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Protein transport across the ER membrane
TIBS 15- SEPTEMBER1990
THE MOLECULAR MECHANISM by which signal sequences direct polypeptides to the ER membrane is still poorly understood. In particular, it is puzzling that signal sequences do not share any sequence homology; their only common property is an uninterrupted stretch of at least six hydrophobic amino acids. Other signals involved in the intracellular protein transport, such as those for the import of proteins into mitochondria, chloroplasts or peroxisomes, appear to be equally vague (for review see Ref. 1).
Initiation of protein transport: the SRP cycle In the case of translocation across the ER membrane, signal sequences in general are initially recognized by the signal recognition particle (SRP)z. The SRP initiation or targeting cycle is schematically depicted in Fig. 1. The synthesis of a polypeptide to be translocated across the ER membrane starts with an actively translating ribosome that is free in the cytoplasm. As soon as the signal sequence of the nascent chain has emerged from the ribosome, it is recognized and bound by SRP, which also has an affinity for the ribosome. This association slows down the elongation of the polypeptide chain (elongation arrest) 2. In the next step, the SRP-ribosome-nascent chain complex is bound to the ER membrane via a specific interaction of SRP with its receptor (also termed docking protein) 3,4. The latter then binds GTP, which perhaps replaces previously bound GDPE After docking of the complex, both the signal sequence and the ribosome are released from SRP. The initiation cycle is completed by the dissociation of SRP from its receptor, a process that is triggered by GTP hydrolysis (R. Gilmore, pers. commun.). Several features of this scheme should be emphasized. (1) SRP and its receptor are not involved in the actual process of protein translocation through the membrane. (2) The interactions of SRP with a ribosome and a signal sequence are cooperative, i.e. one interaction strengthens the other.
T.A. Rapoportis at the Zentralinstitutffir Molekularbiologieder Akademieder Wissenschaftender DDR,1115 Berlin-Buch, Robert-R6ssle-Strasse10, DDR.
Protein transport across the ER membrane Protein transport across the endoplasmic reticulum (ER) membrane may be divided into two phases: an initiation or targeting cycle, which has been fairly well characterized, and the actual transfer of the polypeptide chain through the membrane, the mechanism of which is still unknown. In this review, the initiation cycle is discussed with emphasis on the mechanism of signal sequence recognition by the 54 kDa polypeptide of the signal recognition particle (SRP) and on the efficiency of targeting of nascent chains. Recent results are reviewed suggesting the transfer of the polypeptide chain by means of a translocation complex, a constituent of which appears to be the signal sequence receptor protein (SSR). As a consequence, SRP does not bind to completed polypeptide chains released from ribosomes 6. (3) Nucleotide hydrolysis is only required to start the next round of initiation5.
SRP functions as an 'antifolding device' by segregating the bound signal sequence from the rest of the polypeptide chain. In this respect, it resembles proteins called 'molecular chaperones'
ribosome , 5
~ r
mRNA ~
SRP SRP- receptor
ER-membrane
Pi
i
~.
GT P
,
Figure 1 The initiation (SRP)cycle of protein translocation. The scheme shows the first steps in protein translocation across the ER membrane after the signal sequence(hydrophobic portion indicated) has emerged from the ribosome and has been bound to the signal recognitionparticle (SRP).
© 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00
355
TIBS 1 5 - SEPTEMBER1990
or 'polypeptide chain-binding proteins '7,s. Other members of this family, such as the heat shock protein hsp70, may keep precursor polypeptides in a translocation-competent conformation in the exceptional cases where SRP and its receptor are not neededs-~°. These factors, however, unlike SRP, do not seem to recognize signal sequences specifically (at least in eukaryotes), nor are they bound to the membrane by specific receptors. It is therefore as yet unclear how the signal sequence is recognized in SRP-independent translocation pathways.
Signal sequence recognition by the SRP Photocrosslinking studies have shown that signal sequences of different proteins interact with the 54 kDa polypeptide component of SRP (SRP54)H,12. The sequence of SRP54 indicates the existence of two domains, an N-terminal G domain with sequence elements typical of a GTP-binding protein (GTP binding has indeed been demonstrated; J. Miller and P. Walter, pers. commun.), and a C-terminal methionine-rich M domain~3,14.Whereas the G domain was implicated by RSmisch et al. 13 in the binding of signal sequences, Bernstein et al. ~4 have assigned this function to the M domain. Bernstein et al. postulated that the methionine residues reside on one face of three amphipathic it-helices. These would form or contribute to a hydrophobic binding groove on the surface of SRP54 which could accommodate the hydrophobic core of signal sequences. Although a hydrophobic pocket that induces identical helical conformations with intra-chain saturation of all hydrogen-bond donors and acceptors would explain the binding of signal sequences of different primary structures ~s, the M domain appears not to be the only site involved. It has recently been found that the N-terminal part of a signal sequence can be crosslinked to the G domain and that the M domain links the protein to the rest of the particle (P. Walter and B. Dobberstein, pers. commun.). It therefore appears that the G domain, either alone or in conjunction with the M domain, provides the binding site for signal sequences. A recent report ~6 of homologues to the mammalian two-domain SRP54 in both yeast and E. coli suggests that the proteins involved in signal sequence recognition may be more conserved in evolution than hitherto assumed. 356
Most surprisingly, SRP54 shares further sequence homology with the SRP receptor in the G domain in addition to the short sequence elements typical for GTP-binding proteins, suggesting that the SRP receptor may also bind signal sequences ~3,~4. However, experimental tests of this proposal so far have been negative. A provocative alternative would be that the homologous regions of both SRP54 and the SRP receptor compete for a common binding site. It appears possible that SRP54 is transiently displaced from the SRP particle, together with the bound signal sequence, on interaction with the membrane receptor. SRP54 could be bound by mp30, a membrane protein that has been shown to have an affinity for SRP17. Alter release of the signal sequence into the membrane, SRP54 would rejoin the core particle to reform the intact SRP. Indeed, SRP54 is the only component of SRP that can dissociateTM. It is conceivable that free SRP54 is the signal sequence receptor for those proteins that do not require SRP for translocation9.
How is the efficient and specific targeting of nascent chains achieved? It has been proposed that GTP hydrolysis plays a role in enhancing the specificity of signal sequence recognition~4,19. Rothman 19 has argued that 'the incorrectly matched peptides would have an opportunity to dissociate from SRP54 before the switch of GTP hydrolysis is thrown', while the bound signal peptide would be fixed in place. Such a 'proof-reading' mechanism is nothing else but a rapid and reversible ligand-binding reaction preceding a slow and irreversible step (not necessarily coupled to GTP hydrolysis). A rapid binding and release of signal sequences is indeed required for fidelity of recognition. However, efficient targeting of nascent chains to the ER membrane is mainly achieved by a different mechanism that involves repetitive binding steps. Nascent chains have a certain size range (or 'window') in which they are able to interact with SRP2°.2LThe lower limit is determined by the length of the chain when the signal sequence has just completely emerged from the ribosome, the upper one by the point at which the signal sequence is buried in the folding chain. Alter each elongation step within these limits, a certain percentage of nascent chains will bind SRP (depend-
ing on the binding constant and the SRP concentration) and will be targeted irreversibly to the membrane. Membranebound chains no longer have a chance to bind SRP and are thus withdrawn from the equilibrium. The number of chains not yet translocated will therefore decrease as the end of the window region is approached and that bound to the membrane will increase accordingly. Consequently, even if the percentage of chains that interacts with SRP is low at each step, the overall targeting efficiency may be high due to the cumulative nature of the process 2°. The targeting efficiency will depend both on the binding constant of the signal sequence for SRP and on the size of the SRP window which is a function of the folding properties of the nascent chain. We have estimated that even the change of a single residue in the hydrophobic core of a signal sequence to a polar, uncharged one would be expected to reduce the binding constant by a factor of 103-105 (see Ref. 15; the binding constants have been estimated 2° to be in the range of 0.25-2.5nM). Thus, a high degree of discrimination in the recognition of signal sequences may be achieved.
Is there further signal recognition at the membrane? Photocrosslinking has been used to study the fate of the signal sequence after disengagement of SRP. With short nascent polypeptide fragments (70 residues long), the signal sequence was found in close proximity to an approximately 35 kDa integral membrane glycoprotein of the ER, provisionally termed the signal sequence receptor (SSR)=. It is unlikely that random encounters between the nascent chain and the membrane protein were responsible for the observed crosslinking; otherwise one should have expected a far more complex pattern of crosslinked species than was actually observed. Of course, proximity of the signal sequence and SSR does not necessarily imply specific binding, to which several objections can be raised. First, longer nascent chains that carried photoreactive groups exclusively in the mature portion still gave rise to crosslinks with SSR23,24. Secondly, the yield of crosslinked product was significantly lower for a polypeptide fragment with 70 residues as compared to one with 86 residues, although interaction of the signal sequence with a putative receptor should have been at least equal for
TIBS 1 5 - SEPTEMBER1990
the shorter fragment23,24. Finally, the sequence of SSR makes it unlikely that it could bind hydrophobic signal sequences since its cytosolic tail is very hydrophilic2s. Further experiments are thus needed to find out whether SSR actually binds to signal sequences. Membrane passage of the polypeptide: is there a translocation tunnel? The mechanism by which a polypeptide is transferred across a membrane is an issue of a long-standing debate. According to one viewpoint, transport occurs directly through the hydrophobic phospholipid bilayer without participation of proteins 26-28. The translocation of some polypeptides indeed appears to be independent of the presence of membrane proteins2E The ability of signal peptides of different structures to insert into protein-free phospholipid bilayers strongly correlates with their functionality in the overall process of translocation29. It is also possible that the transfer of the polypeptide occurs through a hexagonal lipid phase that would provide a hydrophilic environment in the interior of the membrane 3°,3~. Such a structure is favoured by certain phospholipids which are indeed abundant in natural membranes3L It is conceivable that the occurrence of a hexagonal lipid phase is also influenced or triggered by membrane proteins. According to alternative models, polypeptides are transported through a hydrophilic or amphiphilic tunnel that is assembled from transmembrane proteins 32,33. A protein environment for the nascent polypeptide chain during its membrane transfer was indeed suggested by the fact that aqueous perturbants such as urea remove the nascent chain from the membrane 34. Furthermore, after solubilization of ER membranes, the nascent polypeptide chains were found to be resistant to proteases. The C-terminal portion of the nascent chain was protected by the ribosome and the N-terminal one presumably by membrane proteins 3s. Fusion of microsomal vesicles with planar lipids has provided evidence for large aqueous channels of high conductivity that presumably represent the translocation tunnels 36. The involvement of one or more glycoproteins in the translocation process has been demonstrated recently with reconstituted proteoliposomes produced from detergent-solubilized microsomes 37. Finally, SSR has been found in close proximity to nascent chains dur-
SSRoc(34kDo)
5kDat
Cytoso[
c°rb°hydr°t ~ e
SSR ![3(22kDo)
lkDQ{t($)..chor gecluster ;b[°cked ~ N-terminus ~ ~0
k> chorgcleuster Figure 2 Membrane topology and sequence features of ~- and ~-SSR. Scheme showing the membrane-spanning regions, carbohydrate chains and charge clusters for the two subunits of the signal sequence receptor (SSR) (based on Ref. 25 and D. G6rlich eta/., unpublished).
ing membrane transfer and it may therefore be a constituent of the putative tunnel 23'24. SSR: a component of the translocation complex? To get more insight into the role of SSR in protein translocation, its purification was attempted. An integral 34 kDa glycoprotein was purified from canine pancreatic microsomes 38. Identity with SSR was suggested by the fact that antibodies raised against the intact protein or against a C-terminal peptide partially immunoprecipitate6 the crosslinked products of nascent secretory protein and SSR23. That the extent of immunoprecipitation was rather low may be due to the large amounts of non-crosslinked 34 kDa pro-
tein in the ER membrane and/or to the altered antigenicity of the protein after crosslinking. SSR is a major ER membrane protein (about 1.5% of total membrane protein in canine microsomes). It is present in a more than tenfold excess over SRP and SRP receptor and is even in excess over membrane-bound ribosomes 38, as would be expected if SSR indeed participates in the actual translocation process; each membrane-bound ribosome would be in the process of transferring a nascent chain through the membrane. Furthermore, SSR is largely restricted to the rough portion of the ER (F. Vogel et al., unpublished). In agreement with the assumed general function of SSR, it has been immunologically detected in various
Table I. ER-membrane proteins implicated in protein translocation Proteina
Source
Function
SRP-receptor ct-subunit ~subunit
mammals
Signal peptidasecomplex
mammals yeast (sec11)
Cleavageof signal peptide
SSR-complex
mammals
Tunnelconstituent, signal sequencebinding?
SRP-binding,GTP-binding Membrane-insertionof ~-subunit
ct-subunit Fsubunit Ribophorins I and II
mammals
?
sec61
yeast
?
sec62
yeast
?
sec63
yeast
?
sec65
yeast
?
aForreferencessee Ref. 39. 357
TIBS 15- SEPTEMBER1990
tissues of mammals and in birds. Antibodies against SSR and Fab-fragments prepared from the antibodies block the in vitro translocation of several secretory proteins ~. Thus, SSR appears to be essential for protein translocation. The sequence of SSR indicates that it has one hydrophobic membrane-spanning segment; the subsequent 56 amino acids are located in the cytosol (Fig. 2) 25. The luminal portion contains two carbohydrate chains. The protein has a striking charge distribution: a region close to the N-terminus is highly negatively charged (23 out of 35 residues) and the cytosolic tail carries a net positive charge. SSR does not share any sequence homology with other known proteins. It is made with a cleavable Nterminal signal peptide and is inserted into the ER membrane in an SRP-dependent manner. Both the sequence and the size exclude the possibility that a single molecule of the 34 kDa SSR can form a tunnel. Using bifunctional crosslinking reagents we have indeed found that other proteins are located in its close proximity in the ER membrane. Among them is a 22 kDa glycoprotein which remains tightly bound to the 34 kDa polypeptide even after solubilization of the membrane with detergent and which forms with it a stable, stoichiometric complex (D. G6rlich et al., unpublished). The two polypeptides have thus been named a- (34 kDa) and (22 kDa) subunits of SSR. The crosslinking experiments have also indicated the proximity of two 0~SSR molecules in the ER membrane, suggesting that SSR heterodimers associate with each other. The two subunits of the SSR share several features: a cleavable N-terminal signal peptide, a single hydrophobic membrane-spanning segment close to the C-terminus, and two carbohydrate chains in the luminal portion (Fig. 2). Like e~SSR, [~SSR also has no resemblance to any other protein so far registered in any data bank (D. G6rlich et aL unpublished).
Except for the signal peptidase complex, a function has not yet been demonstrated for any of them. In addition to the listed proteins, a ribosome receptor probably exists. Unfortunately, with the exception of one subunit of the signal peptidase complex (sec11), so far the proteins identified in yeast by genetic means have no correspondence to those found in mammals by biochemical methods and vice versa. On the basis of our current knowledge, the existence of a translocation tunnel remains equivocal. However, the recent breakthrough in reconstituting the translocation activity in proteoliposomes after complete solubilization of the ER membrane has provided a method to assess the function of purified membrane proteins in the translocation process 37.
Acknowledgements l thank M. Wiedmann, E. Hartmann, M. Shia, and particularly S. M. Rapoport, for critical reading of the manuscript.
References 1 Pugsley, A. P. (1989) Protein Targeting, Academic Press 2 Walter, P. and Blobel, G. (1981) J. Cell Biol. 91, 557-561 3 Gilmore, R., Walter, P. and Blobel, G. (1982) J. Cell Biol. 95, 470-477 4 Meyer, D. I., Krause, E. and Dobberstein, B. (1982) Nature 297,503-508 5 Connolly, T. and Gilmore, R. (1989) Cell 57, 599-610 6 Wiedmann, M., Kurzchalia, T. V., Bielka, H. and Rapoport, T. A. (1987) J. Cell Biol. 104, 201-208 7 Hemmingsen, S. M., Woolford, C., van der Vies, S. M., Tilly, K., Dennis, D. T., Georgopoulos, C. P., Hendrix, R. W. and Ellis, R. J. (1988) Nature 333, 330-334 8 Rothman, J. E. (1989) Cell 59, 591-601 9 Zimmermann, R. and Meyer, D. I. (1986) Trends Biochem. Sci. 11, 512-515 10 Bernstein, H. D., Rapoport, T. A. and Walter, P. (1989) Cell 58, 1017-1019 11 Kurzchalia, T. V., Wiedmann, M., Girshovich, A. S., Bochkareva, E. S., Bielka, H. and Rapoport, T. A. (1986) Nature 320, 634-636 12 Krieg, U. C., Walter, P. and Johnson, A. E.
(1986) Proc. Natl Acad. Sci. USA 83, 8604-8608 13 R6misch, K., Webb, J., Herz, J., Prehn, S., Frank, R., Vingron, M. and Dobberstein, B. (1989) Nature 340, 478-482 14 Bernstein, H. D., Poritz, M. A., Strub, K., Hoben, P. J., Brenner, S. and Walter, P. (1989) Nature 340, 482-486 15 Finkelstein, A. V., Bendzko, P. and Rapoport, T. A. (1983) FEBS Lett. 161, 176-179 16 Hann, B. C., Poritz, M. A. and Walter, P. (1989) J. Cell Biol. 109, 3223-3230 17 Tajima, S., Lauffer, L., Rath, V. L. and Walter, P. (1986) J. Cell Biol. 103, 1167-1178 18 Siegel, V. and Walter, P. (1988) Cell 52, 39-49 19 Rothman, J. E. (1989) Nature 340, 433-434 20 Rapoport, T. A., Heinrich, R., Walter, P. and Schulmeister, T. (1987) J. Mol. Biol. 195, 621-636 21 Siegel, V. and Walter, P. (1988) EMBO J. 7, 1769-1775 22 Wiedmann, M., Kurzchalia, T, V., Hartmann, E. and Rapoport, T. A. (1987) Nature 328, 830-833 23 Wiedmann, M., G6rlich, D., Hartmann, E., Kurzchalia, T. V. and Rapoport, T. A. (1989) FEBS Lett. 257, 263-268 24 Krieg, U. C., Johnson, A. E. and Walter, P. (1989) 3. Cell Biol. 109, 2033-2043 25 Prehn, S., Herz, J., Hartmann, E., Kurzchalia, T. V., Frank, R., R6misch, K., Dobberstein, B. and Rapoport, T. A. (1990) Eur. J. Biochem. 188, 439-445 26 Wickner, W. (1979) Annu. Rev. Biochem. 48, 23-45 27 Engelman, D. M. and Steitz, T. A. (1981) Cell 23, 411-422 28 yon Heijne, G. and Blomberg, C. (1979) Eur. J. Biochem. 97,175-181 29 Briggs, M., Cornell, D., Dlahy, R. A. and Gierasch, L. (1986) Science 233, 206-208 30 Nesmeyanova, M. A. (1982) FEBS Lett. 142, 184-193 31 de Vrije, G. J., Batenburg, A. M., Killian, J. A. and de Kruiff, B. (1990) in Dynamics and Biogenesis of Membranes, (NATOASI Series, Vol. H40) (Op den Kamp, J. A. F., ed.), pp. 247-258, Plenum Press 32 Blobel, G. and Dobberstein, B. (1975) J. Cell Biol. 67,852-862 33 Rapoport, T. A. (1985) FEBS Lett. 187, 1-10 34 Gilmore, R. and Blobel, G. (1985) Ce1142, 497-505 35 Connolly, T., Collins, P. and Gilmore, R. (1989) J. Cell Biol. 108, 299-307 36 Simon, S., Blobel, G. and Zimmerberg, J. (1989) Proc. Natl Acad. Sci. USA 86, 6176-6180 37 Nicchitta, C. and Blobel, G. (1990) Cell 60, 259-269 38 Hartmann, E., Wiedmann, M. and Rapoport, T. A. (1989) EMBO J. 8, 2225-2229 39 Rapoport, T. A. (1990) in Dynamics and Biogenesis of Membranes, (NATOASI Series, Vol. H40) (Op den Kamp, J. A. F., ed.), pp. 231-245, Plenum Press
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Other candidates for constituents of the translocation complex
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It appears unlikely that SSR is the only component of the translocation complex, as already indicated by the crosslinking experiments. Other membrane proteins have been suggested, on various grounds, to be involved in the translocation process (see Table l).
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THE MOLECULAR MECHANISM by which signal sequences direct polypeptides to the ER membrane is still poorly understood. In particular, it is puzzling that signal sequences do not share any sequence homology; their only common property is an uninterrupted stretch of at least six hydrophobic amino acids. Other signals involved in the intracellular protein transport, such as those for the import of proteins into mitochondria, chloroplasts or peroxisomes, appear to be equally vague (for review see Ref. 1).
Initiation of protein transport: the SRP cycle In the case of translocation across the ER membrane, signal sequences in general are initially recognized by the signal recognition particle (SRP)z. The SRP initiation or targeting cycle is schematically depicted in Fig. 1. The synthesis of a polypeptide to be translocated across the ER membrane starts with an actively translating ribosome that is free in the cytoplasm. As soon as the signal sequence of the nascent chain has emerged from the ribosome, it is recognized and bound by SRP, which also has an affinity for the ribosome. This association slows down the elongation of the polypeptide chain (elongation arrest) 2. In the next step, the SRP-ribosome-nascent chain complex is bound to the ER membrane via a specific interaction of SRP with its receptor (also termed docking protein) 3,4. The latter then binds GTP, which perhaps replaces previously bound GDPE After docking of the complex, both the signal sequence and the ribosome are released from SRP. The initiation cycle is completed by the dissociation of SRP from its receptor, a process that is triggered by GTP hydrolysis (R. Gilmore, pers. commun.). Several features of this scheme should be emphasized. (1) SRP and its receptor are not involved in the actual process of protein translocation through the membrane. (2) The interactions of SRP with a ribosome and a signal sequence are cooperative, i.e. one interaction strengthens the other.
T.A. Rapoportis at the Zentralinstitutffir Molekularbiologieder Akademieder Wissenschaftender DDR,1115 Berlin-Buch, Robert-R6ssle-Strasse10, DDR.
Protein transport across the ER membrane Protein transport across the endoplasmic reticulum (ER) membrane may be divided into two phases: an initiation or targeting cycle, which has been fairly well characterized, and the actual transfer of the polypeptide chain through the membrane, the mechanism of which is still unknown. In this review, the initiation cycle is discussed with emphasis on the mechanism of signal sequence recognition by the 54 kDa polypeptide of the signal recognition particle (SRP) and on the efficiency of targeting of nascent chains. Recent results are reviewed suggesting the transfer of the polypeptide chain by means of a translocation complex, a constituent of which appears to be the signal sequence receptor protein (SSR). As a consequence, SRP does not bind to completed polypeptide chains released from ribosomes 6. (3) Nucleotide hydrolysis is only required to start the next round of initiation5.
SRP functions as an 'antifolding device' by segregating the bound signal sequence from the rest of the polypeptide chain. In this respect, it resembles proteins called 'molecular chaperones'
ribosome , 5
~ r
mRNA ~
SRP SRP- receptor
ER-membrane
Pi
i
~.
GT P
,
Figure 1 The initiation (SRP)cycle of protein translocation. The scheme shows the first steps in protein translocation across the ER membrane after the signal sequence(hydrophobic portion indicated) has emerged from the ribosome and has been bound to the signal recognitionparticle (SRP).
© 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00
355
TIBS 1 5 - SEPTEMBER1990
or 'polypeptide chain-binding proteins '7,s. Other members of this family, such as the heat shock protein hsp70, may keep precursor polypeptides in a translocation-competent conformation in the exceptional cases where SRP and its receptor are not neededs-~°. These factors, however, unlike SRP, do not seem to recognize signal sequences specifically (at least in eukaryotes), nor are they bound to the membrane by specific receptors. It is therefore as yet unclear how the signal sequence is recognized in SRP-independent translocation pathways.
Signal sequence recognition by the SRP Photocrosslinking studies have shown that signal sequences of different proteins interact with the 54 kDa polypeptide component of SRP (SRP54)H,12. The sequence of SRP54 indicates the existence of two domains, an N-terminal G domain with sequence elements typical of a GTP-binding protein (GTP binding has indeed been demonstrated; J. Miller and P. Walter, pers. commun.), and a C-terminal methionine-rich M domain~3,14.Whereas the G domain was implicated by RSmisch et al. 13 in the binding of signal sequences, Bernstein et al. ~4 have assigned this function to the M domain. Bernstein et al. postulated that the methionine residues reside on one face of three amphipathic it-helices. These would form or contribute to a hydrophobic binding groove on the surface of SRP54 which could accommodate the hydrophobic core of signal sequences. Although a hydrophobic pocket that induces identical helical conformations with intra-chain saturation of all hydrogen-bond donors and acceptors would explain the binding of signal sequences of different primary structures ~s, the M domain appears not to be the only site involved. It has recently been found that the N-terminal part of a signal sequence can be crosslinked to the G domain and that the M domain links the protein to the rest of the particle (P. Walter and B. Dobberstein, pers. commun.). It therefore appears that the G domain, either alone or in conjunction with the M domain, provides the binding site for signal sequences. A recent report ~6 of homologues to the mammalian two-domain SRP54 in both yeast and E. coli suggests that the proteins involved in signal sequence recognition may be more conserved in evolution than hitherto assumed. 356
Most surprisingly, SRP54 shares further sequence homology with the SRP receptor in the G domain in addition to the short sequence elements typical for GTP-binding proteins, suggesting that the SRP receptor may also bind signal sequences ~3,~4. However, experimental tests of this proposal so far have been negative. A provocative alternative would be that the homologous regions of both SRP54 and the SRP receptor compete for a common binding site. It appears possible that SRP54 is transiently displaced from the SRP particle, together with the bound signal sequence, on interaction with the membrane receptor. SRP54 could be bound by mp30, a membrane protein that has been shown to have an affinity for SRP17. Alter release of the signal sequence into the membrane, SRP54 would rejoin the core particle to reform the intact SRP. Indeed, SRP54 is the only component of SRP that can dissociateTM. It is conceivable that free SRP54 is the signal sequence receptor for those proteins that do not require SRP for translocation9.
How is the efficient and specific targeting of nascent chains achieved? It has been proposed that GTP hydrolysis plays a role in enhancing the specificity of signal sequence recognition~4,19. Rothman 19 has argued that 'the incorrectly matched peptides would have an opportunity to dissociate from SRP54 before the switch of GTP hydrolysis is thrown', while the bound signal peptide would be fixed in place. Such a 'proof-reading' mechanism is nothing else but a rapid and reversible ligand-binding reaction preceding a slow and irreversible step (not necessarily coupled to GTP hydrolysis). A rapid binding and release of signal sequences is indeed required for fidelity of recognition. However, efficient targeting of nascent chains to the ER membrane is mainly achieved by a different mechanism that involves repetitive binding steps. Nascent chains have a certain size range (or 'window') in which they are able to interact with SRP2°.2LThe lower limit is determined by the length of the chain when the signal sequence has just completely emerged from the ribosome, the upper one by the point at which the signal sequence is buried in the folding chain. Alter each elongation step within these limits, a certain percentage of nascent chains will bind SRP (depend-
ing on the binding constant and the SRP concentration) and will be targeted irreversibly to the membrane. Membranebound chains no longer have a chance to bind SRP and are thus withdrawn from the equilibrium. The number of chains not yet translocated will therefore decrease as the end of the window region is approached and that bound to the membrane will increase accordingly. Consequently, even if the percentage of chains that interacts with SRP is low at each step, the overall targeting efficiency may be high due to the cumulative nature of the process 2°. The targeting efficiency will depend both on the binding constant of the signal sequence for SRP and on the size of the SRP window which is a function of the folding properties of the nascent chain. We have estimated that even the change of a single residue in the hydrophobic core of a signal sequence to a polar, uncharged one would be expected to reduce the binding constant by a factor of 103-105 (see Ref. 15; the binding constants have been estimated 2° to be in the range of 0.25-2.5nM). Thus, a high degree of discrimination in the recognition of signal sequences may be achieved.
Is there further signal recognition at the membrane? Photocrosslinking has been used to study the fate of the signal sequence after disengagement of SRP. With short nascent polypeptide fragments (70 residues long), the signal sequence was found in close proximity to an approximately 35 kDa integral membrane glycoprotein of the ER, provisionally termed the signal sequence receptor (SSR)=. It is unlikely that random encounters between the nascent chain and the membrane protein were responsible for the observed crosslinking; otherwise one should have expected a far more complex pattern of crosslinked species than was actually observed. Of course, proximity of the signal sequence and SSR does not necessarily imply specific binding, to which several objections can be raised. First, longer nascent chains that carried photoreactive groups exclusively in the mature portion still gave rise to crosslinks with SSR23,24. Secondly, the yield of crosslinked product was significantly lower for a polypeptide fragment with 70 residues as compared to one with 86 residues, although interaction of the signal sequence with a putative receptor should have been at least equal for
TIBS 1 5 - SEPTEMBER1990
the shorter fragment23,24. Finally, the sequence of SSR makes it unlikely that it could bind hydrophobic signal sequences since its cytosolic tail is very hydrophilic2s. Further experiments are thus needed to find out whether SSR actually binds to signal sequences. Membrane passage of the polypeptide: is there a translocation tunnel? The mechanism by which a polypeptide is transferred across a membrane is an issue of a long-standing debate. According to one viewpoint, transport occurs directly through the hydrophobic phospholipid bilayer without participation of proteins 26-28. The translocation of some polypeptides indeed appears to be independent of the presence of membrane proteins2E The ability of signal peptides of different structures to insert into protein-free phospholipid bilayers strongly correlates with their functionality in the overall process of translocation29. It is also possible that the transfer of the polypeptide occurs through a hexagonal lipid phase that would provide a hydrophilic environment in the interior of the membrane 3°,3~. Such a structure is favoured by certain phospholipids which are indeed abundant in natural membranes3L It is conceivable that the occurrence of a hexagonal lipid phase is also influenced or triggered by membrane proteins. According to alternative models, polypeptides are transported through a hydrophilic or amphiphilic tunnel that is assembled from transmembrane proteins 32,33. A protein environment for the nascent polypeptide chain during its membrane transfer was indeed suggested by the fact that aqueous perturbants such as urea remove the nascent chain from the membrane 34. Furthermore, after solubilization of ER membranes, the nascent polypeptide chains were found to be resistant to proteases. The C-terminal portion of the nascent chain was protected by the ribosome and the N-terminal one presumably by membrane proteins 3s. Fusion of microsomal vesicles with planar lipids has provided evidence for large aqueous channels of high conductivity that presumably represent the translocation tunnels 36. The involvement of one or more glycoproteins in the translocation process has been demonstrated recently with reconstituted proteoliposomes produced from detergent-solubilized microsomes 37. Finally, SSR has been found in close proximity to nascent chains dur-
SSRoc(34kDo)
5kDat
Cytoso[
c°rb°hydr°t ~ e
SSR ![3(22kDo)
lkDQ{t($)..chor gecluster ;b[°cked ~ N-terminus ~ ~0
k> chorgcleuster Figure 2 Membrane topology and sequence features of ~- and ~-SSR. Scheme showing the membrane-spanning regions, carbohydrate chains and charge clusters for the two subunits of the signal sequence receptor (SSR) (based on Ref. 25 and D. G6rlich eta/., unpublished).
ing membrane transfer and it may therefore be a constituent of the putative tunnel 23'24. SSR: a component of the translocation complex? To get more insight into the role of SSR in protein translocation, its purification was attempted. An integral 34 kDa glycoprotein was purified from canine pancreatic microsomes 38. Identity with SSR was suggested by the fact that antibodies raised against the intact protein or against a C-terminal peptide partially immunoprecipitate6 the crosslinked products of nascent secretory protein and SSR23. That the extent of immunoprecipitation was rather low may be due to the large amounts of non-crosslinked 34 kDa pro-
tein in the ER membrane and/or to the altered antigenicity of the protein after crosslinking. SSR is a major ER membrane protein (about 1.5% of total membrane protein in canine microsomes). It is present in a more than tenfold excess over SRP and SRP receptor and is even in excess over membrane-bound ribosomes 38, as would be expected if SSR indeed participates in the actual translocation process; each membrane-bound ribosome would be in the process of transferring a nascent chain through the membrane. Furthermore, SSR is largely restricted to the rough portion of the ER (F. Vogel et al., unpublished). In agreement with the assumed general function of SSR, it has been immunologically detected in various
Table I. ER-membrane proteins implicated in protein translocation Proteina
Source
Function
SRP-receptor ct-subunit ~subunit
mammals
Signal peptidasecomplex
mammals yeast (sec11)
Cleavageof signal peptide
SSR-complex
mammals
Tunnelconstituent, signal sequencebinding?
SRP-binding,GTP-binding Membrane-insertionof ~-subunit
ct-subunit Fsubunit Ribophorins I and II
mammals
?
sec61
yeast
?
sec62
yeast
?
sec63
yeast
?
sec65
yeast
?
aForreferencessee Ref. 39. 357
TIBS 15- SEPTEMBER1990
tissues of mammals and in birds. Antibodies against SSR and Fab-fragments prepared from the antibodies block the in vitro translocation of several secretory proteins ~. Thus, SSR appears to be essential for protein translocation. The sequence of SSR indicates that it has one hydrophobic membrane-spanning segment; the subsequent 56 amino acids are located in the cytosol (Fig. 2) 25. The luminal portion contains two carbohydrate chains. The protein has a striking charge distribution: a region close to the N-terminus is highly negatively charged (23 out of 35 residues) and the cytosolic tail carries a net positive charge. SSR does not share any sequence homology with other known proteins. It is made with a cleavable Nterminal signal peptide and is inserted into the ER membrane in an SRP-dependent manner. Both the sequence and the size exclude the possibility that a single molecule of the 34 kDa SSR can form a tunnel. Using bifunctional crosslinking reagents we have indeed found that other proteins are located in its close proximity in the ER membrane. Among them is a 22 kDa glycoprotein which remains tightly bound to the 34 kDa polypeptide even after solubilization of the membrane with detergent and which forms with it a stable, stoichiometric complex (D. G6rlich et al., unpublished). The two polypeptides have thus been named a- (34 kDa) and (22 kDa) subunits of SSR. The crosslinking experiments have also indicated the proximity of two 0~SSR molecules in the ER membrane, suggesting that SSR heterodimers associate with each other. The two subunits of the SSR share several features: a cleavable N-terminal signal peptide, a single hydrophobic membrane-spanning segment close to the C-terminus, and two carbohydrate chains in the luminal portion (Fig. 2). Like e~SSR, [~SSR also has no resemblance to any other protein so far registered in any data bank (D. G6rlich et aL unpublished).
Except for the signal peptidase complex, a function has not yet been demonstrated for any of them. In addition to the listed proteins, a ribosome receptor probably exists. Unfortunately, with the exception of one subunit of the signal peptidase complex (sec11), so far the proteins identified in yeast by genetic means have no correspondence to those found in mammals by biochemical methods and vice versa. On the basis of our current knowledge, the existence of a translocation tunnel remains equivocal. However, the recent breakthrough in reconstituting the translocation activity in proteoliposomes after complete solubilization of the ER membrane has provided a method to assess the function of purified membrane proteins in the translocation process 37.
Acknowledgements l thank M. Wiedmann, E. Hartmann, M. Shia, and particularly S. M. Rapoport, for critical reading of the manuscript.
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Other candidates for constituents of the translocation complex
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It appears unlikely that SSR is the only component of the translocation complex, as already indicated by the crosslinking experiments. Other membrane proteins have been suggested, on various grounds, to be involved in the translocation process (see Table l).
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