negative charge close to the NLS presumably inactivates it, in a fashion analogous to that of the cdk/CDC28 phosphorylation-mediated inactivation of the SWI5 NLS described above. Cofilin, Nuclear translocation of the actin-binding protein cofilin occurs upon heat shock treatment and is accompanied by dephosphorylation at a consensus multifunctional calmoduUn-dependent protein kinase site adjacent to the putative cofilin NLS (see Table 1; Nishida et al..
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1987; Ohta et al., 1989). Phosphorylation at this site is proposed to mask or inactivate the function of the NLS (Ohta et al., 1989). C. The CcN Motif: Positive and Negative Regulation by Dual Phosphorylation We have established that the CKII and cdc2-K. phosphorylation sites appear to function completely independently of one another in terms of both regulating nuclear transport and influencing phosphorylation at the other site (Jans et al., 1991); CKII phosphorylation does not enhance nuclear import by reducing phosphorylation at the inhibitory cdc2-K site; nor does Thr^^"^ phosphorylation inhibit nuclear transport by impairing Ser^^^ phosphorylation (Jans et al., 1991). Both phosphorylations almost certainly occur in the cytoplasm (see Scheidtmann et al., 1982; Jans et al., 1991; Jans and Jans, 1994), implying that the regulatory events determining nuclear import kinetics largely take place in the cytoplasm (see Figure 4). This is consistent with the cytosolic requirement for signal dependent nuclear transport in vitro (see Section III). Nuclear phosphorylation at either kinase site to effect retention in the nucleus is clearly not the mechanism of nuclear accumulation. Aspartic acid can substitute functionally for phospho-serine/threonine in the case of both phosphorylation sites of the T-ag CcN motif (Jans et al., 1991; Jans and Jans, 1994; see Figure 3), implying that the phosphorylated sites may represent signals in themselves that are recognized by components of the cellular nuclear transport machinery (see also Jans and Jans, 1994). The results with aspartic acid substituted proteins clearly demonstrate that dephosphorylation/phosphatase activity at either of the CcN motif kinase sites can have no direct role in the nuclear transport process; i.e., nuclear dephosphorylation at either the CKII or cdcl-k site is not involved in T-ag retention in the nucleus (Jans and Jans, 1994). Whereas cytoplasmic retention (see SV40 Large T Antigen, above) would appear to be the basis of the inhibition of T-ag nuclear transport via the cell-cycle-regulated ct/c2-K, the mechanism of CKII-phosphorylation-mediated enhancement of the rate of nuclear transport is not clear. Phosphorylation (or aspartic acid substitution) at the CKII site increases negative charge at the site, and it is intriguing to speculate that the combination of concentrations of negative (phosphorylated or Asp-substituted CKII site) and positive (NLS) charge has a mechanistic role. The molecular basis of this may be analogous to that proposed for eukaryotic proteasome component proteins, which possess both putative NLS and
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"cNLS" (series of Asp and Glu residues complementary to the NLS) sequences, whereby a masking interaction between the two regions is proposed (Grziwa et al., 1992). It may be a worthwhile exercise to examine the sequences of nuclear proteins that lack a CKII site for regions of negative charge in the vicinity of the NLS, which may conceivably represent, in conjunction with the NLS, a signal for constitutively rapid nuclear transport similar to Asp^^^-substituted T-ag fusion proteins (Jans and Jans, 1994). Another possible explanation of how the CKII site exerts its effect on the NLS-dependent nuclear import of SV40 T-ag proteins is that it facilitates the interaction between the T-ag NLS and NLSBPs, possibly HSP70/HSC70 (Imamoto et al., 1992; Shi and Thomas, 1992). Interestingly in this regard, the spacing between the CKII site and NLS in T-ag is 10 amino acid residues, which coincides with the 10-12-amino acid spacer between the two arms of basic residues required for efficient binding to bipartite NLSs (Dingwall and Laskey, 1992). It is intriguing to speculate that the CKII site, in conjunction with the T-ag NLS, is directly involved in NLSBP interaction, whereby phosphorylation may strongly regulate the affinity of binding. Preliminary evidence (unpublished) from our in vitro system implies that CKII site phosphorylation may increase the rate of interaction ("docking") at the nuclear envelope/nuclear pore complex, which is likely to be NLSBP mediated, and hence is consistent with this idea. Figure 4 is a simplistic representation of some of the events envisaged to be involved in the regulation of T-ag nuclear import by the CcN motif, involving cdc2-K regulation of cytoplasmic retention and CKH regulation of the kinetics of docking at the pore complex. Vll. VARIANTS OF THE CcN MOTIF: OTHER KINASES ABLE TO REGULATE NLS-DEPENDENT NUCLEAR PROTEIN IMPORT A. Signal Transduction-Responsive NLSs CKII clearly regulates T-ag nuclear import kinetics (Rihs et al., 1991; Jans and Jans, 1994), and the presence of CKII sites in the CcN motifs identified in other nuclear localized proteins implies significance in regulating the nuclear uptake of many other proteins. Since its activation is regulated by growth and proliferative signals, it is possible that one of the major physiological roles of CKII is to regulate protein transport to
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the nucleus in response to such hormonal signals. However, this putative central role of CKII does not preclude the possibility that the kinases, which participate in hormonally regulated signal transduction pathways, may play a similar role. Evidence that this may indeed be the case already exists for PK-A, PK-C, and the multifunctional calmodulin dependent protein kinase, where phosphorylation sites for these kinases, close to or within the NLS of the respective proteins, regulate NLS function. PK-A is directly implicated in positively regulating nuclear locahzation of the c-rel proto-oncogene (see Table 1), with spacing (22 amino acids) between the PK-A site and NLS having been demonstrated to be of importance (Gilmore and Temin, 1988; Garson and Kang, 1990). In a further parallel with T-ag and CKII, aspartic acid at the PK-A site in c-rel simulates PK-A phosphorylation at the site in terms of the effect on subcellular localization (Mosialos et al., 1991). Results indicate that phosphorylation at the PK-A site enhances nuclear translocation of c-rel. It is noteworthy in this regard that all of the xtMdorsal family TFs possess a homologous region including the NLS and PK-A site (see Schmitz et al., 1991), and significantly, the region of dorsal to which the cytoplasmic retention factor cactus binds has been localized to this region (Kidd, 1992). PK-A phosphorylation of c-rel may decrease its affinity for its specific cytoplasmic retention factor and thereby effect its nuclear translocation. It is an intriguing possibility that the apparent nuclear transport regulatory function of the PK-A site near the NLS of c-rel may be conserved in other rel/dorsal family members. A further example of putative PK-A regulation of nuclear localization is that of rNFIL-6, which translocates to the nucleus upon the elevation of intracellular cAMP levels in rat PC 12 pheochromocytoma cells (Metz and Ziff, 1991). A consensus PK-A site resides adjacent to the putative rNFIL-6 NLS (see Table 1). As for PK-A, evidence thus far implies that both PK-C and the multifunctional calmodulin-dependent protein kinase are signal transduction-responsive kinases able to regulate nuclear protein import (see NLS Masking by Phosphorylation). NLS-dependent nuclear transport of lamin B2 is inhibited by PK-C phosphorylation at sites N-terminally adjacent to the NLS (see Table 1) (Hennekes et al., 1993), whereas nuclear localization of the actin-binding protein cofilin is similarly negatively regulated by a multifunctional calmodulin-dependent protein kinase site N-terminally adjacent to the NLS (see Table 1) (Ohta et al., 1989).
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Clearly, kinases other than CKII can regulate NLS-dependent nuclear transport in response to hormonal signals in a fashion mechanistically similar to the T-ag CcN motif. One can regard these kinase sites together with the NLSs as variants of the CcN motif, where a hormonally regulated signal other than those activating CKII can enhance or inhibit nuclear localization of the appropriate TF or other proteins of a signal transduction pathway. Many other kinases will presumably be demonstrated to be able to function in the same way to regulate nuclear protein transport according to mitogenic, differentiation, and developmental signals. Such constellations of signal transduction responsive kinase sites and NLSs ("N") can be called SRNs (signal transduction-responsive NLSs). A number of SRN variants may well reside in the list of proteins known to exhibit regulated (conditional) nuclear transport in Table 1, where demonstration of this will require direct experimentation of a quantitative nature. B. Cell-Cycle-Dependent NLSs As listed in Table 1, cdks regulate the nuclear transport of a variety of proteins, the best demonstrated examples being cdc2'k and T-ag and CDC28 kinase and SWI5. This regulation appears to affect the maximal amount of nuclear protein accumulated rather than its rate of transport to the nucleus, although an exception appears to be v-jun transport to the nucleus, where cdk (?)-mediated phosphorylation within the NLS is responsible for determining the rate of nuclear transport in cell-cycle-dependent fashion (Chida and Vogt, 1992). Cell cycle dependence of nuclear entry may also be effected directly by kinases other than cdks, although their respective activities may well be directly regulated by cdks. CKII, for example, may be the kinase responsible for nuclear exclusion of the Drosophila lodestar protein through multiple phosphorylation sites in the vicinity of a putative NLS, whereas dorsal nuclear relocalization during development probably involves a Ca^^-regulated kinase and possibly PK-A (see Section VILA; Table 1). NLSs whose function appears to be modulated by cell cycle phosphorylation can be called CDNs (cell-cycle-dependent NLSs). Clearly, the combination of a variety of SR and (directly or indirectly) CD kinases provides a plethora of possible ways to regulate the nuclear translocation of proteins very precisely, in terms of both the amount of protein transported to the nucleus and the rate at which it is imported, as
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required during cellular processes such as development, differentiation, transformation, and regulation of cellular metabolism.
VIIL CONCLUSION AND FUTURE PROSPECTS It is quite clear now from a number of studies that the structure and function of NLSs is essentially the same across the plant and animal kingdom, with NLSs from plants, yeast, and higher mammals being essentially identical. NLSs functional in plants are active in nuclear targeting in yeast cells; yeast NLSs are functional in mammalian cells; and the SV40 T-ag NLS appears able to confer nuclear localization on appropriate, normally cytoplasmic carrier proteins in mammalian, insect, yeast, and plant cells. Furthermore, there is the implication that the regulation of NLS activity, primarily by phosphorylation, also appears to be universal in eukaryotic cells. Phosphorylation regulates NLS-dependent nuclear localization of many different proteins and in a variety of cell types. The clear implication is that the signals for conditional NLS-dependent nuclear protein transport are potentially as universal as the NLS signals themselves. Hand in hand with this is the fact that the mechanisms of regulating nuclear protein transport, such as cytoplasmic retention factors, intra- and intermolecular NLS masking, the masking of NLSs by phosphorylation, etc., also appear to be conserved across eukaryotes (e.g., the conservation of structure and function of the IKB, p40'^^', and cactus cytoplasmic retention factor proteins, as well as their specific TF partners, despite their phylogenetically diverse origins). The nuclear targeting regulatory module of the CcN motif, comprising the NLS, CKII site, and cdk site, appears to be conserved in many nuclear localizing proteins from mammals, yeast, insects, and plants (see Table 2). Several other regulatory module variants for NLS-dependent nuclear protein transport appear to exist, whereby kinase sites close to the NLS, other than those for CKII and cdk, modulate NLS function through signal transduction-regulated (SRNs) and cell-cycle-regulated (CDNs) NLSs. Both of these are likely to be as universal as CcN motifs in terms of distribution and function. In terms of future prospects, the rapidly increasing knowledge with respect to regulated nuclear protein transport may be applied to medically pertinent problems, such as the possibility of using NLSs to target molecules of interest to the nucleus. Signals for tightly regulated, conditional nuclear localization responding to hormonal or cell-cycle-dependent stimulation in a variety of cell types (e.g., CcN motifs, CDNs,
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SRNs) may enable precise cueing of the nuclear localization of relevant proteins and other molecules according to need. This has potential application in gene therapy and gene targeting through facilitating the directed transport of DNA molecules to the nucleus in mammalian (or plant) cells, and thereby potentially increasing transfection/homologous recombination efficiencies (see Rosenkranz et al., 1992; Jans, 1994). Alternatively, toxic molecules such as photosensitizers might be efficiently targeted to sensitive subcellular sites such as the nucleus in order to kill tumor cells (Akhlynina et al., 1993, 1995). The apparent universality of NLSs, and more importantly, the apparent universaUty of the mechanisms of regulation of nuclear protein targeting and CcN motifs and variants thereof, would imply that these approaches are assured of success, above all through their potential widespread appUcability in terms of cell type and protein/gene.
ACKNOWLEDGMENTS Patricia Jans is thanked for helpful discussions, and the Clive and Vera Ramaciotti Foundation is gratefully acknowledged for supporting the work on the CcN motif.
NOTES 1. Several proteins larger than 45 kDa, although lacking an identifiable NLS, have been shown to be predominantly localized in the nucleus. The mechanism appears to be through association with NLS-bearing proteins and cotransport into the nucleus ("piggy back" transport) (e.g., Zhao and Padmanabhan, 1988). 2. Using reconstituted nuclear transport systems, a role in nuclear envelope binding ("docking") has been proposed for a 97-kDa protein together with an NLSBP (Adam and Adam, 1994); and a role in active import into the nucleus has been implicated for the 25-kDa GTP-binding protein Ran/TC4 (and possibly the interacting components RCCl and RanBP-1; see Moore and Blobel, 1993,1994; Melchior et al., 1993). 3. Deletion of either the regulatory or kinase domains of PK-Ca leads to its constitutive nuclear localization (Eldar et al., 1992), purported to be through the unmasking of normally inaccessible NLS sequences in the intact molecule (see Section VLB for examples of NLS masking). Activation of PK-Ca upon phorbol ester binding effects conformational changes that appear to relieve the intramolecular NLS masking in the nonactivated molecule and regulate kinase activity. 4. CKII is regulated by proliferative (as well as other) stimuli such as epidermal growth factor and insulin (Krebs et al., 1988; Meisner and Czech, 1991). 5. Physiological stimuli for NF-KB activation/nuclear localization include the cytokine interleukin (IL)-la. This is interesting in the context of NF-KB, rel, and dorsal
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homologies since the toll receptor in the dorsal pathway has homology to the IL-1 receptor. Common nuclear signaling mechanisms are clearly implicated.
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MEMBRANE PROTEIN TOPOGENESIS IN ESCHERICHIA COLI
Gunnar von Heijne
I. Intrcxiuction II. Mechanisms of Membrane Protein Assembly A. The "Positive Inside" Rule B. The Sec Machinery C. The Membrane Potential III. Helix-Helix Packing in a Lipid Environment IV. Predictionof Topology and Structure V. Artificial Membrane Proteins VI. Conclusions and Outlook References
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I. INTRODUCTION How does the amino acid sequence of a membrane protein dictate its insertion into the membrane and how does it determine the final threedimensional fold? This deceptively simple question has gradually moved
Membrane Protein IVansport Volume 2, pages 201-214 Copyright © 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-983-4
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to the forefront of membrane protein research, and it now excites workers vyith interests ranging from bacterial genetics and cell biology to protein structure prediction and modeling. As will be reviewed in this chapter, our own work has largely focused on membrane protein assembly in the inner membrane of the bacterium Escherichia coli and has led to the formulation of the "positive inside" rule—a shorthand intended to capture the essential role played by positively charged amino acids in determining the topology of proteins spanning the cytoplasmic membrane. Although all of the mechanistic details of the membrane protein assembly processes in various cell types and organelles are still far from being worked out, our understanding is already sufficiently advanced to allow good predictions of protein topology to be made directly from the amino acid sequence, and to suggest how "artificial" membrane proteins of a predetermined topology may be designed. II. MECHANISMS OF MEMBRANE PROTEIN ASSEMBLY A. The "Positive Inside" Rule At the most simple level, the structure of an integral membrane protein can be characterized by its membrane topology, that is, by a diagram showing which parts are exposed on each side of the membrane. The first question that must be addressed when trying to understand the entire assembly process is thus whether the formation of the topology corresponds to a distinct step in the assembly pathway, or whether the formation of the topology and the formation of the entire 3D structure are inseparable processes. Fortunately, there is now ample evidence in support of the former view, both from biophysical studies on the reformation of the native structure of the multi-spanning protein bacteriorhodopsin from fragments that individually retain the same topology as in the intact molecule (Popot et al., 1987; Kahn and Engelman, 1992; Kahn et al., 1992; Kataoka et al., 1992), and from molecular genetic studies where pieces of inner membrane proteins have been expressed and shown to insert into the membrane separately while still being able to ultimately assemble into functional molecules (Zen et al., 1994). Thus, the concept of a two-step folding pathway (Popot and de Vitry, 1990; Popot and Engelman, 1990), where the membrane-spanning segments are first inserted individually and fold into stable transmembrane a-heli-
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ces and only later pack together to form the full 3D structure, is now well established. If the topology is thus more than an abstract representation of the structure and in fact corresponds to a real intermediate on the folding pathway, it may be fruitful to ask whether there is some simple relation between the primary amino acid sequence and the topology. Indeed, statistical studies of membrane proteins of known topology from a range of different cellular membranes have demonstrated that there is a strong correlation between the location of positively charged amino acids and the topology; such residues abound in segments of the chain that are not translocated across the membrane, but are scarce in translocated segments (von Heijne, 1986; von Heijne and Gavel, 1988; Gavel et al., 1991; Gavel and von Heijne, 1992). In particular, this "positive inside" rule holds exceptionally well for E. coli inner membrane proteins, an observation that prompted us to undertake a series of experimental studies intended to clarify the role of basic amino acids in controlling the topology of bacterial inner membrane proteins in vivo. Partly by serendipity, our first studies were conducted using E. coli leader peptidase (Lep), a protein that spans the inner membrane twice with an NQ^J-CQU^ topology (Figure 1). It proved possible to redesign this molecule such that it inserted in the opposite topology (Nij^-C^j^) by
periplasm
Lep wt
Figure 1. Topology of wild-type Lep [left), a deletion mutant where most of the charged residues in the cytoplasmic loop have been removed {middle), and a mutant with an "inverted'' topology [right) that was constructed by adding four positively charged lysine residues to the N-terminus of the deletion mutant (von Heijne, 1989).
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periplasm
cytoplasm
Figure 2. Topology of a "frustrated" construct where three positively charged residues have been placed in the first and second loops. This forces the molecule to adopt a "leave-one-out" topology with only three transmembrane segments. The construct was made by fusing two gene fragments encoding the membrane domains of two Lep mutants in tandem (Gafvelin and von Heijne, 1994).
simply removing most of the positively charged amino acids normally present in the loop connecting the two transmembrane segment while at the same time adding a few positively charged lysines to the N-terminal tail (von Heijne, 1989). This result opened the way for studies where the topological effects of both positively and negatively charged residues could be analyzed in detail (Nilsson and von Heijne, 1990; Andersson and von Heijne, 1991; Andersson etal., 1992; Andersson and von Heijne, 1993a), and ultimately, together with data from other groups (Boyd and Beck with, 1990; Dalbey, 1990), provided ample experimental verification of the positive inside rule. More recently, we have extended our studies of the topological effects of positively charged residues to proteins with up to four transmembrane segments (Gafvelin and von Heijne, 1994). An interesting result was that molecules where the location of the positively charged residues was intentionally chosen to "frustrate" the molecule (Figure 2) inserted into the inner membrane with "leave-one-out" topologies, that is, the requirement that all highly charged loops should remain in the cytoplasm forced at least one putative transmembrane segment to stay out of the membrane. From these and other results, we concluded that the insertion process is locally determined and probably proceeds by the independent
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insertion of individual "helical hairpins" (Engelman and Steitz, 1981) composed of two neighboring transmembrane segments and a connecting loop. B. The Sec Machinery Periplasmic and outer membrane E. coli proteins normally make use of the so-called Sec machinery for translocation across the inner membrane. The secretory pathway involves cytoplasmic chaperones such as Ffh, GroES/EL, DnaK, and SecB; the SecAAf/E/G complex in the inner membrane; and the additional inner membrane components SecD and SecF (Schatz and Beckwith, 1990; Wickner et al., 1991; Luirink and Dobberstein, 1994; Nishiyama et al., 1994; Pogliano and Beckwith, 1994). However, certain inner membrane proteins are able to insert efficiently into the membrane even under conditions when the function of one or the other of the Sec components is severely compromised. In particular, this is true for the "inverted" form of Lep (von Heijne, 1989). Translocation of the C-terminal periplasmic domain in wild-type Lep, in contrast, requires a fully functional Sec machinery (Wolfe and Wickner, 1984; Wolfe etal., 1986). A possible relation between the length of a translocated segment and its dependence on the Sec machinery for translocation was suggested by a statistical study of the content of positively charged residues in periplasmic loops as a function of loop length (von Heijne and Gavel, 1988; von Heijne, 1994). Although most periplasmic loops tend to be short and contain only few arginines and lysines, loops longer than -60 residues often contain a much higher frequency of such residues, similar to the overall frequency found in periplasmic proteins. A possible explanation would be that the mechanism of translocation is different for short and long loops, and that this leads to different restrictions on the amino acid composition of the loops. This hypothesis was confirmed in a study where the length of the periplasmic loop in "inverted" Lep molecules was successively increased from -25 to -65 residues (Andersson and von Heijne, 1993b). For this series of molecules, the SecA dependence of translocation was found to increase in proportion to the loop length, up to a limiting value of about 55 residues. A qualifying remark should be added at this point: different loops of similar lengths may show different degrees of Sec dependence, depending on their amino acid composition and on the surrounding transmem-
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brane segments. Thus, when the positively charged residues in a long periplasmic loop placed in an "inverted" Lep construct were successively mutated to uncharged residues, the degree of SecA dependence dropped proportionately (Andersson and von Heijne, 1994b). In another series of experiments, we recendy tested the SecA dependence of the translocation of a 180-residue-long periplasmic loop in the MalF protein and found that whereas translocation of this loop when in its normal sequence context is only marginally affected by perturbation of SecA function, the SecA dependence is dramatically increased when the loop is placed out of context in an "inverted" Lep construct (Saaf et al, 1994). In all but one of the cases that we have looked at, the translocation of long loops (>60 residues) has been clearly affected by perturbation of the Sec machinery, although to varying degrees. We have encountered only one exception to the rule that the translocation of long periplasmic segments is Sec dependent: the N-terminal tail of the ProW inner membrane protein (Whitley et al., 1994b). ProW spans the inner membrane seven times, and its N-terminal, 100-residuelong tail is located in the periplasm. Interestingly, the N-tail contains only three positively charged residues (but 12 negatively charged ones), suggesting that this particular tail, although long, is under the same selective pressure as the normally much shorter. Sec-independent loops. Indeed, translocation of the ProW N-tail was found to be independent of SecA within the accuracy of our measurements, and it was completely blocked by the introduction of extra positively charged residues. Generalizing from this one example, it is possible that the Sec machinery can only translocate polypeptide stretches located C-terminally to a translocation signal, and that N-terminally located segments may only be translocated by a Sec-independent mechanism regardless of their length. So far, we have thus found that Sec dependence tends to correlate with the length of the translocated segment, with its content of positively charged residues, and with its location within the protein. The issue of what a high versus low Sec dependence means in mechanistic terms is entirely unclear, however, and may only be possible to address using reconstituted in vitro systems where the different Sec components can be physically removed. C. The Membrane Potential What, finally, may be the mechanistic basis for the positive inside rule? An attractive possibihty is that the membrane electrochemical
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periplasm
cytoplasm
Figure 3. A Lep mutant that adopts different topologies depending on whether a transmembrane electrochemical potential is present during the insertion step. In the presence of a potential (i.e., in the absence of the protonophore CCCP), the negatively charged N-terminal tail is translocated in preference to the loop carrying one positively and one negatively charged residue (left). In the absence of a potential, in contrast, the loop is more easily translocated than the N-terminal tail {middle). If the function of SecA is blocked by the addition of sodium azide, translocation of the Sec-dependent C-terminal domain is prevented but the topology is not affected (right) (Andersson and von Heijne, 1994a).
potential (acidic and positive toward the periplasm) may act against the translocation of positively charged residues. To test this notion, we expressed various Lep-derived constructs both in normal cells and under conditions where the electrochemical potential had been dissipated by addition of the protonophore CCCP (Andersson and von Heijne, 1994a). To our surprise, certain constructs were found to insert with different orientations when expressed in the absence or the presence of CCCP, (Figure 3), and their insertion behavior could in all cases be explained by a model which postulates that the electrochemical potential promotes the translocation of negatively charged residues and works against the translocation of positively charged ones, thus introducing an asymmetry into the topological effects of positively and negatively charged amino acids. Although this study must be followed up, it provides a first hint that interactions between the membrane potential and charged residues in the nascent polypeptide may explain the positive inside rule.
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IIL HELIX-HELIX PACKING IN A LIPID ENVIRONMENT According to the two-step folding model, the transmembrane helices are first formed individually in the membrane, and only then pack together into the final helix-bundle structure. The energetics of helix-helix interactions in a lipid environment are largely unknown, and hence prediction of the 3D structure of integral membrane proteins is in its infancy. In part, this is the result of the lack of high-resolution 3D structures for membrane proteins; to date, such information is available only for three helix-bundle membrane proteins: the photosynthetic reaction center (Deisenhofer et al., 1985), bacteriorhodopsin (Henderson et al, 1990), and a light-harvesting polypeptide (Kiihlbrandt et al., 1994). These difficulties have prompted the development of altemative means of obtaining 3D structure information. In particular, methods based on molecular genetic techniques seem quite promising. The most complete analysis of this kind has so far been carried out on the glycophorin A homo-dimer, a single-spanning plasma membrane protein where dimerization is driven by interactions between the two transmembrane helices. Saturation mutagenesis has identified seven critical residues on one face of the helix that are necessary and sufiicient for dimerization (Lemmon et al, 1992,1994); all of the other residues in the helix can be replaced by leucines with only minor effects on the monomer-dimer equilibrium. Both small nonpolar and large hydrophobic residues are found among the critical residues, and it seems that dimerization is driven by the tight fit between the two complementary helix surfaces rather than by a few specific interactions between, for example, charged or polar residues embedded in the hydrophobic helices. Another recent method is based on the idea that cysteine residues introduced into neighboring transmembrane helices by site-directed mutagenesis may form disulfide bridges if located close to each other. This approach has so far been tried on two E. coli inner membrane proteins, the Tar chemoreceptor (Pakula and Simon, 1992), and leader peptidase (Whitley et al, 1993). In both cases it was found that the observed pattern of disulfides could be interpreted in terms of a 3D model, but the method has yet to be tested on a known 3D structure. Other techniques that can yield distance information are, for example, various energy transfer measurements (Lakey et al, 1993; Adair and Engelman, 1994), and the analysis of second-site revertants of nonfunctional point mutations in transmembrane helices (Sahin-Toth and Kaback, 1993).
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IV. PREDICTION OF TOPOLOGY AND STRUCTURE To predict the topology of a membrane protein from its amino acid sequence, one needs (i) to predict the approximate location of all the transmembrane segments, and (ii) to decide on the orientation of the molecule in the membrane. Transmembrane segments are usually identified by the aid of a hydrophobicity plot, where the average hydrophobicity over a 15-20-residue-long window is plotted as a function of position in the chain. Various optimization schemes intended to perfect the underlying hydrophobicity scale have been proposed (Klein et al., 1985; Edelman and White, 1989; Edelman, 1993), but many of the early rough-and-ready scales perform almost as well, and the choice of scale is more a matter of taste than rational deliberation. None of the available hydrophobicity analysis algorithms allow a clear distinction between transmembrane and nontransmembrane segments to be made, however. Thus, in many cases one is left with one or more uncertain candidate transmembrane segments that can be combined into a number of different topologies. In such cases, the positive inside rule can be of help in deciding which of the possible topologies is the most likely one; for each topology, one simply calculates the difference in the number of positively charged residues between the two sides of the structure (counting only short loops of 60 residues or less, but always including the N-terminal tail, irrespective of its length; see above) and then chooses the topology with the highest difference as the best guess. This procedure has been automated (Claros and von Heijne, 1994) and works very well for bacterial inner membrane proteins (von Heijne, 1992) and somewhat less well for eukaryotic membrane proteins (Sipos and von Heijne, 1993). Amore refined version of this basic approach has recently been developed (Jones et al., 1994); to what extent this method performs better than the simple version is not clear. As noted above, the prediction of optimal helix-helix packing interactions is still an unsolved problem, although a few early attempts have been made. A systematic study of all possible ways of arranging the seven transmembrane helices in bacteriorhodopsin on a lattice, with an optimization criterion based on the maximization of contacts between polar residues and the minimization of contacts between evolutionarily highly variable residues, places the observed arrangement near the top (Taylor et al., 1994). Amore detailed calculation on bacteriorhodopsin where the helical axes are kept fixed in space but where each helix is allowed to
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rotate around and slide along its axis also produces an optimal structure close to the correct one (Tuffery et al., 1994). The arguably most realistic prediction attempted so far has been carried out for the glycophorin A dimer, where a simulated annealing molecular dynamics protocol was used to identify low-energy structures (Treutlein et al, 1992; Lemmon et al, 1994). Again, the structure identified by the mutagenesis approach described above was among the ones of lowest energy. Taken together, these studies suggest that good, generally applicable methods of making predictions of the 3D structure of integral membrane helix-bundle proteins may not be far off.
V. ARTIFICIAL MEMBRANE PROTEINS As is clear from the previous sections, a rather simple picture of the sequence factors dictating membrane protein insertion is emerging: hydrophobic stretches of amino acids drive the insertion and end up forming transmembrane helices, and positively charged residues flank-
periplasm
cytoplasm
Figure 4. Topology of a de novo designed inner membrane protein with four highly simplified transmembrane segments. Each transmembrane segment is composed only of leucine and alanine residues, and the topology is controlled by appropriately placed lysine residues (Whitley et a l , 1994a).
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ing the hydrophobic segments control their orientation in the membrane. As a stringent test of this idea, and as a first step toward the construction of completely novel membrane proteins, we recently decided to attempt the design and expression of artificial membrane proteins composed of highly simplified transmembrane segments and appropriately located, positively charged residues. So far, proteins with up to four transmembrane segments composed only of leucines and alanines have been successfully expressed in E, coli and have been shown to insert efficiently and with the predicted topology into the inner membrane (Figure 4) (Whitley et al., 1994a). These proteins do not appear to be toxic to the cell and can be produced in reasonable yields, providing the first indication that the in vivo expression of de novo designed membrane proteins is a viable idea.
VI. CONCLUSIONS AND OUTLOOK Over the past few years, studies reviewed here and elsewhere (von Heijne, 1994) have led to a fairly advanced understanding of how integral membrane proteins insert into membranes, both from the point of view of the sequence determinants that encode the topogenic information and in terms of the mechanism of integration. Based on this understanding, rather reliable prediction schemes that allow the topology of a protein to be deduced directly from its amino acid sequence have been developed, and completely novel membrane proteins have been successfully designed and expressed in vivo. In the next few years, it should be possible to further decipher the role of the Sec machinery during the insertion process by studies both in vitro and in vivo. Also, the corresponding processes in eukaryotic cells (e.g., integration into the ER membrane and into the inner membrane of mitochondria) must be studied in a systematic way. Perhaps the most exciting prospect for the coming years, however, is the possibility of designing artificial membrane proteins that may provide new and powerful tools for studying important biological processes such as ion channel function and electron transport phenomena. REFERENCES Adair, B.D. & Engelman, D.M. (1994). Glycophorin A helical transmembrane domains dimerize in phospholipid bUayers: a resonance energy transfer study. Biochemistry 33, 5539-5544.
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Andersson, H. & von Heijne, G. (1991). A 30-residue-long "export initiation domain" adjacent to the signal sequence is critical for protein translocation across the inner membrane of Escherichia coli. Proc Natl. Acad. Sci. USA 88,9751-9754. Andersson, H. & von Heijne, G. (1993a). Position-specific Asp-Lys pairing can affect signal sequence-function and membrane protein topology. J. Biol. Chem. 268, 21389-21393. Andersson, H. & von Heijne, G. (1993b). 5^c-dependent and sec-independent assembly of E. coli inner membrane proteins: the topological rules depend on chain length. EMBO J. 12,683-691. Andersson, H. & von Heijne, G. (1994a). Membrane protein topology: effects of A^H^ on the translocation of charged residues explain the "positive inside" rule. EMBO J. 13,2267-2272. Andersson, H. & von Heijne, G. (1994b). Positively charged residues influence the degree of sec-dependence in protein translocation across the E. coli inner membrane. FEBS Lett. 347,169-172. Andersson, H., Bakker, E., & von Heijne, G. (1992). Different positively charged amino acids have similar effects on the topology of a polytopic transmembrane protein in Escherichia coli. J. Biol. Chem. 267, 1491-1495. Boyd, D. & Beckwith, J. (1990). The role of charged amino acids in the localization of secreted and membrane proteins. Cell 62,1031-1033. Claros, M.G. & von Heijne, G. (1994). TopPred II: an improved software for membrane protein structure prediction. CABIOS 10, 685-686. Dalbey, R.E. (1990). Positively charged residues are important determinants of membrane protein topology. Trends Biochem. Sci. 15,253-257. Deisenhofer, J., Epp, O., Miki, K., Huber, R., & Michel, H. (1985). Structure of the protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3A resolution. Nature 318, 618-624. Edelman, J. (1993). Quadratic minimization of predictors for protein secondary structure: application to transmembrane a-helices. J. Mol. Biol. 232,165-191. Edelman, J. & White, S.H. (1989). Linear optimization of predictors for secondary structure: application to transbilayer segments of membrane proteins. J. Mol. Biol. 210, 195-209. Engelman, D.M. & Steitz, T.A. (1981). The spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis. Cell 23,411-422. Gafvelin, G. & von Heijne, G. (1994). Topological "frustration" in multi-spanning E. coli inner membrane proteins. Cell 77,401-412. Gavel, Y. & von Heijne, G. (1992). The distribution of charged amino acids in mitochondrial inner membrane proteins suggests different modes of membrane integration for nuclearly and mitochondrially encoded proteins. Eur. J. Biochem. 205,12071215. Gavel, Y., Steppuhn, J., Herrmann, R., & von Heijne, G. (1991). The positive-inside rule applies to thylakoid membrane proteins. FEBS Lett. 282,41-46. Henderson, R., Baldwin, J.M., Ceska, T.A., Zemlin, P., Beckmann, E., & Downing, K.H. (1990). A model for the structure of bacteriorhodopsin based on high resolution electron cryo-microscopy. J. Mol. Biol. 213, 899-929.
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Jones, D.T., Taylor, W.R., & Thornton, J.M. (1994). A model recognition approach to the prediction of all-helical membrane protein structure and topology. Biochemistry 33, 3038-3049. Kahn, T. & Engelman, D. (1992). Bacteriorhodopsin can be refolded from two independently stable transmembrane helices and the complementary five-helix fragment. Biochemistry 31, 6144-6151. Kahn, T.W., Sturtevant, J.M., & Engelman, D.M. (1992). Thermodynamic measurements of the contributions of helix-connecting loops and of retinal to the stability of bacteriorhodopsin. Biochemistry 31, 8829-8839. Kataoka, M., Kahn, T.W., Tsujiuchi, Y, Engelman, D.M., & Tokunaga, F. (1992). Bacteriorhodopsin reconstituted from two individual helices and the complementary 5-helix fragment is photoactive. Photochem. Photobiol. 56, 895-901. Klein, P., Kanehisa, M., & DeLisi, C. (1985). The detection and classification of membrane-spanning proteins. Biochim. Biophys. Acta 815,468^76. Kiihlbrandt, W., Wang, D.N., & Fujiyoshi, Y. (1994). Atomic model of plant light-harvesting complex by electron crystallography. Nature 367, 614-621. Lakey, J.H., Duche, D., Gonzalezmanas, J.M., Baty, D., & Pattus, F. (1993). Ruorescence energy transfer distance measurements: the hydrophobic helical hairpin of colicinA in the membrane bound state. J. Mol. Biol. 230,1055-1067. Lemmon, M.A., Flanagan, J.M., Treutlein, H.R., Zhang, J., & Engelman, D.M. (1992). Sequence specificity in the dimerizatibn of transmembrane a-helices. Biochemistry 31,12719-12725. Lemmon, M. A., Treutlein, H.R., Adams, RD., Briinger, A.T., & Engelman, D.M. (1994). A dimerization motif for transmembrane a-helices. Nature Struct. Biol. 1,157-163. Luirink, J. & Dobberstein, B. (1994). Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11,9-13. Nilsson, I.M. & von Heijne, G. (1990). Fine-tuning the topology of apolytopic membrane protein role of positively and negatively charged residues. Cell 62, 1135-1141. Nishiyama, K., Hanada, M., & Tokuda, H. (1994). Disruption of the gene encoding pl2 (SecG) reveals the direct involvement and important function of SecG in the protein translocation of Escherichia coli at low temperature. EMBO J. 13, 32723277. Pakula, A.A. & Simon, M.I. (1992). Determination of transmembrane protein structure by disulfide cross-linking: the Escherichia coli Tar receptor. Proc. Natl. Acad. Sci. USA 89, 4144-4148. Pogliano, J.A. & Beckwith, J. (1994). SecD and SecF facilitate protein export in Escherichia coli. EMBO J. 13, 554-561. Popot, J.-L. & de Vitry, C. (1990). On the microassembly of integral membrane proteins. Annu. Rev. Biophys. Biophys. Chem. 19, 369-403. Popot, J.-L., Gerchman, S.-E., & Engelman, D.M. (1987). Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process. J. Mol. Biol. 198,655-676. Popot, J.L. & Engelman, D.M. (1990). Membrane protein folding and oligomerization: the 2-stage model. Biochemistry 29,4031-4037. Saaf, A., Andersson, H., Gafvelin, G., & von Heijne, G. (1994). SecA-dependence of the translocation of a large periplasmic loop in the E. coli MalF inner membrane protein is a function of sequence context. Mol. Memb. Biol. 12, 209-215.
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Sahin-Toth, M. & Kaback, H.R. (1993). Properties of interacting aspartic acid and lysine residues in the lactose permease of Escherichia coli. Biochemistry 32, 1002710035. Schatz, P.J. & Beckwith, J. (1990). Genetic analysis of protein export in Escherichia coli. Annu. Rev. Genet. 24, 215-248. Sipos, L. & von Heijne, G. (1993). Predicting the topology of eukaryotic membrane proteins. Eur. J. Biochem. 213, 1333-1340. Taylor, W.R., Jones, D.T., & Green, N.M. (1994). A method for a-helical integral membrane protein fold prediction. Proteins Struct. Funct. Genet. 18, 281-294. Treutlein, H.R., Lemmon, M.A., Engelman, D.M., & Briinger, A.T. (1992). The glycophorin A transmembrane domain dimer: sequence-specific propensity for a righthanded supercoil of helices. Biochemistry 31, 12726-12732. Tuffery, P., Etchebest, C., Popot, J.L., & Lavery, R. (1994). Prediction of the positioning of the seven transmembrane a-helices of bacteriorhodopsin: a molecular simulation study. J. Mol. Biol. 236, 1105-1122. von Heijne, G. (1986). The distribution of positively charged residues in bacterial inner membrane proteins correlates with the transmembrane topology. EMBO J. 5, 3021-3027. von Heijne, G. (1989). Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues. Nature 341,456-458. von Heijne, G. (1992). Membrane protein structure prediction: hydrophobicity analysis and the positive-inside rule. J. Mol. Biol. 225,487-494. von Heijne, G. (1994). Membrane proteins: from sequence to structure. Annu. Rev. Biophys. Biomol. Struct. 23, 167-192. von Heijne, G. & Gavel, Y. (1988). Topogenic signals in integral membrane proteins. Eur. J. Biochem. 174, 671-678. Whitley, P., Nilsson, L., & von Heijne, G. (1993). 3-Dimensional model for the membrane domain oiEscherichia coli leader peptidase based on disulfide mapping. Biochemistry 32, 8534-8539. Whitley, P., Nilsson, I., & von Heijne, G. (1994a). De novo design of integral membrane proteins. Nature Struct. Biol. 1, 858-862. Whitley, P, Zander, T, Ehrmann, M., Haardt, M., Bremer, E., & von Heijne, G. (1994b). Sec-independent translocation of a l(X)-residues long periplasmic N-terminal tail in the E. coli inner membrane proteins ProW EMBO J. 13,4658-4661. Wickner, W, Driessen, A.J.M., & Hartl, F.U. (1991). The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu. Rev. Biochem. 60, 101-124. Wolfe, P.B. & Wickner, W. (1984). Bacterial leader peptidase, a membrane protein without a leader peptide, uses the same export pathway as pre-secretory proteins. Cell 36, 1067-1072. Wolfe, P.B., Rice, M., & Wickner, W (1985). Effects of two sec genes on protein assembly into the plasma membrane of Escherichia coli. J. Biol. Chem. 260,1836-1841. Zen, K.H., Mckenna, E., Bibi, E., Hardy, D., & Kaback, H.R. (1994). Expression of lactose permease in contiguous fragments as a probe for membrane-spanning domains. Biochemistry 33, 8198-8206.
MODEL FOR INTEGRATING P-TYPE ATPases INTO ENDOPLASMIC RETICULUM
Randolph Addison and Jialing Lin
I. II. III. IV. V.
Introduction An Overview ofA^^Mrc?^/76>ra Plasma Membrane H"*"ATPase . . . Experimental Design Integration Model ofA^^Mra5/7ora Plasma Membrane H'^ATPase . . A General Integration Mechanism for P-Type ATPases and for Porters Acknowledgment References
215 216 218 226 231 232 233
r. INTRODUCTION The Neurospora plasma membrane proton-translocating ATPase (H*^ ATPase) transduces the chemical energy of ATP hydrolysis into a transmembrane proton-motive force (Scarborough, 1976). The latter in turn
Membrane Protein IVansport Volume 2, pages 215-235 Copyright © 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-983-4
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functions to drive the uptake and extrusion of various ions, metabolites, and nutrients through chemiosmotic coupling devices called porters (Mitchell, 1967). The Neurospora W ATPase forms a kinetically competent phosphoryl-enzyme intermediate during the catalytic cycle (Dame and Scarborough, 1980). Thus, this enzyme shares properties with the NaVK"", W/K", and the Ca^"" ATPases of higher eukaryotic cells. The purposes of this article are to discuss how manipulations at the cDNA level have been used to gain insights into the topogenesis of the Neurospora W ATPase, to propose a plausible model for its integration into the membrane, and to propose how this model can be applied to the integration of other polytopic integral membrane proteins.
II. AN OVERVIEW OF NEUROSPORA PLASMA MEMBRANE H^ ATPase The physiological role of the plasma membrane H"*^ ATPase from Neurospora and yeast was reviewed previously (Serrano, 1989). C. L. Slay man and associates (1970) provided the first experimental evidence for an electrogenic ATPase in the plasma membrane of Neurospora, They postulated that this electrogenic ATPase is a proton pump. It became possible to demonstrate this directly when Neurospora plasma membrane was isolated in high yield and purity by using a concanavalin A method (Scarborough, 1978). Experimental results with everted vesicles of the plasma membrane containing fluorescein isothiocyanate-labeled dextran provided direct proof that the electrogenic ATPase is a proton pump (Scarborough, 1980). Subsequently, the plasma membrane H^ ATPase was solubilized by detergent and purified to homogeneity (Addison and Scarborough, 1981; Bowman et al., 1981). The H"" ATPase has a molecular weight of approximately 105,000 when resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Biochemical characterization of the H"^ ATPase showed that the catalytic sequence involves a phosphoryl-enzyme intermediate (Dame and Scarborough, 1980), that vanadate is a potent inhibitor of ATP hydrolysis by the ATPase (Addison and Scarborough, 1981), and that the enzyme undergoes significant conformational changes during its catalytic cycle (Addison and Scarborough, 1982). Reconstituted proteoliposomes containing only the hydrolytic moiety of the H"^ ATPase provided evidence that the 105-kDa moiety coupled ATP hydrolysis to the generation of a proton-motive force (Scarborough and Addison, 1984).
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Biochemical experiments dearly established that the Neurospora and yeast plasma membrane H"^ ATPase have features in common with the NaVK^ and Ca^"^ ATPases, implying that cation-motive ATPases function via a similar mechanism (Dame and Scarborough, 1981; Mitchell, 1981). During recent years, the genomic DNA and the cDNA of the cation-motive ATPases were cloned and sequenced (Hesse et al., 1984; MacLennan et al., 1985; ShuU et al., 1985; Addison, 1986). A comparison of the deduced amino acid sequences of the ATPases revealed highly conserved regions (Addison, 1986). This observation combined with the biochemical data strongly supported the earlier conjectures that cation-motive ATPases operate through a common mechanism. This implies that all ion-motive ATPases evolved from a common ancestral cation-motive pump. To understand the molecular mechanism of these remarkable cationmotive ATPases, membrane topological models were proposed that were based mainly on hydropathy plots of the ATPases' primary sequence. These models predict that these proteins contain either 8- or 10-transmembrane segments with approximately 80% of the polypeptide chain on the cytoplasmic side of the membrane and less than 5% localized to the exoplasmic side of the membrane. By using different techniques, the putative energy-transduction domain, the ATP-binding and hydrolytic domains, and the amino and carboxyl termini have been localized to the cytoplasmic side of the membrane (Green, 1989; Clarke et al., 1990; Scarborough and Hennessy, 1990). The latter observation implies that these enzymes have an even number of transmembrane segments. Site-directed mutagenesis of the cDNAof the cation-motive ATPases is being used to generate mutants that are in turn used to gain insights into the structure-function relationships of these energy transducers (MacLennan, 1990). The membrane topological models as predicted by the hydropathy plots are used to select specific residues to mutate to determine their role as specific ligand-binding residues and their role in the energy transduction mechanism in general. These plots are, however, ambiguous for regions that span the membrane (Fasman and Gilbert, 1990; Jahnig, 1990). For instance, three topological models of the Neurospora plasma membrane H"*^ ATPase exist that have either 8,10, or 12 segments (Addison, 1986; Hager et al, 1986; Rao et al., 1991). Clearly, x-ray diffraction analysis of crystals of these cation-motive ATPases will resolve these problems and will provide reliable models of these enzymes. However, a major impedance to achieving this goal is the inability to obtain crystals of cation-motive ATPases that are suitable for
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x-ray structural analysis. Nevertheless, reliable models are needed to assist in designing and interpreting mutagenesis studies. To achieve this, an experimental technique is required that can verify whether a predicted transmembrane segment is within the lipid bilayer.
Hi. EXPERIMENTAL DESIGN An approach to verifying whether a stretch of residues is within the lipid bilayer is to study the topogenesis of polytopic integral membrane proteins. The molecular mechanism by which proteins obtain their characteristic arrangement across the osmotic barrier is poorly understood, however. Two fundamentally different models of protein integration have been proposed. One model proposes that a stretch of hydrophobic residues inserts directly into the hydrophobic interior of the lipid bilayer (Engelman and Steitz, 1981). Experimental observations have demonstrated that a stretch of hydrophobic residues is capable of partitioning spontaneously into liposomes or detergent micelles. This model, however, cannot explain the membrane selectivity of proteins— why are proteins inserted only into certain membranes and not others? Nor can this model explain the role of integral membrane proteins of the endoplasmic reticulum that are apparently essential for the integration event. The other model proposes that protein integrates into membranes by the decoding of topogenic sequences within proteins by proteinaceous effectors (Blobel, 1980). The characteristic asymmetrical arrangement of proteins in the membrane is achieved by the decoding of topogenic signals that are able to initiate and stop translocation across the membrane. This model, referred to as the sequential insertion model, was originally proposed by Blobel. It has been modified by experiments that unveiled proteins which are involved in the integration event, as well as by insights provided from in vitro studies of the integration of proteins into membranes. Blobel classified integral membrane proteins on the basis of the number of times the polypeptide chain spanned the membrane: (a) monotopic integral membrane proteins have their hydrophilic residues exposed on only one side of the membrane; (b) bitopic integral membrane proteins span the membrane once and have their hydrophilic residues exposed to both sides of the membrane; (c) polytopic integral membrane proteins span the membrane multiple times. In the sequential insertion model, a topogenic signal (termed a "starttransfer signal") would initiate translocation of carboxyl terminal resi-
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dues and a stop-transfer signal (another topogenic signal) would halt the translocation process. The topogenic signals are subsequently displaced (unspecified) from the putative integration machinery into the lipid bilayer. This model is attractive because it allows for the manipulation of the cDNA of a poly topic integral membrane protein by cut-and-paste types of experiments to assay for the presence of topogenic sequences as well as designing on the cDNA level a fusion protein to test the ability of a stretch of hydrophobic residues to function as a topogenic signal. That is, it is possible to engineer into the cDNA of a particular protein a DNA fragment coding a presumed topogenic signal. RNA transcripts generated frorn this template are then used to program an in vitro translation system supplemented with translocation competent microsomes. Integration or translocation of the resultant proteins into microsomes is readily determined by fractionation methods or by monitoring processing of the product, that is, N-linked glycosylation. Therefore, to study the topogenesis of the Neurospora H^ ATPase, RNA transcripts of the H^ ATPase were translated in a Neurospora in vitro system supplemented with microsomes isolated from Neurospora (Addison, 1987,1990). The H^ ATPase was integrated into microsomes. This was demonstrated by cosedimentation of the in vitro synthesized fj+ ATPase with microsomes after extracting the translation mixture with 0.1 M Na2C03 (pH .11.5). Although the H"" ATPase was integrated into microsomes, the results provided no insights into the integration process and provided no insights into the role, if any, of topogenic signals. To localize putative topogenic signals, the cDNA of the H"^ ATPase was restricted by restriction endonucleases within codons to generate RNA transcripts coding truncated H"^ ATPase of different lengths (Addison, 1991, 1993). These transcripts were translated in a Neurospora in vitro system. The products integrated into microsomes in a process that required nucleotides and microsomal proteins. Although these early results early established the presence of topogenic signals in the H^ ATPase, they provided no information about the exact position of or the number of signals involved in the integration of the truncated proteins into the microsomes. It proved difficult to remove putative transmembrane segments and test them individually or in tandem because of the absence of unique restriction endonuclease sites at suitable positions within the cDNA of the H"^ ATPase. To overcome this problem, we used the polymerase chain reaction (PCR) to construct on the cDNA level fusion proteins of H^ ATPase (Lin and Addison, 1994a).
RANDOLPH ADDISON and JIALING LIN
220 H+ATPase cDNA
T3 promoter
T3RNA polymerase
RNA transcripts I Neurospora in-vitro translation i system Fusion Proteins
Figure 1. Constructing the fusion proteins. cDNA fragments encoding the hydrophilic residues of the amino (rectangular box with diagonal lines) or carboxyl (rectangular box with dots) terminus and the putative transmembrane segments (solid rectangular boxes) were obtained using PCR and the Neurospora H"^ ATPase cDNA as a template. cDNA fragments encoding the invertase segment with consensus N-linked glycosylation sites (rectangular box with two dash lines) were obtained using PCR and yeast invertase cDNA as a template. In the cDNA of invertase, the gray box represents the region encoding the signal sequence. cDNA fragments were ligated into pBluescript II KS+ plasmid. The recombinant plasmid digested with PvuW was used as a template to generate RNA transcripts using T3 RNA polymerase. The transcripts were translated in a Neurospora in vitro system. Other details are in the text.
Figure 1 depicts the construction of fusion proteins of the Neurospora plasma membrane H^ ATPase. As diagrammed, DNA fragments were generated from the cDNA of the H"^ ATPase by using oligonucleotide primers that were complementary to the first 15 bases at the ends of the region that is to be amplified by PCR. The cDNA fragment coding
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hydrophilic residues 1-115 of the H^ ATPase and 170 bp of the 5' untranslated sequence was obtained using an oligonucleotide sense primer with a recognition sequence for HindlU and an antisense primer with recognition sequences for Pstl/BamHl. The DNA fragment coding hydrophilic residues 879-920 of the H"^ ATPase, a stop codon, and 15 bp of the 3' untranslated sequence was obtained using an oligonucleotide sense primer with a recognition sequence for Xbal and an antisense primer with a recognition sequence for Sacll. cDNA fragments coding the putative transmembrane segments were obtained using oligonucleotide sense primers with recognition sequences for Pstl/BamHl and antisense primers that had recognition sequences for EcoRVBamHl, cDNA fragment coding residues 73-132 of the secretory form of invertase was obtained using an antisense primer with a recognition sequence for Xbal and a sense primer that had recognition sequences for EcoRl/BamHL This invertase segment contains three consensus Nlinked glycosylation sites that will serve as a reporter of translocation into the microsomes. To ensure complete digestion of the cDNA fragments by the restriction endonucleases, three base pairs were added to the 5' end of each oligonucleotide primer. The addition of two recognition sequences in some primers gave us flexibility in generating certain combinations of putative transmembrane segments. For the PCR, singlestranded DNA of the cDNA was generated from the recombinant phagemid pBluescript II KS4- (Stratagene). PCR products were generated using the Perkin Elmer's GeneAmp kit and the Thermal cycler 880 following standard protocols. PrimerErase Quick push columns (Stratagene) were used to remove excess primers. The products were digested by restriction endonucleases, resolved on an agarose gel, and excised and eluted from the gel using Glassmilk or Glassfog (BIO 101, La JoUa, CA). cDNA fragments were ligated into pBluescript II KS+. DNA sequence of the constructs was confirmed by DNA sequencing using chain-terminating dideoxyl nucleotides. As mentioned, topological models of the Neurospora plasma membrane H"^ ATPase are based almost exclusively on hydropathic plots of the primary sequence that predict either 8- or 10-transmembrane segments (Addison, 1986; Hager et al., 1986). This method identifies stretches of hydrophobic residues that are long enough to span the membrane as a-helices. Another model of the H^ ATPase based on the exhaustive trypsin digestion of the reconstituted H^ ATPase proposes that the enzyme has 12-transmembrane segments (Rao et al., 1991). The 8and 10-segment models agree that there are four transmembrane seg-
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Table 1. Amino Acid Sequences of the Neurospora H^ ATPase Used in Constructing the Fusion Proteins N-terminus sequence: MADHSASGAPALSTNIESGKFDEKAAEAAAYQPKPKVEDDEDEDIDALIEDL ESHDGHDAEEEEEEATPGGGRVVPEDMLQTDTRVGLTSEEVVQRRRKYGLN 1-115 QMKEEKENHFLKPA AM 116-138 M1
GSFLGFFVGPIQFVMEG A AVLA AGLEF
M2
EWVDFGVICGLLLLNAVVGFV
M3
GSGIGTILLILVIFTLLIVWVSSFYEF * * ** AILEFTLAITIIGVPVGLPAVVTTTMAVGAAYLA *
141-160 292-314
M4
322-354 688-713
M5
AMYAYVVYRIALSIHLEIFLGLWIAIL
M6
ASLNIELVVFIAIFADVATLAIAYDNA
M7
ALWGMSVLLGVVLAVGTWITVTTMYA *
807-826
M8
AWLIFITRANGPFWSSIPSWQ
827-847
M9
ALSGAIFLVDILATCFTIWGWF
852-878
716-741 755-779
M10 ATSIVAV VRIWIFSFGIFCIMGG V Y YIL Invertase sequence: GSFWGHATSDDLTNWEDQPIAIAPKRNDSGAFSGSMVVEYNNTSGFF ** NDTIDPRQRCVAIWTSR
73-132
C-terminus sequence: QDSVGFDNLMHGKSPKGNQKQRSLEDFVVSLQRVSTQHEKSQ
879-920
Notes: The hydrophilic residues of the amino terminus of the H"^ ATPase are followed by the sequence of a putative transmembrane segment (M), which in turn is followed by the invertase segment and the hydrophilic residues of the carboxyl terminus of the enzyme. Amino acids marked with an asterisk were introduced during construction of the fusion proteins. The numbers of inclusive residues are given at the end of each sequence.
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ments in the amino terminal third, but disagree about the number of segments in the carboxyl terminal third of the protein. The 12-segment model has six transmembrane segments in the amino terminal third and six in the carboxyl third of the enzyme. With the protein fusion method it now became possible to verify the number of transmembrane segments in H"^ ATPase. This method should also provide means for accurately determining the nature of the topogenic signals required for integrating H"^ ATPase into the membrane. The amino acid sequences used in the fusion proteins are listed in Table 1. Constructs with a putative transmembrane segment were designated pM followed by a number that indicated the position of the segment within a model. As mentioned, in the sequential insertion model, oddnumbered transmembrane segments initiate translocation of the carboxyl terminal residues across the membrane, and even-numbered segments stop the transfer process. Therefore, if the transmembrane segments are correctly predicted, an odd-numbered segment engineered into the fusion protein should initiate transfer of the invertase segment into the lumen of the microsomes, and when this segment is combined with an evennumbered segment, glycosylation of the invertase segment should be blocked (see Figure 2). Using transmembrane segments as predicted in the 10-segment model (Table 1), we generated constructs containing M3, M5, or M7. The resultant proteins were glycosylated. These transmembrane segments functioned as predicted by the sequential insertion model. Experimental results from digestion of the membrane-integrated products by proteases, from fractionation of the translation mixtures on step-sucrose gradient under alkaline conditions, and from immunoprecipitation of the digested membrane-integrated products using polyclonal antibodies directed against the amino- or carboxyl-terminal residues showed that the proteins were integrated into microsomes with the amino terminus on the cis side and the carboxyl terminus on the trans side of the membrane (Lin and Addison, 1995a,b). In contrast, constructs containing Ml or M9 were not glycosylated. These constructs were also not associated with microsomes after extracting the translation mixtures at pH 11.5. Constructs containing M2, M4, M6, or M8 did not associate with microsomes. These segments are predicted to function as stop-transfer signals. Only MIO, a predicted stop-transfer segment, initiated translocation of the invertase segment into microsomes. Stop-transfer signals can sometime function as start-transfer signals. Therefore, it was not surprising that MIO could do so in this in vitro.system.
RANDOLPH ADDISON and JIALING LIN
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I
II
Figure2. Membranetopology of fusion proteins. Fusion proteins with one transmembrane segment (so//drecfangu/ar box) have the membranetopology of a bitopic integral membrane protein (I) with the carboxyl terminus and the invertase segment localized in the lumen of the microsomes. In the lumen, the invertase segment (rectangular box with diagonal lines) will be glycosylated. The arrows represent oligosaccharide side chains. Fusion proteins with two transmembrane segments will have a membrane topology of a polytopic integral membrane protein (II). In this membrane topology, the invertase segment remains on the cytosolic side of the membrane.
We noticed, however, that any putative transmembrane segments containing a negatively charged residue did not initiate transfer of the invertase segment into microsomes. These segments include Ml, M2, M4, M6, and M9 (see Table 1). M8 and MIO each contain a positively charged residue. MIO initiated transfer of the carboxyl terminal residues into microsomes; M8 did not. The latter also contains two prolines. It is possible that this combination of residues impaired the ability of this segment to function individually in the construct. One transmembrane segment, M5, functioned individually in the construct although it contains a negatively and a positively charged residue. These charged residues are seven residues apart. It is possible that these residues are able to form a salt bridge that would mask the ionic charges and create a functional topogenic signal. Charged residues in the transmembrane segments are presumed to function as cation-binding sites (Clarke et al., 1989). If so, these residues serve an essential function for cation-motive
Topogenesis of Neurospora tt ATPase
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ATPases. However, the presence of these charged residues in the segments impaired their ability to integrate into the membrane. Insight into this apparent paradox was provided when we engineered additional constructs containing two or more transmembrane segments. For instance, neither Ml nor M2 functioned in the constructs. In contrast, construct pMlM2 integrated into microsomes as a polytopic integral membrane protein. This implies that combining these segments created a topogenic signal for integration. The segments apparently acted concertedly in the integration event. Identical results were observed for the combination of M3, M4; M5, M6; M7, M8; and M9, MIO. In the aqueous milieu, these closely juxtaposed transmembrane segments must associate. This association is favored by the short stretch of residues between the segments and by hydrophobic force that gives a hydrophobic interaction energy of about 20 kcal/mol of paired segments (Engelman and Steitz, 1981). In the case of pMlM2, when the number of residues between the segments was gradually lengthened, the integration effectiveness of the resultant products decreased (Lin and Addison, 1995a). This implies that the proximity of the segments is crucial in forming the topogenic signal. In contrast, when residues between the segments in pM3M4 and in pM7M8 were lengthened, the intervening residues were transferred to the lumen of the microsomes and the termini of the proteins were localized to the cis (cytosolic) side of the membrane. Although some of the transmembrane segments of the H^ ATPase behaved as predicted by the sequential insertion model, others did not. Are these other segments integrated into the membrane by a novel mechanism? If so, this implies that integrating the H"^ ATPase into the membrane requires multiple integration machineries. In pM3M4 and pM7M8 the transmembrane segments functioned as predicted by the sequential insertion model; these segments are separated, however, by a short stretch of hydrophilic residues in the native H"^ ATPase and in the constructs. In this closely juxtaposed position, will the segments act concertedly or individually in integrating the H"^ ATPase into the membrane? When we replaced M2 in the construct pMlM2 by M4, the resultant protein integrated into microsomes as a polytopic integral membrane protein. This implies that the putative integration machinery recognizes some general structural feature in the paired segments. In pMlM2, three hydrophilic residues separate the segments; in pMlM4 and in pM3M4, eight residues separate the segments. Therefore, it is likely that M3 and M4 form a topogenic signal similar to that formed by M1 and M2. If this
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is true, this implies that integrating the H"^ ATPase into microsomes uses only one type of topogenic signal for integration and only a single integration machinery. This topogenic signal consists of two transmembrane segments. Since this topogenic signal is required for integrating the H"*^ ATPase into microsomes, we will refer to it as an integration signal. Also, our experimental results localized four transmembrane segments in the amino-terminal third and six in the carboxy-terminal third of H^ ATPase. These data support a 10-segment model for H"*^ ATPase.
IV. INTEGRATION MODEL OF NEUROSPORA PLASMA MEMBRANE H^ ATPase Before continuing to a plausible integration model for H^ ATPase, it might be useful to make a few remarks about two main types of molecular recognition mechanisms in living organisms. One type is described for enzymatic reactions: enzymes bind stereospecific substrates and catalyze the rearrangement of chemical groups or atoms. The other type is described for receptor binding: a receptor binds stereospecific molecules or stereospecific areas of biological polymers. Information is transmitted in the form of conformational changes to other region(s) of the receptor, or the resultant signal is transduced into another signal through the interactions of the receptor-ligand complex with other biological molecules. Chemical equilibrium in the cell is expressed using the Gibbs free energy AG' at constant pressure and temperature. Generally, the difference between AG° measured from standard states of reactants and products and AG' measured from the conditions under which the reaction is conducted is termed the thermodynamic drive, or driving force (AG*), of a particular reaction. This is expressed by the simple relationship: AG* = jdG The change in G* is the integral or sum over all increments of dG in different states of the reaction. This is best explained by giving as an example the transfer of a hydrophobic polymer from an aqueous milieu (phase 1) to a hydrophobic milieu (phase 2). The product of the moles of polymer in phase 1 times the difference in the chemical potential of the polymer in the standard states and in the states from which the reaction is conducted will be termed dG^^^ ^ The same type of calcula-
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227
tion for the polymer in phase 2 will give dG^^^^ 2- Therefore, the difference in G* would be the difference between Gphase2 ^^^ Gp^ase i- This AG* represents a measure of the hydrophobicity of the polymer and measures the system's ability to adopt a thermodynamically stable structure in phase 2 by minimizing interactions with water in phase 1. Integration of transmembrane segments (TS) into the membrane from the aqueous phase is viewed as a receptor-mediated process: TS
^^
^^
• TS
RTS
In this scheme, the receptor (R) is situated between the aqueous compartment (on the left), that is, the cytosol or the aqueous channel of the putative integration machinery, and the lipid bilayer (on the right), the hydrophobic phase. For the reaction to go from left to right, the overall sign of AG* must be negative. The difference between TS on the left and TS on the right of the diagram need involve only changes in the configuration or the conformation of secondary and ionic or noncovalent bonds such as those participating in the ionization or hydration of TS (these reactions are not indicated above). These changes may require an input of energy. This will make an intermediate dG positive. As stated, the overall sign of AG* must be negative for the reaction to go from left to right. This can be accomplished if the dG from the configuration changes is much smaller than the overall AG* from transferring a pair of a helices from one phase to another. Alternatively, the reaction could be coupled to an exergonic reaction. Our objective in introducing the foregoing arguments is to emphasize that integrating proteins into the lipid bilayer is similar to other basic biological recognition processes in living organisms. To integrate proteins into membranes, an effector of this process must function at the interface of and within two distinct compartments. Although integration has not received as much interest and investigation as the translocation process, we feel that an understanding of the details of the integration mechanism is just as interesting as that of translocation and that integration can be analyzed also by dissecting the integration process into
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separate catalytic and structural units. Studying components of the integration process may provide insights into novel protein-protein interactions that may be interesting in a much broader context. Based on the experimental results reviewed briefly in the above sections, a plausible model of how the Neurospora H^ ATPase is integrated into membrane can be formulated. The key features of this model are shown in Figure 3. The nature of the topogenic signal in this model is based on experimental evidence. But the binding of this signal to the integration machinery and its subsequent spatial displacement to the lipid bilayer are yet unsupported by any data. Therefore, this part of the model must remain conjectural. Since trypsin treatment of microsomes blocked integration (Lin and Addison, 1995a), we postulate that integration of H^ ATPase into microsomes is a receptor-mediated event. Integration begins with the binding of a pair of transmembrane segments to a binding cleft positioned at the interface of subunits of a multisubunit complex localized in the membrane. Reasons for placing the binding site here will be given below. This multisubunit complex could be identical to that used for translocating proteins across the membrane. When the pair of segments is integrated into the membrane, there is no translocation. Therefore, a ribosome engaged in synthesizing H^ ATPase will not form a tight ribosome-membrane junction. The osmotic barrier across the membrane is maintained because binding of the topogenic signal to the integration machinery will not gate the opening of a channel in the membrane. After binding to the receptor in the membrane, the next major consideration is the spatial displacement of the bound topogenic signal from the machinery to the lipid bilayer. The bound ligand is a pair of transmembrane segments that apparently remain unmodified upon binding to the putative receptor. Therefore, why would the receptor release a substrate for which it has a high affinity? As mentioned, enzymes (E) have a high affinity for their substrates (S). There is a finite amount of substrate released after binding. The overall reaction is forced through the ES complex to the formation of the transition state (Segel, 1975). The substrate is eventually modified. The resultant product(s) is released from the enzyme because the product(s) has a lower binding affinity with the enzyme than the substrate. So why would a pair of bound and apparently unmodified transmembrane segments be released from the receptor? Insight into one possibility of how the segments are released is provided by experimental evidence. Integration into microsomes requires ATP and GTP (Lin and Addison, 1995a). The latter may be required for a SRP-SRP receptor-
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Cytosol Membrane Lumen Figure 3. Integration nnodel of P-Type ATPases. Pair of transmembrane segments (solid rectangular boxes) is decoded by a soluble effector (R1), which in turn targets the resultant complex at the membrane, where R1 interacts with its cognate receptor (R2). The integration signal is transferred to the integration machinery (X/Y). Other proteins, represented by a question mark, may be required for this step. Alternatively, the integration signal interacts directly with the machinery, bypassing R1. The binding site for the integration signal in the machinery is depicted at the interface of subunits X and Y. The binding site could be composed of identical subunits of the complex. This possibility is not depicted in the above diagram. The pair of segments is channeled to the membrane (two horizontal lines).
mediated targeting event, analogous to that found in canine pancreas microsomes (Figure 3). But what is the ATP used for? One possibility is that binding and/or hydrolysis of ATP could be used to drive the channeling of the ligand from the receptor to the lipid bilayer. Alternatively, another, more attractive possibility is that in the aqueous milieu the pair of transmembrane segments has a secondary or tertiary structure that is different form that in a hydrophobic milieu. This conjecture is supported
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by experimental evidence demonstrating a milieu-specific configuration of hydrophobic peptides (Li and Deber, 1993). If this is true, we postulate that the pair of segments that binds to the putative receptor either directly from the cytosolic compartment or through a SRP-SRP receptor-mediated targeting pathway has a specific conformation that is subsequently altered at the receptor-binding site. This could occur in an ATP-dependent fashion, analogous to the action of heat shock proteins, or the putative receptor itself could induce specific alteration in the conformation of the segments, analogous to the binding of antigens to antibodies. In either case, the net effect of these conformational maturations is to alter the affinity of the substrate, a pair of transmembrane segments, for the receptor. This will drive the ligand from the receptor down a hydrophobic gradient to the lipid bilayer. Since we have posited that the integration machinery will have an aqueous channel, the receptor will be positioned between two phases: the aqueous phase of the channel and the hydrophobic phase of the lipid bilayer. This will create a hydrophobic potential difference across the receptor, and this will serve as the driving force for channeling the pair of segments from the receptor to the lipid bilayer. This integration mechanism, however, will explain the integration of four of the possible five pairs of transmembrane segments of H^ ATPase into the membrane. The exception to this model is the pair M7, M8. These segments are separated by 27 residues. In the 10-segment model, these residues are localized on the exoplasmic side of the membrane. Translocation of these across the membrane can be accommodated within the framework of the above model, however. We have postulated that the substrate for the receptor is a pair of segments. Therefore, the binding of a single segment to the receptor will not elicit reactions as outlined above. M7 will bind and remain associated with the receptor. Consequently, the ribosomes will become tightly associated with the putative machinery and eventually will form a tight membrane junction. Polypeptide synthesis will continue. The growing chain will evoke the opening of the gated channel and will be transferred to the lumenal side of the membrane. Upon entering the aqueous compartment of the channel, M8 will bind to the receptor next to M7, concomitantly evoking specific conformation changes in the resultant pair of segments and the channeling of these to the lipid bilayer. As mentioned, in the sequential insertion model (Blobel, 1980), the odd-numbered transmembrane segments initiate translocation of the carboxy-terminal residues into the lumen of the endoplasmic reticulum
Topogenesis of Neurospora H^ ATPase
2 31
and the even-numbered segments stop the translocation event. A series of these events resuhs in the asymmetrical arrangement of the protein in the membrane. This model is a direct function of the decoding of individual transmembrane segments. The integration model outlined herein differs from the sequential insertion model in three major ways: (a) the topogenic signal for integration is a pair of transmembrane segments; (b) the putative integration machinery does not disassemble in order to release the segments into the lipid bilayer; and (c) a putative start-transfer signal does not gate the channel, but instead simply holds the ribosomes in proximity to the machinery. Subsequent events will lead to the formation of a tight ribosome membrane junction. The growing polypeptide chain opens the channel to the lumenal side of the membrane. This conjecture is supported by the fact that proteins without a signal sequence are translocated across the endoplasmic reticulum (Wiedmann et al., 1994) and by the fact that the segment of nascent secretory proteins localized in the aqueous compartment of the gated channel is not accessible from the lumenal compartment to probes (Crowley et al., 1994). Taken together, this suggests that the channel is not gated by the signal sequence but by a segment of the nascent polypeptide chain.
V. A GENERAL INTEGRATION MECHANISM FOR P-TYPE ATPases AND FOR PORTERS Since the Neurospora plasma membrane H"^ ATPase, as mentioned in the introduction, is a member of a family of cation-motive ATPases, it is reasonable to assume that all P-type ATPases will have a common integration mechanism. That is, the topogenesis of these energy transducers is achieved by a receptor-mediated, energy-dependent integration of pairs of transmembrane segments into the membrane. Can the results obtain from studying Neurospora H^ ATPase be applied to the integration of other polytopic membrane proteins? Porters, that is, uniporters, antiporters, and symporters, are polytopic integral membrane proteins involved in the import or export of sugars, metabolites, ions, and peptides across membranes. They have an even number of transmembrane segments, with most of these containing charged residues that function as specific ligand-binding sites. They have few residues on the exoplasmic side of the membrane and few residues between the transmembrane segments. Accordingly, porters share fundamental features with P-type ATPases. It is reasonable to assume that pairs of transmem-
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brane segments play a fundamental role in integrating porters into the membrane. If this is true, it is possible that all polytopic integral membrane proteins have a fundamentally similar integration mechanism that varies only in the length of the polypeptide chain translocated across the membrane and in the number of segments integrated into the membrane. The concepts that we have tried to convey in our description of how a P-type ATPase is integrated into the membrane are the key role of a pair of transmembrane segments in the integration event, the role of the receptor in mediating the conformational maturation of the bound transmembrane segments, and the channeling of the modified segments down a hydrophobic potential gradient. Integration into the membrane occurs as the natural consequent of the intrinsic asymmetrical arrangement of the receptor in the membrane that leads to a vectorial nature in the way the substrate, a pair of transmembrane segments, binds to and is released from the binding site. We emphasized, also, that effectors of the integration process are thermodynamically stable structures and are not assembled from various components to form the integration machinery, nor are they disassembled to release the segments into the membrane. Whether this model is ultimately proved correct, it does provide a reasonable explanation of how transmembrane segments containing charged and/or proline residues integrate into the lipid bilayer. In contrast, the sequential insertion model cannot provide a reasonable explanation of the cooperative interaction of transmembrane segments in the integration event or of how segments containing charged residues can integrate into the membrane. It has been hoped by some that a structural description of polytopic integral membrane proteins would provide valuable insight into the nature of the topogenesis signals that are essential for their integration into the lipid bilayer. So far this type of data has provided no insight into the nature of the signals used to integrate these proteins into the membrane. Instead, the information that is currently available suggests specific interactions among the transmembrane segments and has shown that only a small percentage of the surface of the segments is in direct contact with the lipid bilayer (Rees et al., 1989). It is hoped that attempts to confirm or deny the principles as outlined herein will lead to progress in understanding the molecular mechanism of integration.
ACKNOWLEDGMENT Supported in part by National Science Foundation grant DCB-9108460.
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REFERENCES Addison, R. (1986). Primary structure of the Neurospora plasma membrane H"^ ATPase deduced from the gene sequence. J. Biol. Chem. 261,14896-14901. Addison, R. (1987). Secretory protein translocation in a Neurospora crassa in vitro system: hydrolysis of a nucleoside triphosphate is required for posttranslational translocation. J. Biol. Chem. 262, 17031-17037. Addison, R. (1990). Studies on the sedimentation behavior of the Neurospora crassa plasma membrane H"^ ATPase synthesized in vitro and integrated into homologous microsomal membranes. Biochim. Biophys. Acta 1030,127-133. Addison, R. (1991). GTP is required for the integration of a fragment of the Neurospora crassa H"*" ATPase into homologous microsomal vesicles. Biochim. Biophys. Acta 1065, 130-134. Addison, R. (1993). The initial association of a truncated form of the Neurospora plasma membrane H"*" ATPase and of the precursor of yeast invertase with microsomes are distinct processes. Biochim. Biophys. Acta 1152,119-127. Addison, R. & Scarborough, G.A. (1981). Solubilization and purification of the Neurospora plasma membrane H"*" ATPase. J. Biol. Chem. 256,13165-13171. Addison, R. & Scarborough, G.A. (1982). Conformational changes of the Neurospora plasma membrane H"*" ATPase during its catalytic cycle. J. Biol. Chem. 257, 10421-10426. Blobel, G. (1980). Intracellular protein topogenesis. Proc. Natl. Acad. Sci. USA 77, 1496-1500. Bowman, B.J., Blasco, F., & Slayman, C.W. (1981). Purification and characterization of the plasma membrane ATPase of Neurospora crassa. J. Biol. Chem. 256,1234312349. Clarke, D.M., Loo, T.W., Inesi, G., & Maclennan, D.H. (1989). Location of high affinity Ca "^-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca^'^-ATPase. Nature 339,476^78. Clarke, D.M., Loo, T.W., & Maclennan, D.H. (1990). The epitope for monoclonal antibody A20 (amino acids 870-890) is located on the lumenal surface of the Ca^"*" ATPase of sarcoplasmic reticulum. J. Biol. Chem. 265,17405-17408. Crowley, K.S., Liao, S., Worrell, V.E., Reinhart, G.D., & Johnson, A.E. (1994). Secretory proteins move through the ER membrane via an aqueous, gated pore. Cell 78, 461-471. Dame, J.B. & Scarborough, G.A. (1980). Identification of the hydrolytic moiety of the Neurospora plasma membrane H"*" ATPase and demonstration of a phosphoryl-enzyme intermediate in its catalytic mechanism. Biochemistry 19, 2931-2937. Dame, J.B. & Scarborough, G.A. (1981). Identification of the phosphorylated intermediate of the Neurospora plasma membrane H"^ ATPase as P-aspartyl phosphate. J. Biol. Chem. 256,10724-10730. Engelman, D.M. & Steitz, T.A. (1981). The spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis. Cell 23,411-422. Fasman, G.D. & Gilbert, W.A. (1990). The prediction of transmembrane protein sequences and their conformation: an evaluation. Trends Biochem. Sci. 15, 89-92. Green, N.M. (1989). ATP-driven cations pumps: alignment of sequences. Biochem. Soc. Trans. 17,972-974.
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Hager, K.M., Mandala, S.M., Davenport, J.W., Speicher, P.W., Benz, EJ., & Slayman, C.W. (1986). Amino acid sequence of the plasma membrane ATPase of Neurospora crassa: deduction from genomic and cDNA sequences. Proc. Natl. Acad. Sci. USA 83, 7693-7697. Hesse, J.E., Wieczorek, L., Altendorf, K., Reicin, A.S., Dorus, E., & Epstein, W. (1984). Sequence homology between two membrane transport ATPases, the Kdp-ATPase of Escherichia coli and the Ca ATPase of sarcoplasmic reticulum. Proc. Natl. Acad. Sci. USA 81,4746-4750. Jahnig, F. (1990). Structure predictions of membrane proteins are not that bad. Trends Biochem. ScL 15,93-95. Li, S-C. & Deber, CM. (1993). Peptide environment specifies conformation: helicity of hydrophobic segments compared in aqueous, organic, and membrane environments. J. BioL Chem. 268, 22975-22978. Lin, J. & Addison, R. (1994a). Topology of the Neurospora plasma membrane H"*" ATPase: localization of a transmembrane segment. J. Biol. Chem. 269,3887-3890. Lin, J. & Addison, R. (1995a). A novel integration signal that is composed of two transmembrane segments is required to integrate the Neurospora plasma membrane H"*" ATPase into microsomes. J. Biol. Chem. 270,6935-6941. Lin, J. & Addison, R. (1995b). The membrane topology of the carboxyl terminal third of the Neurospora plasma membrane H"*" ATPase. J. Biol. Chem. 270,6942-6948. MacLennan, D.H. (1990). Molecular tools to elucidate problems in excitation contraction coupling. Biophys. J. 58, 1355-1365. MacLennan, D.H., Brandle, C.J., Korezak, B., & Green, N.M. (1985). Amino-acid sequence of a Ca + Mg -dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature 316,696-7(X). Mitchell, P. (1967). Translocation through natural membranes. Adv. Enzymol. 29,33-87. Mitchell, R (1981). In: Of Oxygen, Fuels, and Living Matter (Semenza, G., ed.). Part I, pp. 1-56. John Wiley and Sons, Chichester, England. Rao, U.S., Hennessey, J.P., & Scarborough, G.A. (1991). Identification of the membraneembedded regions of the Neurospora crassa plasma membrane H"*" ATPase. J. Biol. Chem. 266, 14740-14746. Rees, D.C., Komiya, H., Yeates, TO., Allen, J.R, & Feher, G. (1989). The bacterial photosynthetic reaction center as a model for membrane proteins. Annu. Rev. Biochem. 58, 607-633. Scarborough, G.A. (1976). Proton translocation catalyzed by the electrogenic ATPase in the plasma membrane of Neurospora. Proc. Natl. Acad. ScL USA 73,1485-1488. Scarborough, G.A. (1978). Isolation of plasma membrane from Neurospora. Methods CellBiol. 20, 117-133. Scarborough, G.A. (1980). The Neurospora plasma membrane ATPase is an electrogenic pump. Biochemistry 19, 2925-2931. Scarborough, G.A. & Addison, R. (1984). On the subunit composition of the Neurospora plasma membrane H"^ ATPase. J. Biol. Chem. 259,9109-9114. Scarborough, G.A. & Hennessy, J.R, Jr. (1990). Identification of the major cytoplasmic regions of the Neurospora crassa plasma membrane H"*" ATPase using protein chemical techniques. J. Biol. Chem. 265, 16145-16149. Segel, I.H. (1975). In: Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems. John Wiley & Sons, New York.
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Serrano, R. (1989). Structure and function of plasma membrane ATPase. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 61-94. Shull, G.E., Schwartz, A., & Lingrel, J.B. (1985). Amino-acid sequence of the catalytic subunit of the (Na"*" + K'^)ATPase deduced from a complementary DNA. Nature 316,691-695. Slayman, C.L., Lu, C.Y-H., & Shane, L. (1970). Correlated changes in membrane potential and ATP concentrations in Neurospora. Nature 226, 274-276. Wiedmann, B., Sakai, H., Davis, T.A., & Wiedmann, K. (1994). A protein complex required for signal-sequence-specific sorting and translocation. Nature 370,434440.
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NUCLEAR TRANSPORT AS A FUNCTION OF CELLULAR ACTIVITY
Carl M. Feldherr and Debra Akin
I. Introduction II. Macromolecular Transport across the Nuclear Envelope III. Regulation of Transport across the Nuclear Envelope A. Regulation of Specific Proteins B. Changes in Transport Capacity IV. Conclusions Acknowledgments References
237 238 240 240 241 254 255 256
I. INTRODUCTION Considering the strategic location of the nuclear envelope in eukaryotic cells, it could play an important role in regulating the transcription and translation of genetic information by modulating the nucleocytoplasmic exchange of macromolecules. In this report, we discuss the data relating to this fundamental question, specifically, whether the capacity for
Membrane Protein Transport Volume 2, pages 237-259 Copyright © 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-983-4
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CARL M. FELDHERR and DEBRA AKIN
signal-mediated nuclear transport varies as a function of cellular activity. Before considering these results, however, it is necessary to review the basic mechanisms by which large molecules or molecular aggregates are able to cross the nuclear envelope. Since the overall transport process as well as specific elements of the transport machinery have been the subject of numerous recent reviews (e.g., Forbes, 1992; Izaurralde and Mattaj, 1992; Newmeyer, 1993; Fabre and Hurt, 1994; Feldherr and Akin, 1994a; Pante and Aebi, 1994), only those aspects of the exchange process relevant to regulation are summarized in the next section.
II. MACROMOLECULAR TRANSPORT ACROSS THE NUCLEAR ENVELOPE The exchange of macromolecules across the envelope occurs through the nuclear pores, by either passive diffusion or signal-mediated transport. Diffusion occurs through a 90-100-A channel located at the center of each pore (Paine et al., 1975; Peters, 1986) and, perhaps, at peripheral sites along the pore margins (Hinshaw et al., 1992). The rates of diffusion are inversely related to the dimensions of the permeant molecules; for example, dextran with a hydrodynamic diameter of 24 A will equilibrate between the nucleoplasm and cytoplasm within 30 minutes after being injected into Xenopus oocytes, whereas 46 A dextran requires approximately 15 hours for equilibration. When the diameter increases to 71 A, the equilibration time increases to approximately 7 days (Paine et al., 1975). Signal-mediated transport of macromolecules across the envelope is a multistep process that is initiated by specific targeting signals. Signals that direct nuclear import are generally referred to as nuclear localization signals (NLSs) and have been identified in over 50 proteins (Richter and Standiford, 1992; BouHkas, 1994). Although there is no consensus sequence, NLSs can be divided into two general categories, simple and bipartate. The simple signals are short basic domains containing fewer than ten amino acids, whereas the bipartate signals comprise two basic regions separated by ten or more spacer amino acids. The large T antigen NLS (PKKKRKV; Kalderon et al, 1984) and the nucleoplasmin NLS (AVKRPAATKKAGQAKKKKLDGG; Robbins et al., 1991), respectively, are prototypes of these two signal categories. There is reason to believe that the signal content of specific proteins can play a regulatory role in nuclear import. In this regard, it has been demonstrated that NLSs are not all equally effective in transport. Fur-
Nuclear Transport and Cellular Activity
239
thermore, both the rate of nuclear import and the effective size of the transport channel (see below) are dependent on the number of available signals, which can vary among different proteins. The significance of signal content in nuclear protein uptake has been reviewed in detail by Feldherr and Akin (1994a). The NLSs function by interacting with cytoplasmic transport factors that are thought to direct the signal-containing permeant molecules to docking sites along the cytoplasmic surface of the pores. The nature of the cytoplasmic factors involved in this process is currently under investigation in several laboratories. So far, the following proteins have been identified as transport components; a 54~55-kDa polypeptide that functions in NLS binding (Adam and Gerace, 1991), a 97-kDa polypeptide (Adam and Adam, 1994), and Ran/TC4, which is a 25-kDa GTPase (Melchior et al., 1993; Moore and Blobel, 1993). Whether the complex between the permeant and cytoplasmic factors dissociates at the pore surface or enters the nucleus prior to dissociation is not known. After binding to the pore surface, translocation across the envelope occurs through the central region of the pore complex, the same area that functions in diffusion. However, in the presence of an NLS the central channel is able to dilate from approximately 90 A to over 230 A in diameter in normal proliferating cells. The existence of a central transport channel is supported by both functional and morphological studies (i.e., Feldherr et al., 1984; Akey and Radermacher, 1993). Other than the fact that the translocation step is energy dependent (Newmeyer and Forbes, 1988; Richardson et al., 1988) and the degree of dilation of the central channel is variable and related to signal number (Dworetzky et al., 1988), there is no information regarding the mechanism of exchange through the pore complex. Based largely on its molecular mass (approximately 125 x 10^ kDa), it has been estimated that the pore complex is made up of over 100 different proteins, referred to as nucleoporins. Of this hypothetical number, at least 12 vertebrate nucleoporins have been identified. These include (1) a family of 0-linked glycoproteins that bind wheat germ agglutinin (WGA) and contain FXFG repeat sequences, (2) transmembrane proteins that extend from the pore complex into the lumen of the nuclear envelope, and (3) at least one protein that is associated with the fibrillar elements that extend from the cytoplasmic surface of the pores (Starr and Hanover, 1992; Fabre and Hurt, 1994). At least three nucleoporins in yeast also contain FXFG repeats; however, it is not known whether these homologs of the vertebrate proteins are also glycosylated.
240
CARL M. FELDHERR and DEBRA AKIN
In addition, a second subfamily of yeast nucleoporins has been identified; the proteins that make up this group contain up to 33 copies of a GLFG motif (Wente et al, 1992). It has been shown in vertebrate cells that WGA or antibodies against the pore glycoproteins block signal-mediated nuclear import, but not passive diffusion. Based on a report by Steme-Marr et al. (1992) that 0-linked glycoproteins can bind essential cytoplasmic transport factors, it is possible that these substances act as receptors. In yeast, at least two of the nucleoporins (one each in the FXFG and GLFG subfamilies) are nonessential, although deletions are lethal when accompanied by mutations of other nucleoporins. These results suggest that specific pore proteins may have overlapping functions. There are also indications that the transmembrane proteins can modulate both mediated and passive transport through the pores (Greber and Gerace, 1992). Hi. REGULATION OF TRANSPORT ACROSS THE NUCLEAR ENVELOPE A. Regulation of Specific Proteins Cells have developed several strategies for controlling the nucleocytoplasmic distribution of specific regulatory proteins. These include (1) anchoring to cytoplasmic elements, (2) masking the NLS by binding to transport inhibitors, and (3) phosphorylation at sites flanking the NLS. These mechanisms are illustrated by the following examples. In the inactive state, the catalytic subunits of the cAMP-dependent kinase (PKA) are anchored to regulatory polypeptides associated with the Golgi. In the presence of cAMP, the catalytic subunits, but not the regulatory elements, are released from their anchorage sites and enter the nucleus. As the cAMP concentration decreases, the inactive PKA complex reforms at the Golgi (Nigg et al., 1985). Masking of the NLSs is required for the cytoplasmic retention of the transcription factors NF-KB, Rel, and dorsal (Govind and Steward, 1991; Schmitz et al., 1991; Blank et al., 1992; Grimm and Baeuerle, 1993). The inhibitors that bind these proteins can be released by several different stimuli (e.g., kinases or second messengers), thus initiating the nuclear import process. The cell-cycle-dependent transcription factor SWI5 in yeast is localized in the cytoplasm of cells in S, G2, and M. During these phases of division, three serine residues associated with the NLS are phosphorylated. Translocation of SWI5 into the nucleus, which occurs during Gj, is
Nuclear Transport and Cellular Activity
241
accompanied by dephosphorylation of the serines (Moll et al., 1991). Similarly, there is evidence that the effectiveness of the SV40 large T NLS is related to the phosphorylation of casein kinase II sites (which increases transport) or cdc2 kinase sites (which decreases the level of import) in the region flanking the signal (Rihs and Peters, 1989; Jans et al., 1991; Rihsetal., 1991). B. Changes in Transport Capacity
In addition to the above mechanisms that affect the nucleocytoplasmic exchange of specific macromolecules, it is conceivable that more general changes in signal-mediated transport, which are the direct result of alterations in the transport machinery itself, occur during different stages of cellular activity. This possibility was initially considered by Feldherr and Akin (1990), who investigated the nuclear import of nucleoplasmincoated gold (NP-gold) in proliferating, quiescent, and differentiating 3T3-L1 cells. The NP-gold was introduced into the cytoplasm by microinjection. The cells were fixed 30 minutes later and subsequently examined with a transmission electron microscope (Figure 1). Nucleoplasmin is a karyophilic protein found in Xenopus oocytes. It is com-
^' -
;1
Figure 1. An electron micrograph of a proliferating 3T3-L1 cell showing the intracellular distribution of NP-gold (arrows) 30 minutes after injection into the cytoplasm. By this time, the mean N/C gold ratio was 0.67. N, nucleus; C, cytoplasm. Bar, 0.5 |xm.
CARL M. FELDHERR and DEBRA AKIN
242
• Cytoplasm C3 Nuclei, proliferating 0 Nuclei, confluent
40-80
80-120
120-160
200 +
Particle Size (A) Figure 2. The size distribution of NP-gold particles that entered the nuclei of proliferating and confluent 3T3-L1 cells 30 minutes after injection. The size of the particles that were initially injected into the cytoplasm and were available for transport is also shown. From Feldherr and Akin (1990).
posed of five 22 kDa subunits, each of which contains one copy of a well-characterized bipartate NLS (see above), which is highly effective in nuclear targeting. By adsorbing the nucleoplasmin to colloidal gold, it is possible not only to determine the relative rates of nuclear import (by counting gold particles in equal areas of nucleoplasm (N) and cytoplasm (C) and calculating the N/C ratio), but also to establish the functional size of the transport channels (by measuring the particles that entered the nucleus). It was found in these studies that the N/C gold ratio obtained for 110-270-A gold particles (total diameter including the 30-A protein coat) was significantly greater in proliferating as compared to quiescent cells (0.67 vs. 0.09, respectively). In addition to the decrease in relative import, there was a significant decrease in the size of the particles that were able to enter the nuclei of quiescent cells (see Figure 2), reflecting a change in the functional diameter of the transport channel from approximately 230 A to 190 A. After differentiation of the quiescent cells to adipocytes, nuclear import of NP-gold returned to the proliferating cell level. It could be demonstrated that the results were not due to either
Nuclear Transport and Cellular Activity
243
changes in pore number or differences in the intracellular migration patterns of the larger gold particles during different physiological states, leading to the conclusion that alterations had occurred in the transport process itself. A significant decrease in the relative import of large NP-gold particles was also observed when proliferating BALB/c 3T3 cells entered GQ after growing to confluence or after serum depletion (Feldherr and Akin, 1991). In fact, the functional decrease in pore size in these cells was as great as 100 A, compared to approximately 40 A in 3T3-L1 cells. In these studies it was also found that the decrease in import rate that occurs during growth arrest, at least in BALB/c cells, is size dependent; thus, the N/C ratio of small NP-gold particles (50-80 A total diameter) did not change significantly as proliferating cells became quiescent. These results (i.e., the reduced uptake of large but not small gold particles) are consistent with the interpretation that transport in quiescent cells is limited primarily by a decrease in the exclusion size limit of the exchange channels. The above results show that nuclear transport capacity can vary as a function of cellular activity. It has been suggested (Feldherr and Akin, 1991) that one of the more important consequences of the observed changes would be a decrease in the efflux of RNP particles, since they have dimensions that can exceed the functional size of the pores in quiescent cells. This would explain, at least in part, the decrease in both cytoplasmic rRNA and protein synthesis that is known to occur during quiescence (Levine et al., 1965; Johnson et al., 1974, 1976). Since growth factors are required to maintain the proliferating state, it is possible that they are also involved in modulating nucleocytoplasmic transport. This was investigated by studying the effects of PDGF, IGF-1, and epidermal growth factor (EGF) on the recovery of nuclear transport capacity as quiescent BALB/c cells reentered the growth cycle (Feldherr and Akin, 1993). PDGF induces competence in GQ cells, and subsequent treatment with EGF and IGF-1 is necessary for progression through the cell cycle (Yang and Pardee, 1986; Ross et al., 1987). Nuclear transport in serum-depleted cells (cultured in 0.5% calf serum for 4 days) was assayed, using NP-gold, at intervals of 6, 12,18, and 24 hours after the addition of either growth factors or 10% serum. The results can be summarized as follows. Recovery of transport activity, after the addition of 10% calf serum, occurred gradually and was complete after 18-24 hours. Partial recovery was achieved by adding growth factors individually at physiological concentrations in either 0% or 0.5% calf serum.
244
CARL M. FELDHERR and DEBRA AKIN
Complete recovery in the absence of serum required both EGF and IGF-1. Surprisingly, PDGF acted as an antagonist when added to this mixture, suggesting that in combination these factors could function as either positive or negative regulators of transport. Whether growth factors act by altering the levels of translation of specific transport components or by causing posttranslational modifications (e.g., by activating kinases or phosphatases) remains to be determined. Cell Fusion
Experiments
To determine whether the differences in nuclear transport capacity between proliferating and quiescent cells are due to cytoplasmic agents (specifically, ATP or transport factors that are known to be necessary for nuclear import) or variations in the properties of the nuclear envelope, Feldherr and Akin (1993) studied nuclear import of NP-gold in mechanically fused cells. Proliferating and serum-starved BALB/c 3T3 cells were superimposed and simultaneously punctured with a microneedle. The cells were left undisturbed for 50 to 60 minutes after fusion to allow thorough mixing of the cytoplasmic components. They were then injected with an NP-gold fraction containing 110-270-A particles (total diameter), in order to analyze nuclear import. The results are shown in Table 1. It was initially found, in control studies, that the experimental procedure had no detectable effect on nuclear import; thus, when growtharrested cells were fused together, nuclear transport remained at a level normally observed for quiescent cells. Similarly, fusion of proliferating Table 1. Nuclear Uptake of Nucleoplasmin-Gold in Fused BALB/c Cells N/C Ratio (Mean-v SE) Fusion Quiescentquiescent Proliferatingproliferating Proliferatingquiescent
No. of Fusions 2 3 8
Quiescent 0.23 + 0.02(416)*' — 0.30 + 0.02(1727)
Proliferating
Significance^
—
—
1.67 + 0.06(2114)
—
1.36 + 0.06(2531)
s
Notes: ^ s indicates that there is a significant difference {P < 0.01) in NP-gold uptake in quiescent versus proliferating nuclei. From Feldherr and Akin (1993). Number of particles counted in parentheses. Source: From Feldherr and Akin (1993).
Nuclear Transport and Cellular Activity
245
cells did not alter nuclear import of NP-gold. In the heterokaryons, it was found that the difference in transport capacity between proliferating and quiescent nuclei was maintained after cell fusion. Since the nuclei of the fused cells shared the same cytoplasm, it was deduced that the differences in transport were due to variations in the properties of the nuclear envelope (presumably at the level of the pores), rather than changes in the availability or activity of cytoplasmic components. In addition to a reduction in transport capacity, the number of NP-gold particles associated with the pores was also significantly lower in quiescent versus proliferating nuclei, suggesting a difference in the effective number of pore complex receptors. This could be an important factor in regulating transport if the number of receptor-signal interactions determines the functional pore size, as suggested by Dworetzky et al. (1988). The Effects of Cell Shape
A number of investigators have demonstrated a relationship between cell shape and metabolic activity. In anchorage-dependent cells, for example, conversion from an extended to a more spherical state is accompanied by a significant change in nuclear function, specifically a decrease in the synthesis of DNA and RNA (Ingber et al., 1987; Ingber and Folkman, 1989). To determine whether signal-mediated nuclear transport capacity is similarly affected, Feldherr and Akin (1993) investigated the nuclear import of NP-gold in B ALB/c cells as a function of shape. Cells in different configurations were obtained according to the method reported by Ireland et al. (1987). In the initial step of this procedure, coverslips are coated with poly-HEMA (poly-2-hydroxyethyl methacrylate), which forms a nonadhesive coat that is unable to support cell attachment and growth. Palladium is then deposited on the polyHEMA through a copper mask that contains circular openings ranging in diameter from 22 to 80 |Lim. Cells that are plated on these surfaces attach only to the palladium; those adhering to the small domains remain rounded, whereas cells that attach to the larger areas of palladium are extended. The relative uptake rates of two different size NP-gold fractions (50-80 A and 110-270 A, total diameter) into the nuclei of rounded and extended cells is shown in Table 2. These results demonstrate that transport capacity is reduced in rounded cells, and, consistent with the changes detected in quiescent cells, the decrease is size dependent. Assuming that cell geometry is a reflection of cytoskeletal organization, transport capacity could be modulated by dynamic changes in the
246
CARL M. FELDHERR and DEBRA AKIN Table 2. The Effect of Cell Shape on Nuclear Transport N/C
Experiment A. Rounded Extended B. Rounded Extended Notes:
No. of cells NP'GoldSize 19 19 19 19
110-270 110-270 50-80 50-80
Mean ± SE
0.52 + 0.09 (644)*' 1.59 + 0.14(872) 1.51+0.15(2724) 1.48 + 0.11 (2041)
Significance^
s NS
NP-gold import is s, significantly different (P < 0.01) between rounded and extended cells. NS, not significant. Number of particles counted in parentheses.
Source: From Feldherr and Akin (1993).
interactions between the nuclear surface and elements of the cytoskeleton. For example, the functional dimensions, and perhaps the molecular organization of the pore complex could be altered by tension applied to the nuclear envelope. Intermediate filaments might play a key role in this regard, since they are known to interact with the envelope (Goldman et al., 1986) and, perhaps, the pore complex itself (Carmo-Fonseca et al., 1987). Comprehensive models suggesting mechanisms by which the cytoskeleton could alter nuclear activity in general and transport in particular have been proposed by Ingber and Folkman (1989) and Hanson and Ingber (1992), respectively. Alternatively, the transport effects could be dependent on an association between the cytoskeletal elements and regulatory substances such as transcription factors and kinases. It has been suggested that the release and activation of these regulatory agents might result from changes in the molecular organization of the cytoskeleton (Ben-Ze'ev, 1991). If the latter mechanism is functional, the alterations in the transport apparatus might be similar to those produced by growth factors, which would also be expected to involve transcriptional and post-translational changes. In fact, the two processes could be interrelated, since there is evidence that modifications in the assembly of the cytoskeleton can be induced by growth factors (Herman and Pledger, 1985). Increases in Transport Capacity in SV40'Transformed Cells
The results described above established that decreases in metabolic activity (as a result of either growth arrest or shape changes) can be accompanied by a reduction in signal-mediated transport capacity. This
Nuclear Transport and Cellular Activity Table 3.
Experiment SVT2 BALB/c controls pSVSneotransformed BALB/c controls
247
Nucleoplasmin-Gold Uptake in Transformed Cells
% Particles^ No. of No. of Particles 80200Cells Measured 200A 320A
N/C
Mean ± SE
Significance
Size
N/C
12 10
432 675
65.7 95.7
34.3 4.3
1.8010.15(814)'^ 1.0010.07(829)
s
s
25
707
82.8
17.2
2.1110.15(1228)
s
s
22
428
94.6
5.4
0.9910.16(1173) —
—
^ Particle measurements do not include the 30-A protein coat, s, significantly different (P < 0.01) than controls. ^ Number of particles counted in parentheses. Source: From Feldherr et al. (1992). Notes:
raises the possibility that an increase in transport capacity (above the normal proliferating cell level) might occur during periods of enhanced cellular activity. In this regard, Feldherr et al. (1992) analyzed nuclear transport in SV40-transformed BALB/c cells (SVT2 cell line) and BALB/c 3T3 cells that were transfected with the early region of the S V40 genome (pSV3neo plasmid) that encodes both the large T and small t antigens. These cell lines exhibited more rapid growth rates than nontransformed cells. In addition, there were statistically significant increases, compared to normal proliferating BALB/c controls, in both the nuclear uptake of NP-gold and the functional diameter of the transport channel (see Table 3). The latter value increased by approximately 40 A. Overall, the changes in transport triggered by SV40 could function to expedite DNA replication, thereby facilitating the production of virions in permissive cells. In nonpermissive cells, this could contribute to the increased division rate and help bring about the transformed state. Since, in most instances, large T alone is sufficient for transformation, we examined its effect on signal-mediated transport. In these experiments, purified large T was injected into the cytoplasm of proliferating BALB/c cells and nuclear transport was assayed either 1 or 6 hours later with NP-gold. The effect of injecting a mutant form of large T, (cT)-3, was also tested. This mutant, which contains asparagine rather than lysine at position 128, is deficient in nuclear translocation and is retained in the cytoplasm. Control cells were either untreated or preinjected with
248
CARL M. FELDHERR and DEBRA AKIN
bovine serum albumin before the transport capacity was measured. The results, given in Table 4, established that a 6-hour pretreatment with large T can increase transport to the level observed in transformed cells; however, there was no significant change in gold import after 1 hour. Interestingly, the (cT)-3 mutant had no effect on nuclear transport when injected into the cytoplasm, but caused a significant increase when introduced into the nucleoplasm. Considering both the time dependence and the requirement for a functional NLS, it can be concluded that large T does not act directly on the transport apparatus (which is presumably localized in the cytoplasm) but indirectly, by mediating the activity of an effector protein(s). In order to identify the large T domain responsible for enhancing transport, Feldherr et al. (1994) investigated the effects of 12 different mutants on the nuclear uptake of NP-gold. The locations of the lesions, in relation to known functional domains of large T, are shown in Figure 3. Plasmids encoding the mutant proteins were injected into the nuclei of BALB/c cells, and nuclear transport was assayed approximately 18 hours later. In all but two cases, there was a significant increase in nuclear transport. Both of the inactive mutants (1061 and 2809) were also unable to bind the tumor suppressor protein p53, suggesting that the transport activity and p53 binding colocalize to the same domain. This was further supported by the finding that simultaneous injection of a plasmid that overexpresses wild-type p53 (PC53-SN3) prevented the increases in nuclear uptake normally caused by the expression of active forms of the large T antigen. In view of the above findings, additional experiments were performed to establish the effect of p53 on NP-gold import in cells that were not expressing large T. Nuclear injection of the plasmid pC53-SN3 had no effect on transport capacity; however, plasmid pC53-Cx33, which expresses a mutant form of p53, significantly increased NP-gold import (see Table 5, part A). The fact that excess wild-type p53 did not alter nuclear uptake probably indicates that the transport system was already saturated by endogenous p53. The effect of the mutant protein, on the other hand, was likely due to a decrease in the effective concentration of endogenous p53. Thus, Milner and Medcalf (1991) reported that mutant and wild-type p53 form inactive oligomers, the net result being a decrease in the active form of p53. More direct evidence that decreasing the intracellular p53 concentration enhances nuclear transport was obtained by investigating the effects of two anti-p53 monoclonal antibodies, designated pAb421 and pAbl22. In separate experiments, the
Table 4. The Effect of Large T Antigen on Nucleoplasmin-Gold Uptake
NO.
Experiment
BSA injection, 6 hr Large T injection, 1 hr Large T injection, 6 hr (cT)-3 injection, cyto, 6 hr (cT)-3 injection, nuc, 6 hr BALBIc untreated controls Notes:
of
cells 16 16 19 16 18 16
No. of Particles Measured 363 402 545 47 1 454 519
80-200.4 %.4 91.0 75.6 94.5 85.2 95.8
a Particle measurements do not include the 30-A protein coat.
s, significantly different (P < 0.01) than controls; NS, not significant. Number of particles counted in parentheses. Source: Fmm Feldherr et al. (1992).
2~320.4 3.6 9.0 24.4 5.5 14.8 4.2
Mean k SE 0.63 k 0.07 (915)' 0.72 k 0.07 (1369) 2.03 f 0.1 1 (1492) 1.10k0.11 (1188) 1.52 0.10 (479) 0.71 k0.12 (1061)
Size
N/C
NS NS s NS s
NS NS
-
s
NS s -
Pola (1-82)
Rb (102-115) — NLS (126-132)
pBSVcT
DNA binding (131-259)
395HN ,
402DE 402DH
>
p53 binding (351-450)
•
408F4 2809 ATPase (418-627)
p53 binding (533-626)
I Host Range
I (682-708)
Figure 3. A map of large T showing the locations of the mutations (listed on the left) in relation to known functional domains of the antigen (listed on the right). A M 76, 1061, and 2465 are deletion mutations. 395HN, 402DE, 402DH, and pBSVcT are single amino acid substitutions. The remainder are insertion mutations. Further details describing the nature of the lesions are given in Feldherr et al. (1994). 250
Table 5. The Effect of p53 on Nuclear Transport No.of Emeriment bd
2
Cells
No. of
% particlesa
Particles Measured
8&200A
4.8 12.2 3.6
200-320A
A. pC53-SN3 ~C53-Cx3~ BALBIc controls
18 15 15
400 41 1 353
95.2 87.8 96.4
B. pAb421 Mouse IgG2a controls
15 15
372 340
87.5 95.4
12.5 4.6
pAb122 Mouse lgG2a controls
30 16
687 333
85.4 94.9
14.6 5.1
Notes:
'particle measurements do not include the 30-A protein coat. s, significantly different (P < 0.01) than controls; NS, not significant. Number of particles counted in parentheses.
Source: From Feldherr et al. (1994).
N/C Mean _+ SE
significanceb Size
N/C
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CARL M. FELDHERR and DEBRA AKIN
antibodies were injected into the cytoplasm of BALB/c 3T3 cells, and transport was analyzed 1 or 2 hours later. Mouse IgG2a was injected into control cells. As shown in Table 5, part B, both anti-p53 antibodies significantly increased the N/C ratios of NP-gold, as well as the functional diameters of the pores. These results are consistent with, but do not prove, the view that large T modulates nuclear permeability by sequestering p53, which, in turn, acts as a transport inhibitor. p53 could function either by activating genes that encode specific repressors, or by blocking the expression of enhancing factors. The Increase in Transport Is Initiated by a Cytoplasmic Factor In unpublished experiments, Feldherr and Akin prepared a 100,000 x g cytoplasmic supernatant from SV40-transformed cells, using the general procedures described by Adam et al. (1992), and studied its effect on nuclear permeability in normal proliferating B ALB/c 3T3 cells. The supernatant was microinjected into the cells, and NP-gold was injected 10 minutes later. Even after this relatively short time interval, there was a significant increase in both the number and size of particles that were able to enter the nucleoplasm, as compared to untreated controls. Injecting a similar cytoplasmic fraction prepared from nontransformed BALB/c cultures had no effect on NP-gold import. It is unlikely that these results were due simply to the retention of large T antigen in the transformed cell extract. As mentioned above, the injection of purified large T (at concentrations that can easily be detected by immunofluorescence) requires over an hour to significantly alter transport capacity. The extract, on the other hand, causes an increase within 10 minutes, and large T is not present in sufficient amounts to be detected by immunofluorescence procedures. It is also necessary to consider whether the enhancing factor(s) in transformed cells correspond to one or more of the known cytoplasmic factors that normally mediate transport (the 54-56-kDa and 97-kDa proteins and Ran/TC4). This could be the case if one of the factors was rate limiting and was produced in considerably larger amounts after transformation. However, based on the results obtained with transformed cell extracts this seems unlikely. It is doubtful that a sufficient amount of material was injected in these experiments (approximately 5% of the cell volume) to significantly increase the concentration of any of the three transport factors. Although the possible involvement of these proteins cannot be formally ruled out, the results are more consistent with the
Table 6. Nuclear Uptake of Nucleoplasmin-Gold during the Cell Cycle % Particles in Nucleusa
N/C Ratio
Measured
80-200A
2O0-320A
Mean f SE
Size
18 20
473 449
85.5 95.4
14.5 4.6
1.44 + 0.08 (960)' 0.55 + 0.04 (566)
s
2 hr. post-anaphase BALBIc control
8 13
341 535
91.8 95.7
8.2 4.3
0.89 + 0.17 (631) 0.48 + 0.07 (788)
NS
NS
4 hr. post-anaphase
20 11
444
5.0 4.9
0.54 + 0.06 (677) 0.55 + 0.04 (566)
NS
448
95.0 95.1
NS
BALBIc control
6 hr. post-anaphase BALBIc control
18 19
478 561
94.6 95.4
5.4 4.6
0.63 + 0.05 (451 ) 0.57 + 0.06 (1116)
NS
NS
8 hr. post-anaphase
14 19
384 561
94.3 95.4
5.7 4.6
0.48 + 0.06 (412) 0.57+0.06(1116)
NS
NS
BALBIc control
12 hr. post-anaphase BALBIc control
19 13
599 535
95.8 95.7
4.2 4.3
0.31 + 0.03 (1506) 0.48 + 0.07 (788)
NS
NS
18 hr. post-anaphase BALBIc control
9 13
380 481
94.5 95.6
5.5 4.4
0.65 + 0.05 (614) 0.59 + 0.10 (788)
NS
NS
21 hr. post anaphase BALBIc control
20 16
629 573
98.7 95.8
1.3 4.2
0.98 + 0.06 (2635) 0.86 + 0.05 (1754)
s
NS
No. of
No.ofPanicles
cell;
I hr. post-anaphase BALBIc controld
Emerimenr
w
Notes:
a Particle measurements do not include the 30A protein coat.
s, significantly different (P< 0.01) than controls; NS, not significant.
'Number of particles counted in parentheses.
Control cells were randomly selected from non-synchronized, proliferating cultures. Source: From Feldherr and Akin (1994b).
significanceb
N/C
-
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CARL M. FELDHERR and DEBRA AKIN
view that the enhancing factor(s) has a catalytic effect and could possibly be a kinase or phosphatase. Signal-Mediated Transport During the Cell Cycle Studies performed on dividing BALB/c 3T3 cells by Feldherr and Akin (1994b) demonstrated that changes in nuclear transport capacity not only occur during extensive, long-term modifications in metabolic activity (such as those that take place following growth arrest and transformation) but also accompany more transient cellular processes. In this investigation, cells in different stages of the division cycle were obtained by first performing shake-offs to increase the proportion of cells in mitosis. These cells were then plated on coverslips, and those entering anaphase were scored and followed individually for up to 21 hours. At various intervals from 1 to 21 hours after the start of anaphase, nuclear transport was assayed with NP-gold. The nuclear import data are shown in Table 6. There was a significant increase in both the N/C ratio and the diameters of the transport channels 1 hour after the onset of anaphase. EM analysis of the nuclear envelopes in early Gj cells and import studies showing that large gold particles coated with PVP (which lacks an NLS) were excluded from the nucleoplasm demonstrated that the transport increase detected at 1 hour was not due to incomplete reformation of the nuclear envelope, but rather to differences in the properties of the transport machinery. The observed increase in nuclear import at this early time in the cell cycle could play a significant role in the reformation of the nucleus following division. At the 21-hour time point there was a decrease in the size of the particles that were present in the nucleus, but no change in the N/C gold ratio. The effect of these transport changes on the division process is not known, but the results do suggest that nuclear import rates and channel size can be regulated independently under certain circumstances. No differences in transport capacity, compared to control cells, were detected at other times in the cell cycle; however, it is possible that transient changes occurred but were not detected. IV. CONCLUSIONS Signal-mediated nuclear import of macromolecules is a multistep process that is initiated by specific nuclear targeting signals that interact with cytoplasmic receptors. The receptor-substrate complex then migrates to
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255
the cytoplasmic surface of the pore complex, where it binds to a docking protein(s). Docking to the pore surface activates an energy-dependent translocation process that results in the movement of the targeted molecule to the nucleoplasmic surface of the pore. This is followed by release into the nucleoplasm, which represents the final step in the process. It is generally assumed that nuclear efflux utihzes the same general mechanism; however, differences are likely to exist with respect to the specific signals and their receptors. In this review, evidence is presented demonstrating that nuclear transport is a dynamic process that can increase or decrease in effectiveness in response to changes in cell function. Furthermore, it appears that regulation of transport capacity is accomplished by several different mechanisms that can act at different steps in the import process. Consistent with the decrease in protein and RNA synthesis that occur during growth arrest, there is a significant decrease in macromolecular exchanges across the envelope. Cell fusion studies, performed with arrested and proliferating cells, demonstrated that the changes affecting transport occur at the level of the envelope, presumably within the pores. The differences in transport could be related to the number or binding capacity of receptors at the pore surface. A size-dependent decrease in nuclear import also occurred when cells were prevented from spreading, implicating the cytoskeleton in transport regulation. In contrast to the above results, an increase in the rate of cell growth after SV40 transformation was accompanied by an increase in both the number and size of NP-gold particles that are able to enter the nucleus. Preliminary studies indicate that the increase in nuclear import in transformed cells is dependent on cytoplasmic enhancing factors. There are indications that enhancer activity is triggered by large T antigen. Although the evidence is not conclusive, it is likely that large T functions by sequestering p53, which appears to act as a transport suppressor. Since an increase in transport capacity was also detected during the cell cycle, it is interesting to speculate that transport enhancers and suppressors are normal components of cells, and that altering the balance between these factors can significantly alter transport capacity.
ACKNOWLEDGMENTS The authors would like to thank Dr. Robert Cohen for critically reviewing the manuscript.
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Govind, S. & Steward, R. (1991). Dorsoventral pattern formation in Drosophila: signal transduction and nuclear targeting. Trends Genet. 7, 119-125. Greber, U. & Gerace, L. (1992). Nuclear protein import is inhibited by an antibody to a lumenal epitope of a nuclear pore complex glycoprotein. J. Cell Biol. 116,15-30. Grimm, S. & Baeuerle, P. (1993). The inducible transcription factor NF-KB: structurefunction relationship of its protein subunits. Biochem. J. 290, 297-308. Hanson, L. & Ingber, D. (1992). Regulation of nucleocytoplasmic transport by mechanical forces transmitted through the cytoskeleton. In: Nuclear Trafficking (Feldherr, C, ed.), pp. 71-89. Academic Press, San Diego. Herman, B. & Pledger, W. (1985). Platelet-derived growth factor induced alterations in vincuUn and actin distribution in BALB/c-3T3 cells. J. Cell Biol. 100,1031-1040. Hinshaw, J., Carragher, B., & Milligan, R. (1992). Architecture and design of the nuclear pore complex. Cell 69,1133-1141. Ingber, D. & Folkman, J. (1989). Tension and compression as basic determinants of cell form and function: utilization of a cellular tensegrity mechanism. In: Cell Shape: Determinents, Regulation and Regulatory Role (Stein, W., and Bronner, F, eds.), pp. 3-31. Academic Press, San Diego. Ingber, D., Madri, J., & Folkman, J. (1987). Endothelial growth factors and extracellular matrix regulate DNA synthesis through modulation of cell and nuclear expansion. In Vitro Cell. Dev. Biol. 23,387-394. Ireland, G., Dopping-Hepenstal, R, Jordan, R, & O'Neill, C. (1987). Effect of patterned surfaces of adhesive islands on the shape, cytoskeleton, adhesion and behavior of Swiss mouse 3T3 fibroblasts. J. Cell Sci. 8,19-33. Izaurralde, E. & Mattaj, I. (1992). Transport of RNA between nucleus and cytoplasm. Semin. Cell Biol. 3, 279-288. Jans, D., Ackermann, M., Bischoff, J., Beach, D., & Peters, R. (1991). p34^^^^-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins. J. Cell Biol. 115,1203-1212. Johnson, L., Abelson, H., Green, H., & Penman, S. (1974). Changes in RNA in relation to growth of the fibroblast. I. Amounts of mRNA, rRNA, and tRNA in resting and growing cells. Cell 1,95-100. Johnson, L., Levis, R., Abelson, H., Green, H., & Penman, S. (1976). Changes in RNA in relation to growth of the fibroblast. IV. Alterations in the production and processing of mRNA and rRNA in resting and growing cells. J. Cell Biol. 71, 933-938. Kalderon, D., Roberts, B., Richardson, W., & Smith, A. (1984). A short amino acid sequence able to specify nuclear location. Cell 39,499-509. Levine, E., Becker, Y, Boone, C , & Eagle, H. (1965). Contact inhibition, macromolecular synthesis and polyribosomes in cultured human diploid fibroblasts. Proc. Natl. Acad. Sci. USA 53, 350-356. Melchior, F, Paschal, B., Evans, J., & Gerace, L. (1993). Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/rC4 as an essential transport factor. J. Cell Biol. 123,1649-1659. Milner, J. & Medcalf, E. (1991). Cotranslation of activated mutant p53 with wild type drives the wild type p53 protein into the mutant conformation. Cell 65,765-774.
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Moll, T., Tebb, G., Surana, U., Robitsch, H., & Nasmyth, K. (1991). The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S. cerevisiae transcription factor SWI5. Cell 66, 743-758. Moore, M. & Blobel, G. (1993). The GTP-binding protein RanA^C4 is required for protein import into the nucleus. Nature 365, 661-663. Newmeyer, D. (1993). The nuclear pore complex and nucleocytoplasmic transport. Curr. Opin. Cell Biol. 5, 395-407. Newmeyer, D. & Forbes, D. (1988). Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell 52, 641-653. Nigg, E., Hilz, H., Eppenberger, H., & Dutley, F. (1985). Rapid and reversible translocation of the catalytic subunit of cAMP-dependent protein kinase type II from the Golgi complex to the nucleus. EMBO J. 4, 2801-2806. Paine, P., Moore, L., & Horowitz, S. (1975). Nuclear envelope permeability. Nature 254, 109-114. Pante, N. & Aebi, U. (1994). Towards understanding the three-dimensional structure of the nuclear pore complex at the molecular level. Curr. Opin. Struct. Biol. 4, 187-196. Peters, R. (1986). Fluorescence microphotolysis to measure nucleocytoplasmic transport and intracellular mobility. Biochim. Biophys. Acta 864,305-359. Richardson, W., Mills, A., Dilworth, S., Laskey, R., & Dingwall, C. (1988). Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell 52, 655-664. Richter, J. & Standiford, D. (1992). StrucUire and regulation of nuclear localization signals. In: Nuclear Trafficking (Feldherr, C , ed.), pp. 90-121. Academic Press, San Diego. Rihs, H. & Peters, R. (1989). Nuclear transport kinetics depend on phosphorylation-sitecontaining sequences flanking the karyophilic signal of the simian virus 40 T antigen. EMBO J. 8, 1479-1484. Rihs, H., Jans, D., Fan, H., & Peters, R. (1991). The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen. EMBO J. 10, 633-639. Robbins, J., Dilworth, S., Laskey, R., & Dingwall, C. (1991). Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequences. Cell 64, 615-623. Ross, R., Raines, E., & Bowen-Pope, D. (1987). The biology of platelet derived growth factor. Cell 46,155-169. Schmitz, M., Henkel, T, & Baeuerle, P. (1991). Proteins controlling the nuclear uptake of NF-KB, Rel and dorsal. Trends Cell Biol. 1, 130-137. Starr, C. & Hanover, J. (1992). Structure and function of nuclear pore glycoproteins. In: Nuclear Trafficking (Feldherr, C, ed.), pp. 175-202. Academic Press, San Diego. Steme-Marr, R., Blevitt, J., & Gerace, L. (1992). O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import. J. Cell Biol. 116,271-280. Wente, S., Rout, M., & Blobel, G. (1992). A new family of yeast nuclear pore complex proteins. J. Cell Biol. 119, 705-723.
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Yang, H. & Pardee, A. (1986). Insulin-like growth factor regulation of transcription and replicating enzyme induction necessary for DNA synthesis. J. Cell Physiol. 127, 410-416.
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INDEX
ABC proteins, 58-96 {see also "ATP binding cassette...") list of, 60-61 ABC transporters in yeast, 57-96 a-factor pheromone, export of, 6577 ABC-domain, 72 CAAX-box, 65-74 chimeric fusion proteins, 69-74 cytokines, 69 efflux pump, 73 ER-Golgi secretory pathway, 67 farnesyltransferase, 67 Golgi-localization, 76 heterodimers, 76 hydrophilic loops, 72 lipopeptide, 65 MATa2,15 mating pheromones a-factor and a-factor, 65-77 "molecular vacuum cleaner," 73 multidrug resistance phenomena, 77 P-glycoproteins, mammalian, 77 peptide antigens Tapl and Tap2, 76 peptide hormones, 65 pheromone secretion signal, 6774 pheromones, 67-74 261
phospholipid bilayers, 73 plasma membrane receptors, 74 polar localization of Ste6, 75-76 precursors, 65 prenylated proteins, 65, 74 proteasome, 75 resistance phenomena, 77 Saccharomyces cerevisiae, 65-77 "schmoo," 74 secretion signal driving a-factor export, 67-74 Ste6, 65-77 Ste6 a-factor transporter, biosynthesis and turnover of, 74-77 vesicular staining, 76 ABC proteins, yeast, family of, 5964 ALDp, 63 Atml, 63-64 chloroplasts, 63 DHS, 62 HMR, 62 mitochondria, 63-64 MRD, 62 PALI, 63 Pmp70, 63 puromycin, 64 SSHl and SSH2, 62-63 Ste6, 59-77 YKL741, 63
262
ABC-transporters as mediators for multidrug and heavy metal resistance, 77-87 ABC-motif, 80 Candida albicans, 82-83 Cdrl, 82-83 CFTR, 78 cytoplasmic proteasome, 83 dexamethasone, 84 Dos! and Dos2, 81-82 drug substrates, 82 effluxing ligand, 84 endocytosis, 84' facilitator superfamily, 86 Hba2, 78 HMR phenomena, 87 Hmtl,78 LEML 84 major facilitator superfamily, 86 MDR phenotype, 79, 82 Mdr2, 84 membrane fluidity, 85 multidrug transporters, biosynthesis and turnover of, 83-87 non-drug substrates, 82 PDR5. 82 pdrl-3 mutants, 85 permeability, 85 Pmdl,78 sec mutants, 86 SNQ2 gene, 79-87 sporidesmin, 79 squelching, 85 steroid signal transduction pathway, 84 steroid transport, 85 sterol content, 85-86 stress response factors, 79 STSl. 79-87 transcriptional interference, 85 transport of steroids and phospholipids, 86 vesicles, 86
INDEX
Walker A and Walker B, 80 Yap 1,79 YCFl, 78 YDRl, 82 Yef3 elongation factor, 77-79 conclusion and perspectives, 87-88 chimeric transporters, construction of, 87 endogenous ABC proteins, 87 functional reconstitution, 87 heterologous ABC proteins, 87 in human disease, 88 mammalian, 87 number of transporters in yeast, 88 purification, 87 introduction, 58-59 a-factor transporter, 59 ABC transporters, 58-96 adrenoleukodystrophy, 59, 6263 cystic fibrosis transmembrane conductance regulator (CFTR), 58 metal resistance, 58-59 multidrug resistance (MDR) phenotype, 58 P-glycoproteins, 58 peroxisomal diseases, 59 pheromone export, 59 TMS, 58 tumors, 58 Zellweger syndrome, 59, 62-63 multidrug and heavy metal resistance mediated by ABCtransporters, 77-87 {see also ".. .ABC-transporters...") Adrenoleukodystrophy, 59, 62-63 Aspartic acid, 186-187 ATPases, model for integrating into endoplasmic reticulum {see ^/5(9"P-type...")
Index
ATPase activity, 1-28 (see also "Molecular chaperones...") role of in protein translocation, 23-24 pBluescript II KS-f (Stratagene), 221 Caveolae as portals for transmembrane signaling and cellular transport, 111-121 concluding remarks, 118 human disease, role in, 117-118 cholera, 117 dystrophin, 117 insuling receptor, 117 malaria, 118 scrapie, 118 Schuffner's dots, 118 whooping cough, 117 introduction, 111-113 at plasma membrane, 111-112 purified caveolae, 112 in signaling events, 113 transcytosis, 112 as signaling organelles, 113-115 calcium homeostasis, 113 caveolin, 113-115 functions of caveolar membrane domains, 114 G-protein receptor signaling, 113 Src family of nonreceptor tyrosine kinases, 113 v-Src substrate, 113-115 transport processes, 115-117 in endothelial cells, 115-116 GPI-linked molecules, 116 potocytosis, 116 Caveolar clustering and GPI-linked proteins, 97-110 (see also "GPI-linked proteins...") CcN motif, 161-199 (see also "Phosphorylation-mediated regulation...")
263
CDNs, 162, 1^9-190 (see also "Phosphorylation-mediated regulation...") Cellular activity, nuclear transport as function of, 237-259 conclusions, 254-255 docking protein, 255 nuclear efflux, 255 receptor-substrate complex, 254-255 introduction, 237-238 macromolecular transport across nuclear envelope, 238-240 cytoplasmic transport factors, 239 nuclear localization signals (NLS), 238 (see also "Nuclear localization...") through nuclear pores, 238 nucleoporins, 239 passive diffusion, 238 signal-mediated transport, 238 regulation of transport across nuclear envelope, 240-254 alterations in transport machinery, 241 in BALB/c 3T3 cells, 246-252 cell fusion experiments, 244-245 cell shape, effects of, 245-246 changes in transport capacity, 241-254 cytoplasmic factor, increased transport and, 252-254 enhancing factors, 252 epidermal growth factor (EGF), effects of, 243 IGF-1, effects of, 243 NP-gold, investigation of, 241254 p53, effect of on NP-gold import, 248-252 PDGF, effects of, 243 phosphorylation, 240
264
signal-mediated transport during cell cycle, 254 of specific proteins, 240-241 SV40-transformed cells, increased transport capacity in, 246-252 transport inhibitors, 240 CFTR, 58 (see also "ATP binding cassette...") Chaperones, molecular, 1-28 (see also "Molecular chaperones...") CLSM, 165-166 Confocal laser scanning microscopy, 165-166 Cystic fibrosis transmembrane conductance regulator (CFTR), 58 (see also "ATP binding cassette...") Duchenne's muscular dystrophy, 117 {see also "Caveolae...") Escherichia coli, membrane protein topogenesis in, 201-214 (see also "Membrane protein...") Glassfog, 221 Glassmilk, 221 GPI-linked proteins, caveolar clustering and, 97-110 caveolar clustering, of, 103-104 caveolin hetero-oligomers, 104, 105 cholesterol-dependent, 103 plasmalemmal vesicles, 103, 106 Triton insolubility, 104 concluding remarks, 106 epithelia, polarized sorting in, 100103 apical plasma membrane domain, 100
INDEX
basolatera plasma membrane domain, 100 FRT cells, 102 glycosphingolipids, 101, 102 luminal plasma membrane domain, 100 MDCK cells, 102-103 plasma membrane domains, 100 serosal plasma membrane domain, 100 trans-Golgi network (TGN), 101 transfected GPI-linked proteins, 101 zonula occludens, 100 GPI biosynthesis and transfer to protein, 99-100 signal for GPI attachment, 100 introduction, 98-99 abbreviations, 99 glypiation, 99 GPI biosynthesis, 98, 99-100 GPI-synthesis pathway, 98, 106 "greasy foot," 99 inhibitors, 98-99 mutants, 99 PIG tailing, 99 unifying features, 99 model for clustering and assembly, 104-106 hetero-trimeric G-proteins, 106 lipid-modified signaling molecules, 106 protein exclusion-lipid inclusion, 106 tyrosine kinase, 106 Heat shock protein, 34, 182 hsp 70 proteins, 1-28 (see also "Molecular chaperones...") mitochondrial, 5-23 Intermembrane space intermediate, 11
Index
KAR2 alleles, 24 Membrane protein topogenesis in Escherichia coh\ 201-214 artificial, 210-211 conclusions and outlook, 211 helix-helix packing in lipid environment, 208 cysteine residues, 208 energy transfer measurements, 208 glycophorin A homo-dimer, 208 introduction, 201-202 mechanisms of assembly of, 202207 CCCP, 207 "helical hairpins," 204-205 membrane potential, 206-207 peptidase (Lep), 203-204 "positive inside" rule, 202-205 sec machinery, 205-206 topology and structure, prediction of, 209-210 for glycophorin A dimer, 210 hydrophobicity plot, 209 Mitoplasts, 12 Molecular chaperones, role of in protein transport across membranes, 1-28 ATP, role of, 23-24 cytosolic hsp 70, role of, 4-5 SSA proteins, 4-5 endoplasmic reticulum, role of hsp70 of, 24-25 KAR2 allleles, 24 introduction, 2-4 ATPase activity, 2 genetic approach, 4 hsp 70 structure and biochemical properties, 2-3 import into mitochondria, basics of, 4
265
Saccharomyces cerevisiae, hsp 70s of, 3 X-ray crystallography, 2 mitochondrial hsp 70, role of, 5-23 Z?2(167)-DHFR, 15-22 co-immunoprecipitation experiments, 8 components, other, required for protein movement, 22-23 "conservative sorting" model, 22 hemin, 17-18 hsp 70 action in translocation across mitochondrial membranes, model of, 18-20 intermembrane space intermediate, 11 matrix, required for translocation into, 7-14 matrix, translocation of proteins into compared to intermembrane space, 20-22 mitoplasts, 12 mutant, in vivo analysis of, 5-7 and precursor proteins, 8-9 Sscl'2, 5-14 Sscl-3, isolation and analysis of, 9-12 Sscl-2 and Sscl-3 alleles, comparison of, 12-14 Ssclp transport activity, precursor conformation and, 14-18 "stop-transfer" hypothesis, 22 Su9-DHFR, 8-18 summary, 25 NLS-binding proteins (NLSBPs), 33-34, 164 Nuclear envelope lattice (NEL), 31 Nuclear localization signal (NLS), 30, 126-127, 162, 163-164, 238 bipartite, 164, 238 NLS-binding proteins (NLSBPs), 164
266
nucleoplasmin simple, 238 SV40 large tumor antigen, 164, 238 Nuclear pore complex (NPCs) in yeast, 29-56 components, 36-39 coiled-coil interactions, 38, 39 FSFG, 37, 38 GFSFG pentapeptide, 37 GLFG, 38, 45 heptad repeats, 38 immunological approach, 37 Mabl92, 37-38,42 NSP1,37, 38, 39-49 nuclear pore biogenesis, 38 NUP1,37 NUP2, 37 NUP49, 37-38, 42 NUPlOO, 37-38 NUPl 16, 37-38 repeat sequences, 37 conclusions, 49-51 experimental approaches to study of, 39-49 affinity-purification, 40-41 biochemical, 39-42 fluorescence microscopy, use of, 48 genetic, 43-47 GLFG, 45 Mabl92,42 MCM 1,47-48 NIC96, 42, 46 NPL3, 49 NSPl, concentration on, 39-49 NSP49, 42, 45-47 NSPl 16, 45-47 NSP-X, 43-45 NUPl, 46 NUP2, 46 NUP49, 42, 46, 49 NUP116,46
INDEX
p54, 42 protein A fusion proteins, 40 red/ white colony sectoring, 43-45 SDS-PAGE analysis, 41 STE12, 47-48 synthetic lethality, 43-47 thermosensitive mutation, 49 in vitro nuclear transport assays, 47-49 Z domain, 40 introduction, 30-35 digitonin, 33-34 function of, 30 GTP-binding protein, 34 heat shock protein, 34 in higher eukaryotes, structural analysis of, 31 NLS-binding proteins (NBPs), 33-34 nuclear envelope lattice (NEL), 31 nuclear localization signal (NLS), 30 nuclear pore proteins, 32 nuclear transport and in vitro systems, 33-35 nucleoporins, 32-33 NUP153, 33 permeabilized HeLa cells, 34 "plug-spoke" complex, 31 Ran/TC4, 34 Saccharomyces cerevisiae, 35-51 in vertebrates, components of, 32-33 wheat germ agglutinin (WGA), 32-33 in Xenopus oocyte nuclear envelopes, 31, 32 as molecular sieve, 163 structure, 35-36 closed mitosis, 35 nuclear lamina, 36 summary, 51 Nucleoporins, 32-33, 239
Index
P-type ATPases, model for integrating into endoplasmic reticulum, 215-235 experimental design, 218-226 amino acid sequences used in construction of fusion proteins, 222 fusion proteins, construction of, 220-226 integration signal, 226 membrane selectivity, question of, 218 polymerase chain reaction (PCR), use of, 219 proteinaceous effectors, 218 sequential insertion model, 218219 "start-transfer signal," 218-219, 223 "stop-transfer signal," 219, 223 integration mechanism, general, for P-type ATPases and porters, 231-232 transmembrane segments, pairs of, role of, 231-232 integration model of Neurospora plasma membrane H^ATPase, 226-231 for enzymatic reactions, 226 M7, M8 as exception, 230 for receptor binding, 226 introduction, 215-216 H^ATPase, 215 porters, 216 Neurospora H^ATPase, 216-218 concanavalin A method, 216 electrogenic ATPase as proton pump, 216 models, 217 molecular mechanism, 217 vanadate, 216
267
porters, integration mechanism for, 231-232 {see also ".. .integration mechanism...") Perkin Elmer's Gene Amp kit, 221 Phosphorylation-mediated regulation of signal-dependent nuclear protein transport, 161-199 conclusion and future prospects, 190-191 eukaryotic cell, nuclearcytoplasmic processes in, 163-164 {see also ".. .nuclear-cytoplasmic...") future prospects, 190-191 gene therapy, potential for, 191 introduction, 162-163 casein kinase II, 162, 173 CcN motif, 162 cell-cycle-dependent NLSs (CDNs), 162 cyclin-dependent kinase, 162, 173 cytoplasmic retention factors, 162-162 intra-and intermolecular NLS masking, 163 NLS masking, 163 nuclear localization signal (NLS), 162 signal transduction-responsive NLSs (SRNs), 162 SV40 large tumor antigen, 162 mechanisms of regulation of, 180187 aspartic acid, 186-187 c-fos proto-oncogene, 183 cAMP-dependent protein kinase catalytic and regulatory subunits, 182-183 CKII phosphorylation, 186-187 cofilin, 185-186
268
cytoplasmic retention factors, 180-183 dual phosphorylation, positive and negative regulation by, 186-187 glucocorticoid receptor and HSP90, 182 intermolecular masking, 184185 intramolecular masking, 184 lamin B2, 185 NF-(kappa)B, 184-185 NLS masking, 183-186 NLS masking by phosphorylation, 185-186 NLSBP interraction, 187 PK-A R-subunit, 182-183 rel/dorsal family, 180-182 SV large T antigen, 183 SW15. 185 methodological considerations in analysis, 165-168 confocal laser scanning microscopy (CLSM), 165-166 stages, two, 168 in vitro measurement, 166-168 in vivo measurement, 165-166 nuclear-cytoplasmic processes in eukaryotic cell, 163-164 bipartite NLS, 164 NLS-binding proteins (NLSBPs), 164 nuclear localization signals, 163164 nuclear pore complex as molecular sieve, 163 SV40 large tumor antigen, 164 regulated (conditional) nuclear entry, 168-172 SV40 large tumor antigen and CcN motif, 172-180, 181
INDEX
variants on CcN motif, 187-190 cdks, 189-190 CDNs, 189-190 cell-cycle-dependent NLSs, 189190 CKII, 189 PK-A, 188 PK-C, 188-189 rNFIL-6, 188 signal-transduction-responsive NLSs, 187-189 SRNs, 189 Plasmalemmal vesicles, 111-121 {see also "Caveolae...") Polymerase chain reaction (PCR), 219 "Positive inside" rule, 202-205 {see also "Membrane protein...") PrimerErase Quick, 221 Saccharomyces cerevisiae, 35-51 {see also "Nuclear pore complex...") hsp 70s of, 3 {see also "Molecular chaperones...") Schuffner's dots, 118 SDS-PAGE,41 Sec machinery, 205-206 Squelching, 85 SRNs, 162 {see also "Phosphorylation-mediated regulation...") SSA proteins, 4-7 {see also "Molecular chaperones...") Sscl'2 mutant, 5-14 {see also "Molecular chaperones...") Sscl'3, 9-12 {see also "Molecular chaperones...") "Sto-transfer" model, 22 Stratagene, 221 Su9-DHFR, 8-18 {see also "Molecular chaperones...")
Index
Thermal cycler, 221 Trans-Golgi network (TGN), 101, 111-121 Transcriptional interference, 85 U snRNPs, nuclear transport of, 123-159 cap binding activity, nuclear, in U snRNA export, 140-142 cap binding proteins, (CBPs), 141 cap structure, role of in U snRNA export, 139-140 cap structures, role of in signaling RNA subcellular localization, 147-148 import pathways, multiple, 152153 inhibitors, 148-150 anti-gp210 monoclonal, 150 anti-nucleoporin antibodies, 149-150 ATP depletion, 148 chilling, 148-149 lectins, 149 nucleoporins, 149 saturation kinetics, 149 wheat germ agglutinin (WGA), 149-150 introduction, 124-126 classes, two, 126 nucleolar (sno), 126 nucleoplasmic (sn), 126 "spliceosomal," 126, 132 macromolecular assemblies, nuclear import of, 128-132 gag-matrix protein, 131 HlV-1, 131 hsc70, 129 NLS, 129-131 nucleoplasmin, 130 in oligomeric complexes, 129 U snRNA, passage of, 129
269 nuclear export of, 138-139 cap structure, 139 nuclear import of, 142-144 model for role of trimethyl cap and core domain in signaling, 146-147 Sm core domain, 146 nuclear import of U6 snRNP after mitosis, 147 nuclear targeting pathways, multiple, in import, 151-152 proteins, nuclear import of, 126128 ATP-dependent translocation stage, 127-128 hsc70, 128 karyophile, 127-128 NLS receptors, 127-128 nuclear localization signal (NLS), 126-127 nuclear pore complex, 127 spliceosomal, 132-138 assembly pathway of, 137-138 composition and function of, 132 core proteins, 133-135 monomethyl cap structure, 137 protein components, 133-137 proteins, common, 133-135 proteins, specific, 135-137 RNA constituents of, 132-133 Sm binding site, 134-135 Sm core domain, 135, 146 Sm proteins, 133, 142-144 trimethyl cap, 137 trimethyl cap structure for U snRNA nuclear import, 144-145 model for role of with core domain in signaling nuclear import, 146-147
270
Vanadate, 216 Wheat germ agglutinin (WGA), 3233, 149 X-ray crystallography, 2 Xenopus oocyte nuclear envelope, 31,32 nuclei reconstitution system, 32
INDEX
Yeast, ATP binding cassette transporters in, 57-96 {see also "ATP binding cassette...") Yeast, nuclear pore complex in, 2956 {see also "Nuclear pore complex...") Zellweger syndrome, 59, 62-63