- Email: [email protected]
The translocon: more than a hole in the ER membrane?
.... ~
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Additional components. Both nascent secretory and membrane proteins are modified during translation by membrane-bound ER enzymes. In our view, because the nascent polypeptide does not leave the immediate vicinity of the translocon until protein synthesis terminates ]~ any ER enzyme that modifies a polypeptide before translation is complete should be considered a functional component of the translocon (Fig. 1). Thus, both signal peptiIn eukaryotes, the vast majority of secreted and integral membrane prodase (purified as a five-polypeptide teins are targeted to the membrane of the endoplasmic reticulum (ER) complex) and oligosaccharyltransferase early during translation. These polypeptides are then either transported (three polypeptides) are part of the (exacross or inserted into the ER membrane at sites termed translocons. As tended) translocon, because signal protein translocation occurs through an aqueous pore, the minimal recleavage and glycosylation occur before quirement for a translocon is a passive structure that provides a passagetranslation is complete ]2. It seems likely way across the membrane. However, recent data suggest that the translothat the polypeptides responsible for con is a complex structure that orchestrates the localization, orientation, the decoding and cleavage of glycosylmaturation and possibly degradation of nascent chains. phosphatidylinositol (GPI) addition signals are also nearby, even though this modification is necessarily post-transPROTEINS ARE TARGETED to the mRNA-encoded pause in translocation 3. lational. In addition, the translocon is eukaryotic endoplasmic reticulum (ER) Proteins can also be targeted to the ER likely to include components such as membrane either post-translationally as membrane post-translationally. Signal the gate protein(s) described below and full-length polypeptides or co-translation- sequence-dependent post-translational the 11 kDa protein that is crosslinked to ally as nascent polypeptides (Table I). targeting to the ER membrane occurs a nascent chain during translocational Nascent secretory and membrane primarily in yeast 4,5. Another group of pausing ]3. proteins are selected for processing at proteins are anchored at the membrane Topography. The concentration of so the ER membrane w h e n a signal se- following the translocon-independent many ER polypeptides at or near a quence or a signal-anchor sequence insertion of a carboxy-terminal TM se- single locus in the membrane raises emerges from the ribosome ]. By con- quence into the ER membrane 6. Here, the issue of how polypeptide packing trast to a signal sequence, which is we explore the participation of the can be arranged to allow so many ER cleaved from the nascent chain during translocon in several processes that membrane proteins to have direct actranslocation, a signal-anchor sequence occur during co-translational process- cess to the nascent chain. Although serves both to initiate membrane local- ing of nascent chains at the ER the translocon topography remains ization and then to act as an uncleaved membrane. largely a mystery, the long-standing transmembrane (TM) sequence for type controversy about whether transloI or type II membrane proteins. These The translocon cation occurs through an aqueous pore topogenic sequences are usually recogMinimal components. Protein compo- or through the nonpolar interior of nized and bound by the signal recogni- nents of the translocon were first iden- the ER membrane has been resolved. tion particle (SRP), and the resulting tified by crosslinking experiments in The incorporation of fluorescent ribosomal complex then interacts with which photo-reactive probes were probes into nascent secretory proteins the SRP receptor on the ER membrane incorporated into nascent chains permitted the direct demonstration at the translocon. Targeting of these that were undergoing co-translational that the translocating nascent chain proteins is completed by a GTP-depend- translocation and integration. The ER occupies an aqueous pore that spans ent interaction between SRP and its proteins most frequently photo- the entire ER membrane TM. Puromycinreceptor that leaves the ribosome crosslinked were Sec61~ and TRAM];. dependent conductivity data are also bound to an ER site designated the The subsequent reconstitution of puri- consistent with translocation through translocon 2. In at least one case, fied ER membrane proteins into proteo- an aqueous pore ]5. Photo-crosslinking nascent chain targeting of a peripheral liposomes indicated that only Sec61~, studies with nascent secretory proER membrane protein is not SRP- Sec6113, Sec61% SRP receptor (two teins indicate that the inner surface of mediated, but is effected through an polypeptides) and, sometimes, TRAM the aqueous pore is formed primarily were required for co-translational by Sec61~ (Refs 16, 17), but that the translocation or integration of secre- nonpolar region of signal and signalD. W. Andrews is at the Department of tory or membrane proteins 8,9. Although anchor sequences can contact phosBiochemistry, McMasterUniversity,1200 Main St West, Hamilton, Ontario, these six polypeptides constitute the pholipid directly ]8. Finally, it was Canada L8N 3Z5. minimal translocon necessary for sim- shown that the translocon is strucEmail: [email protected] ple SRP-dependent transport processes, turally multi-layered, as the TM doA. E. Johnson is at the College of Medicine, the efficiency of the reconstituted sys- main of a nascent membrane protein TexasA&M University, tem is low, presumably because the moves past Sec61~ to sites adjacent to 116 Reynolds Medical Building, translocon normally contains other pro- TRAM before being released into the College Station, TX 77843-1114, USA. Email: [email protected] teins that optimize its activity. bilayer 1].
The translocon: more than a hole in the ER membrane? David W. Andrews and Arthur E. Johnson
9 1996,ElsevierScienceLtd
PlI: s0968-0004(96)10047-5
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co-translational addition of lipids during apolipoprotein B synthesis. Translation is not arrested during the Involvement of: translocation pause, but the riboTiming SRP Translocon Type of sequence Distinguishing features Refs some-membrane junction is restructured such that the nascent polypepCo-translational + + Signal/signal-anchor SRP-dependent 1 tide is both transiently accessible to Post-translational + Signal/signal-anchor SRP-independent 4, 5 Co-translational SR~ dimerization domain mRNA-encoded pause 3 the cytosol and also moved to a loin translation cation from which it can photo-crosslink Post-translational Insertion sequence Located at carboxyl 6 to an as-yet unknown 11 kDa protein in terminus of protein the translocon13. This restructuring aSRP, signal recognition particle. must maintain the permeability barrier of the membrane while the ApoB Is the translocon dynamic? Although maintaining the permeability barrier nascent chain is exposed to the cytothese data depict a static translocon, across the ER membrane is required. In plasm, although it is not yet clear how the actual translocon structure is dy- the case of co-translational translo- this is accomplished at the molecular namic. This is shown most directly by cation, the binding of the ribosome to level. Recent estimates suggest that the fact that the lumenal end of the the translocon prevents the movement translocation pauses might last several aqueous pore is closed to the lumen of of cytoplasmic ions into the ER lumen by seconds in vivo, which is ample time for the ER immediately after the ribosome creating a tight seal at the cytoplasmic cytoplasmic proteins to modify or interis targeted to the membrane, but is end of the translocon pore ]4,2~ The act with the paused nascent chain. opened after translation increases the lumenal end of the aqueous pore is also Translocation pausing has also been length of the nascent chain to a length closed immediately after targeting, as demonstrated for the prion-related pronear 70 residues 14. This, presumably, noted above, presumably by a pro- tein (PrP), a polypeptide unrelated to constitutes a safety mechanism: the tein(s) that functions to gate the pore 14. apolipoprotein B. As the only apparent pore is opened to the lumen only after a As the tight ribosome-translocon junc- common feature of these proteins is properly-seated ribosome has trans- tion prevents any nascent chain move- that they have a complex pattern of lated sufficient nascent chain to trigger ment into the cytoplasm during the biogenesis, it is possible that pausing the opening of the pore 14. Interestingly, translocation of a simple secreted pro- is a common mechanism for accessing signal sequence-dependent binding of tein, the vectorial movement of such a certain regulated functions of the ribosomes to reconstituted translocons protein is dictated by the co-axial stack- translocon24. was detected at low salt in the absence ing of the ribosomal tunnel on the both of SRP and of SRP receptor when translocon, and no additional energy or Aborted translocation the nascent chain exceeded 70 amino proteins are required ]4,2~ However, When a nascent protein engages the acids in length ]9. The striking coinci- other interactions might be necessary translocation machinery with an aminodence of these results suggests that a to optimize the translocation of some terminal signal sequence, the direction signal sequence--translocon interaction proteins in vivo, as suggested below of polypeptide movement is not set irmight be involved in opening the and by the observation that lumenal revocably from the cytoplasm to the ER lumenal end of the aqueous pore. proteins were required to ensure lumen. In vitro, a fraction of hepatitis B One mechanistic advantage of a trans- translocation after signal sequence pre-core protein initiates translocation, locon that undergoes time-dependent cleavage in vitro 21. but then falls back into the cytosol after changes in conformation, polypeptide As signal peptide cleavage occurs co- signal peptide cleavage 2s. Bidirectional arrangement and even composition is translationally at the lumenal end of the movement was also observed for that the exposure of the nascent chain translocon 1, evidence of any processing transiocation intermediates as long as to different proteins and/or phospho- by signal peptidase shows that a por- 221 residues of an antimicrobial leukolipid can be maximized and pro- tion of the nascent polypeptide has tra- cyte protein26. Both secreted and cytogrammed at a single translocon site. versed the translocon. When produced plasmic forms of plasminogen activaThus, among the most interesting as- in vitro and in BHK cells, connexins are tors inhibitor 2 (PAI-2) are synthesized pects of translocon structure that cleaved incorrectly. However, incorrect from one RNA species using the same remain to be determined are its poss- cleavage is not observed in cells pro- open reading frame27. Treatment of cells ible cyclic assembly and disassembly, ducing connexin normally, which sug- with the phorbol ester PMA increased the magnitude of the conformational gests that the specificity of the signal the fraction of PAI-2 secreted from U937 changes that accompany the reversible peptidase can be controlled at the cells from just a few percent to 50% of conversion of translocon function from translocon22. the PAI-2 synthesized, suggesting that translocation to integration, and the translocation is regulated at the transextent to which peripheral translocon Translocation pausing locon as both cytosoplasmic and secomponents transiently associate with Translocation is transiently arrested creted molecules have the same unthe ER proteins that form the aqueous during the synthesis of apolipoprotein B cleaved signal sequence at the amino pore. by specific sequences within the terminus27. nascent protein termed pause transfer An extreme example of aborted Translocation sequences 23. It has been postulated that translocation was observed for fusion AS secretory proteins are transported by extending the period of time a se- proteins containing the targeting seacross the ER membrane through an quence remains within the translocon, quences of Pseudomonas exotoxin A. aqueous pore ]4, a mechanism for translocation pausing facilitates the This toxin is thought to enter cells by Table I. Protein targeting pathways to the ER membrane a
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retrograde transport through the secretory pathway and escape into the cytoplasm from the ER. Evidence that the exit site from the ER for exotoxin A is the translocon comes from in vitro studies of chimeric proteins in which a secretory signal sequence was fused to exotoxin A sequences. After targeting to the translocon and signal peptide cleavage, the mature portion of the molecule was found only in the cytoso128. When the mRNA lacked a termination codon, the nascent molecule was released back into the cytosol while still attached to the ribosome, indicating that at least in this context, it is released directly from the translocon 2s.
Integration into the membrane During membrane protein integration, the critical mechanistic problem to be overcome at the translocon is not Final folding and release the translocation of hydrophi]ic polypeptide across the Figure 1 lipid bilayer, but the accurate Schematic view of translocon functions. When the nascent polypeptide is targeted to the translocon it recognition and insertion of can be translocated into the ER lumen, integrated in the membrane or retrotranslocated back to the TM sequences into the lipid cytosol. During processing of the nascent polypeptide, the variety of ER enzymes that modify the bilayer. The primary driving nascent polypeptide or regulate translocon function are considered components of the functional force for integration is betranslocon. The nascent chain does not normally leave the translocon until the translation has comlieved to be the hydropleted. Therefore, the enzymes shown are predicted to have at least transient and probably regulated phobicity of the TM sequence, access to the translocation site within the translocon. Some of the functions illustrated are known to be carried out by several polypeptides, while for others the specific polypeptides involved remain based on the creation of artifiuncharacterized. SRP, signal recognition peptide. cial TM domains 29 and the mutagenesis of authentic T M sequences 3~ Although a threshold hydro- amount of polypeptide, the nascent regulates the processing of the molecule phobicity is required for a sequence to chains of such proteins must pass as either a bitopic integral membrane or be thermodynamically stable in a lipid through the ribosome-membrane junc- a completely translocated protein. When bilayer, hydrophobicity is not the only tion at some point during the inte- the PrP STE was fused amino-terminal of mediator of membrane integration. An gration process. It has not yet been es- an otherwise secreted hydrophobic uncleaved mutant signal sequence con- tablished how this is done without amino acid sequence, the STE was taining 23 contiguous, uncharged amino compromising the permeability barrier. shown to initially pause translocation acids did not halt translocation or reTM recognition. A clue to the mecha- and then mediate transmembrane intesult in membrane integration when lo- nism that discriminates translocated gration of the fusion protein 24. By incorcated in the interior of a secreted from non-translocated hydrophobic se- porating the 21-amino acid IgM STE on protein 31. By contrast, some relatively quences comes from the recent identifi- the amino-terminal side of the TM seshort, uncharged sequences (nine Leu cation of non-hydrophobic effectors of quence in fusion proteins that have or 14 alternating Leu/Ala residues) can membrane integration. These so-called either a type I or type II topology, it was function as transmembrane domains stop transfer effector (STE) sequences demonstrated that this STE sequence and integrate into the ER membrane 29. halt translocation and mediate mem- can end up on either side of the ER Another critical mechanistic concern brane integration of relatively non- membrane31. during integration is the maintenance of hydrophobic polypeptide sequences 32. TM orientation. The orientation of a TM the permeability barrier, particularly for The two naturally occurring eukaryotic sequence is determined during intemultiple-spanning membrane proteins STE sequences that have been charac- gration and is widely believed to be and for membrane proteins that have terized are both located amino-terminal dictated by the distribution of charged
large cytoplasmic domains. As the nascent-chain tunnel in the ribosome can only accommodate a limited
of the sequence that is embedded in the ER membrane 3],32. PrP has been shown to contain an STE sequence that
residues surrounding it. Most proteins adopt a topology with the more positively charged TM flanking sequence in
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TALKINGPOINT the cytoplasm. However, much of the experimental data for the 'positiveinside rule' comes from the examination of post-translational and Secindependent integration in Escherichia coil 33. Statistical analysis of protein sequence data from eukaryotes is compatible with the positive-inside rule, but there are a number of exceptions 34, including fusion proteins containing the transmembrane domain of lgM (Ref. 35). Because processing is primarily cotranslational in eukaryotes and usually post-translational in prokaryotes, neither the mechanism nor the amino acid sequence requirements are necessarily similar. When the positive-inside rule was tested directly in a eukaryotic cellfree system by changing the sequences surrounding hydrophobic domains without intrinsic orientational preference, six out of eight molecules adopted an orientation opposite to that predicted by the distribution of charges flanking the TM domain a. Analyses of the topology of ductin suggest that the process that determines orientation and integration is not fixed by sequence, but is actively regulated, as in cells, ductin molecules in different locations have opposite orientations 36. When synthesized in vitro, ductin molecules are inserted into pancreatic microsomes in both orientations. Furthermore, changing the conserved charged residues at the amino terminus did not alter the ratio of the two forms obtained 36. These results and other exceptions to the positive-inside rule 37suggest that orientation is established by an as-yet unidentified balance of properties, and that the distribution of positive charges surrounding the TM domain is only one of the important variables. The translocon component(s) that presumably mediates the orientation of a TM sequence is unknown. Integration Is a regulated, multi-atep process. Thus, co-translational integration is not simply a one-step insertion of the TM sequence into the hydrophobic bilayer. Instead, several choices must be made at and by the translocon during the processing of polypeptides for integration. Experimental evidence for the multistep nature of this process is provided by photo-crosslinking data, which reveal that the TM domain passes sequentially through at least three different proteinaceous environments at the translocon, and that the TM sequence does not leave the translocon until translation terminates u. The movement of a TM sequence from the aqueous 368
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pore into the translocon and its ultimate release into the bilayer therefore appears to be highly regulated, presumably to ensure proper recognition, orientation and insertion of TM sequences, especially in multi-spanning membrane proteins. Moreover, current data suggest that TRAM plays a prominent role in such integration processing n.
Retrotranslocation Recent evidence suggests that rapid ER-associated protein degradation is mediated by proteasomes following movement of the protein from the ER lumen to the cytoplasm38. This movement, which we have termed retrotranslocation, must occur without destroying the permeability barrier between the cytoplasm and the ER lumen. As the translocon transports polypeptides without compromising the ER membrane during translocation, it is reasonable to wonder whether the translocon might also mediate retrotranslocation. Although there is no direct evidence implicating the translocon in this type of protein movement, calnexin is required for the retrotranslocation and degradation of nonglycosylated forms of proalpha factor in a yeast in vitro system 38. Calnexin has also been shown to be involved in the folding of polypeptides in the ER lumen before their translation has been completed TM. Calnexin is therefore a likely component of the extended translocon, and this proximity is consistent with a role for the translocon in retrotranslocation. Translocated MHC class I polypeptides can ratum to the cytoplasm. Newly-synthesized MHC class I molecules are selectively released into the cytoplasm and degraded in cells synthesizing the cytomegalovirus protein US11 (Ref. 40). Full length MHC class I molecules are recovered in an $100 fraction from USllproducing cells when cytoplasmic proteasomes are chemically inactivated, suggesting the surprising possibility that even the transmembrane domain of the MHC class I protein is transported back to the cytoplasm. Much of the new molecule has traversed the translocon before retrotranslocation, as demonstrated by the presence of glycosylated and signal sequence-cleaved MHC class I molecules in cytosoP~ As the only viral protein required to trigger rapid degradation of MHC class I is US11 (itself an integral ER membrane protein) and as extraction of an integrated TM
OCTOBER 1996
sequence would be thermodynamically difficult, it is unlikely that US11 creates an MHC class I specific translocation site that pulls the MHC class I polypeptide out of the lipid bilayer. Instead, we speculate that US11 targets new MHC class I proteins to a translocondependent pathway that is used to return other rapidly degraded molecules to the cytoplasm before integration. Consistent with this possibility, the movement of a polypeptide through the translocon is not necessarily continuous, nor unidirectional, as noted above. The release of exotoxin A fusion proteins, while still attached to the ribosome, is consistent with an export function for the translocon 28. Hence, the translocon might play a role in quality control by preventing the release to the ER lumen or to the lipid bilayer of proteins destined for degradation. The characteristics of some other rapidly degraded integral membrane and lumenal proteins are also consistent with retrotranslocation to the degradative pathway by the translocon. In cells synthesizing apolipoprotein B, rapid degradation has been shown to occur before the molecule is fully translocated across the ER membrane 41. Degradation of mutant cystic fibrosis transmembrane conductance regulator (CFTR) molecules has also been shown to require both cytoplasmic proteasomes and ubiquitination, possibly by Ubc6, a protein that can contact the transiocon 42-44. However, the site of CFTR degradation has not been rigorously examined. Finally, sequences within the transmembrane domains of incorrectly or incompletely assembled oligomeric proteins have been shown to target the polypeptides for rapid degradation 45. These observations, coupled with recent data suggesting that membrane integration is tightly regulated at the translocon 11, are consistent with rapid degradation resulting from retrotranslocation from the translocon to the degradative machinery in the cytoplasm. Concluding remarks A primary function of the translocon in the ER membrane of eukaryotes is to facilitate protein transit from the cytoplasm to the ER lumen. It is also clear that the translocon plays an active role in the selection, orientation and insertion of TM sequences into the bilayer. Furthermore, evidence is accumulating that the translocon participates in processes not conventionally associated
TIBS 21 -
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OCTOBER 1996
with the vectorial cytoplasm-to-lumen transport of nascent polypeptides. The range of activities demonstrated or presumed to occur in the immediate vicinity of the translocon suggests that the translocon is an important regulatory site, and that further surprises are likely to emerge as our understanding of the topography and dynamics of the translocon increases,
Acknowledgements We thank V. Lingappa and H. Ploegh for providing manuscripts before publication, Work in the authors' laboratories was supported by a grant and a scientist award from the Medical Research Council of Canada (D. W. A.), by NIH grant R01 GM 26494 (A. E. J.), and by the Robert A. Welch Foundation (A. E. J.). We apologize to those whose work was not cited because of the limitation in the number of references permitted in this article.
25 Garcia, P. D. et al. (1988) J. Cell Biol.
106, 1093-1105 26 Ooi, C. E. and
Weiss, J. (1992) Cell 71, 87-96 27 Belin, D. et af. (1989) EMBO J. 8, 3287-3294 28 Theuer, C. P. et al. (1993) Proc. Natl. Acad. Sci. U. S. A.
90, 7774-7778 29 Kuroiwa, T. et al. (1991) J. Biol. Chem. 266,
9251-9255 30 Speiss, M.,
31
32
33
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3 Young, J. C. and Andrews, D. W. (1996) EMBO J. 15, 172-181 4 Panzer, S. et al. (1995) Cell 81,561-570 5 Schlenstedt, G., Gudmundsson, G. H., Boman, H. G. and Zimmermann, R. (1990) J. Biol. Chem. 265, 13960-13968 6 Kutay, U., Hartmann, E. and Rapoport, T. A. (1993) Trends Cell Biol. 3, 72-75 7 Martoglio, B. and Dobberstein, B. (1996) Trends Cell Biol. 6,142-147 8 G6rlich, D. and Rapoport, T. A. (1993) Cell 75, 615-630 9 Oliver, J. et al. (1995) FEBS Lett. 362, 126-130 10 Thrift, R. N., Andrews, D. W., Walter, P. and Johnson, A. E. (1991) J. Cell Biol. 112, 809-821 11 Do, H. et al. (1996) Cell 85, 369-378 12 Gilmore, R. (1993) Cell 75, 589-592 13 Hedge, R. S. and Lingappa, V. R. (1996) Celt 85, 217-228 14 Crowley, K. S. et al. (1994) Cell 78, 461-471 15 Simon, S. M. and Blobel, G. (1991) Cell 65, 371-380 16 High, S. et al. (1993) J. Biol. Chem. 268, 26745-26751 17 Mothes, W., Prehn, S. and Rapoport, T. A. (1994) EMBO J. 13, 3973-3982 18 Martoglio, B., Hofmann, M. W., Brunner, J. and Dobberstein, B. (1995) Cell 81, 207-214 19 Jungnickel, B. and Rapoport, T. A. (1995) Cell 82,261-270 20 Crowley, K. S., Reinhart, G. D. and Johnson, A. E. (1993) Cell 73, 1101-1115 21 Nicchitta, C. V. and Blobel, G. (1993) Cell 73, 989-999 22 Falk, M. M., Kumar, N. M. and Gilula, N. B. (1994) J. Cell Biol. 127,343-355 23 Chuck, S. L. and Lingappa, V. R. (1992) Cell 68, 9-21 24 Nakahara, D. H., Lingappa, V. R. and Chuck, S. L. (1994) J. Biol. Chem. 269, 7617-7622
<~IENTI~I"5 To ~,/~N/)oN /'t~E/~L" LA~f.AT~P-'/ AN//4AI-..
34
35
36
37
38
39
Handschin, C. and Baker, K. P. (1989) J. Biol. Chem. 264, 19117-19124 Andrews, D. W. et al. (1992) J. Biol. Chem. 267, 7761-7769 Yost, C. S. et al. (1990) Nature 343, 669-672 Dalbey, R. E., Kuhn,A. and von Heijne, G. (1995) Trends Cell Biol. 5, 380-383 Beltzer, J. P. et aL (1991) J. Biol. Chem. 266, 973-978 Mize, N. K., Andrews, D. W. and Ungappa, V. R. (1986) Cell 47, 711-719 Dunlop, J., Jones, P. C. and Finbow, M. E. (1995) EMBO J. 14, 3609-3616 Speiss, M. (1995) FEBS Lett. 369, 76-79 McCracken, A. A. and Brodsky, J. L. (1996) J. Cell Biol. 132,291-298 Bergeron, J. J. M. et al. (1994)
I
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&A'~eIT~ r A N
~E ~ l ' V l r { l ~
~
Trends Biochem. ScL 40
41
42
43
44
45
19, 124-128 Wiertz, E. J. H. J. et aL (1996) Cell 84, 769-779 Davis, R. A. et aL (1990) J. Biol. Chem. 265, 10005-10011 Ward, C. L., Omura, S. and Kop;to, R. R. (1995) Cell 83, 121-127 Jensen, T. J. et al. (1995) Cell 83, 129-135 Sommer, T. and Jentsch, S. (1993) Nature 365, 176-179 Klausner, R. D. and Sitia, R. (1990) Cell 62, 611-614
Pete Jeffs is a freelancer working in Paris, France 369
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TALKING POINT
TIBS 21 - OCTOBER 1996
Additional components. Both nascent secretory and membrane proteins are modified during translation by membrane-bound ER enzymes. In our view, because the nascent polypeptide does not leave the immediate vicinity of the translocon until protein synthesis terminates ]~ any ER enzyme that modifies a polypeptide before translation is complete should be considered a functional component of the translocon (Fig. 1). Thus, both signal peptiIn eukaryotes, the vast majority of secreted and integral membrane prodase (purified as a five-polypeptide teins are targeted to the membrane of the endoplasmic reticulum (ER) complex) and oligosaccharyltransferase early during translation. These polypeptides are then either transported (three polypeptides) are part of the (exacross or inserted into the ER membrane at sites termed translocons. As tended) translocon, because signal protein translocation occurs through an aqueous pore, the minimal recleavage and glycosylation occur before quirement for a translocon is a passive structure that provides a passagetranslation is complete ]2. It seems likely way across the membrane. However, recent data suggest that the translothat the polypeptides responsible for con is a complex structure that orchestrates the localization, orientation, the decoding and cleavage of glycosylmaturation and possibly degradation of nascent chains. phosphatidylinositol (GPI) addition signals are also nearby, even though this modification is necessarily post-transPROTEINS ARE TARGETED to the mRNA-encoded pause in translocation 3. lational. In addition, the translocon is eukaryotic endoplasmic reticulum (ER) Proteins can also be targeted to the ER likely to include components such as membrane either post-translationally as membrane post-translationally. Signal the gate protein(s) described below and full-length polypeptides or co-translation- sequence-dependent post-translational the 11 kDa protein that is crosslinked to ally as nascent polypeptides (Table I). targeting to the ER membrane occurs a nascent chain during translocational Nascent secretory and membrane primarily in yeast 4,5. Another group of pausing ]3. proteins are selected for processing at proteins are anchored at the membrane Topography. The concentration of so the ER membrane w h e n a signal se- following the translocon-independent many ER polypeptides at or near a quence or a signal-anchor sequence insertion of a carboxy-terminal TM se- single locus in the membrane raises emerges from the ribosome ]. By con- quence into the ER membrane 6. Here, the issue of how polypeptide packing trast to a signal sequence, which is we explore the participation of the can be arranged to allow so many ER cleaved from the nascent chain during translocon in several processes that membrane proteins to have direct actranslocation, a signal-anchor sequence occur during co-translational process- cess to the nascent chain. Although serves both to initiate membrane local- ing of nascent chains at the ER the translocon topography remains ization and then to act as an uncleaved membrane. largely a mystery, the long-standing transmembrane (TM) sequence for type controversy about whether transloI or type II membrane proteins. These The translocon cation occurs through an aqueous pore topogenic sequences are usually recogMinimal components. Protein compo- or through the nonpolar interior of nized and bound by the signal recogni- nents of the translocon were first iden- the ER membrane has been resolved. tion particle (SRP), and the resulting tified by crosslinking experiments in The incorporation of fluorescent ribosomal complex then interacts with which photo-reactive probes were probes into nascent secretory proteins the SRP receptor on the ER membrane incorporated into nascent chains permitted the direct demonstration at the translocon. Targeting of these that were undergoing co-translational that the translocating nascent chain proteins is completed by a GTP-depend- translocation and integration. The ER occupies an aqueous pore that spans ent interaction between SRP and its proteins most frequently photo- the entire ER membrane TM. Puromycinreceptor that leaves the ribosome crosslinked were Sec61~ and TRAM];. dependent conductivity data are also bound to an ER site designated the The subsequent reconstitution of puri- consistent with translocation through translocon 2. In at least one case, fied ER membrane proteins into proteo- an aqueous pore ]5. Photo-crosslinking nascent chain targeting of a peripheral liposomes indicated that only Sec61~, studies with nascent secretory proER membrane protein is not SRP- Sec6113, Sec61% SRP receptor (two teins indicate that the inner surface of mediated, but is effected through an polypeptides) and, sometimes, TRAM the aqueous pore is formed primarily were required for co-translational by Sec61~ (Refs 16, 17), but that the translocation or integration of secre- nonpolar region of signal and signalD. W. Andrews is at the Department of tory or membrane proteins 8,9. Although anchor sequences can contact phosBiochemistry, McMasterUniversity,1200 Main St West, Hamilton, Ontario, these six polypeptides constitute the pholipid directly ]8. Finally, it was Canada L8N 3Z5. minimal translocon necessary for sim- shown that the translocon is strucEmail: [email protected] ple SRP-dependent transport processes, turally multi-layered, as the TM doA. E. Johnson is at the College of Medicine, the efficiency of the reconstituted sys- main of a nascent membrane protein TexasA&M University, tem is low, presumably because the moves past Sec61~ to sites adjacent to 116 Reynolds Medical Building, translocon normally contains other pro- TRAM before being released into the College Station, TX 77843-1114, USA. Email: [email protected] teins that optimize its activity. bilayer 1].
The translocon: more than a hole in the ER membrane? David W. Andrews and Arthur E. Johnson
9 1996,ElsevierScienceLtd
PlI: s0968-0004(96)10047-5
365
TALKINGPOINT
TIBS 21 -
OCTOBER 1996
co-translational addition of lipids during apolipoprotein B synthesis. Translation is not arrested during the Involvement of: translocation pause, but the riboTiming SRP Translocon Type of sequence Distinguishing features Refs some-membrane junction is restructured such that the nascent polypepCo-translational + + Signal/signal-anchor SRP-dependent 1 tide is both transiently accessible to Post-translational + Signal/signal-anchor SRP-independent 4, 5 Co-translational SR~ dimerization domain mRNA-encoded pause 3 the cytosol and also moved to a loin translation cation from which it can photo-crosslink Post-translational Insertion sequence Located at carboxyl 6 to an as-yet unknown 11 kDa protein in terminus of protein the translocon13. This restructuring aSRP, signal recognition particle. must maintain the permeability barrier of the membrane while the ApoB Is the translocon dynamic? Although maintaining the permeability barrier nascent chain is exposed to the cytothese data depict a static translocon, across the ER membrane is required. In plasm, although it is not yet clear how the actual translocon structure is dy- the case of co-translational translo- this is accomplished at the molecular namic. This is shown most directly by cation, the binding of the ribosome to level. Recent estimates suggest that the fact that the lumenal end of the the translocon prevents the movement translocation pauses might last several aqueous pore is closed to the lumen of of cytoplasmic ions into the ER lumen by seconds in vivo, which is ample time for the ER immediately after the ribosome creating a tight seal at the cytoplasmic cytoplasmic proteins to modify or interis targeted to the membrane, but is end of the translocon pore ]4,2~ The act with the paused nascent chain. opened after translation increases the lumenal end of the aqueous pore is also Translocation pausing has also been length of the nascent chain to a length closed immediately after targeting, as demonstrated for the prion-related pronear 70 residues 14. This, presumably, noted above, presumably by a pro- tein (PrP), a polypeptide unrelated to constitutes a safety mechanism: the tein(s) that functions to gate the pore 14. apolipoprotein B. As the only apparent pore is opened to the lumen only after a As the tight ribosome-translocon junc- common feature of these proteins is properly-seated ribosome has trans- tion prevents any nascent chain move- that they have a complex pattern of lated sufficient nascent chain to trigger ment into the cytoplasm during the biogenesis, it is possible that pausing the opening of the pore 14. Interestingly, translocation of a simple secreted pro- is a common mechanism for accessing signal sequence-dependent binding of tein, the vectorial movement of such a certain regulated functions of the ribosomes to reconstituted translocons protein is dictated by the co-axial stack- translocon24. was detected at low salt in the absence ing of the ribosomal tunnel on the both of SRP and of SRP receptor when translocon, and no additional energy or Aborted translocation the nascent chain exceeded 70 amino proteins are required ]4,2~ However, When a nascent protein engages the acids in length ]9. The striking coinci- other interactions might be necessary translocation machinery with an aminodence of these results suggests that a to optimize the translocation of some terminal signal sequence, the direction signal sequence--translocon interaction proteins in vivo, as suggested below of polypeptide movement is not set irmight be involved in opening the and by the observation that lumenal revocably from the cytoplasm to the ER lumenal end of the aqueous pore. proteins were required to ensure lumen. In vitro, a fraction of hepatitis B One mechanistic advantage of a trans- translocation after signal sequence pre-core protein initiates translocation, locon that undergoes time-dependent cleavage in vitro 21. but then falls back into the cytosol after changes in conformation, polypeptide As signal peptide cleavage occurs co- signal peptide cleavage 2s. Bidirectional arrangement and even composition is translationally at the lumenal end of the movement was also observed for that the exposure of the nascent chain translocon 1, evidence of any processing transiocation intermediates as long as to different proteins and/or phospho- by signal peptidase shows that a por- 221 residues of an antimicrobial leukolipid can be maximized and pro- tion of the nascent polypeptide has tra- cyte protein26. Both secreted and cytogrammed at a single translocon site. versed the translocon. When produced plasmic forms of plasminogen activaThus, among the most interesting as- in vitro and in BHK cells, connexins are tors inhibitor 2 (PAI-2) are synthesized pects of translocon structure that cleaved incorrectly. However, incorrect from one RNA species using the same remain to be determined are its poss- cleavage is not observed in cells pro- open reading frame27. Treatment of cells ible cyclic assembly and disassembly, ducing connexin normally, which sug- with the phorbol ester PMA increased the magnitude of the conformational gests that the specificity of the signal the fraction of PAI-2 secreted from U937 changes that accompany the reversible peptidase can be controlled at the cells from just a few percent to 50% of conversion of translocon function from translocon22. the PAI-2 synthesized, suggesting that translocation to integration, and the translocation is regulated at the transextent to which peripheral translocon Translocation pausing locon as both cytosoplasmic and secomponents transiently associate with Translocation is transiently arrested creted molecules have the same unthe ER proteins that form the aqueous during the synthesis of apolipoprotein B cleaved signal sequence at the amino pore. by specific sequences within the terminus27. nascent protein termed pause transfer An extreme example of aborted Translocation sequences 23. It has been postulated that translocation was observed for fusion AS secretory proteins are transported by extending the period of time a se- proteins containing the targeting seacross the ER membrane through an quence remains within the translocon, quences of Pseudomonas exotoxin A. aqueous pore ]4, a mechanism for translocation pausing facilitates the This toxin is thought to enter cells by Table I. Protein targeting pathways to the ER membrane a
366
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. . . . .
TALKINGPOINT
retrograde transport through the secretory pathway and escape into the cytoplasm from the ER. Evidence that the exit site from the ER for exotoxin A is the translocon comes from in vitro studies of chimeric proteins in which a secretory signal sequence was fused to exotoxin A sequences. After targeting to the translocon and signal peptide cleavage, the mature portion of the molecule was found only in the cytoso128. When the mRNA lacked a termination codon, the nascent molecule was released back into the cytosol while still attached to the ribosome, indicating that at least in this context, it is released directly from the translocon 2s.
Integration into the membrane During membrane protein integration, the critical mechanistic problem to be overcome at the translocon is not Final folding and release the translocation of hydrophi]ic polypeptide across the Figure 1 lipid bilayer, but the accurate Schematic view of translocon functions. When the nascent polypeptide is targeted to the translocon it recognition and insertion of can be translocated into the ER lumen, integrated in the membrane or retrotranslocated back to the TM sequences into the lipid cytosol. During processing of the nascent polypeptide, the variety of ER enzymes that modify the bilayer. The primary driving nascent polypeptide or regulate translocon function are considered components of the functional force for integration is betranslocon. The nascent chain does not normally leave the translocon until the translation has comlieved to be the hydropleted. Therefore, the enzymes shown are predicted to have at least transient and probably regulated phobicity of the TM sequence, access to the translocation site within the translocon. Some of the functions illustrated are known to be carried out by several polypeptides, while for others the specific polypeptides involved remain based on the creation of artifiuncharacterized. SRP, signal recognition peptide. cial TM domains 29 and the mutagenesis of authentic T M sequences 3~ Although a threshold hydro- amount of polypeptide, the nascent regulates the processing of the molecule phobicity is required for a sequence to chains of such proteins must pass as either a bitopic integral membrane or be thermodynamically stable in a lipid through the ribosome-membrane junc- a completely translocated protein. When bilayer, hydrophobicity is not the only tion at some point during the inte- the PrP STE was fused amino-terminal of mediator of membrane integration. An gration process. It has not yet been es- an otherwise secreted hydrophobic uncleaved mutant signal sequence con- tablished how this is done without amino acid sequence, the STE was taining 23 contiguous, uncharged amino compromising the permeability barrier. shown to initially pause translocation acids did not halt translocation or reTM recognition. A clue to the mecha- and then mediate transmembrane intesult in membrane integration when lo- nism that discriminates translocated gration of the fusion protein 24. By incorcated in the interior of a secreted from non-translocated hydrophobic se- porating the 21-amino acid IgM STE on protein 31. By contrast, some relatively quences comes from the recent identifi- the amino-terminal side of the TM seshort, uncharged sequences (nine Leu cation of non-hydrophobic effectors of quence in fusion proteins that have or 14 alternating Leu/Ala residues) can membrane integration. These so-called either a type I or type II topology, it was function as transmembrane domains stop transfer effector (STE) sequences demonstrated that this STE sequence and integrate into the ER membrane 29. halt translocation and mediate mem- can end up on either side of the ER Another critical mechanistic concern brane integration of relatively non- membrane31. during integration is the maintenance of hydrophobic polypeptide sequences 32. TM orientation. The orientation of a TM the permeability barrier, particularly for The two naturally occurring eukaryotic sequence is determined during intemultiple-spanning membrane proteins STE sequences that have been charac- gration and is widely believed to be and for membrane proteins that have terized are both located amino-terminal dictated by the distribution of charged
large cytoplasmic domains. As the nascent-chain tunnel in the ribosome can only accommodate a limited
of the sequence that is embedded in the ER membrane 3],32. PrP has been shown to contain an STE sequence that
residues surrounding it. Most proteins adopt a topology with the more positively charged TM flanking sequence in
367
TALKINGPOINT the cytoplasm. However, much of the experimental data for the 'positiveinside rule' comes from the examination of post-translational and Secindependent integration in Escherichia coil 33. Statistical analysis of protein sequence data from eukaryotes is compatible with the positive-inside rule, but there are a number of exceptions 34, including fusion proteins containing the transmembrane domain of lgM (Ref. 35). Because processing is primarily cotranslational in eukaryotes and usually post-translational in prokaryotes, neither the mechanism nor the amino acid sequence requirements are necessarily similar. When the positive-inside rule was tested directly in a eukaryotic cellfree system by changing the sequences surrounding hydrophobic domains without intrinsic orientational preference, six out of eight molecules adopted an orientation opposite to that predicted by the distribution of charges flanking the TM domain a. Analyses of the topology of ductin suggest that the process that determines orientation and integration is not fixed by sequence, but is actively regulated, as in cells, ductin molecules in different locations have opposite orientations 36. When synthesized in vitro, ductin molecules are inserted into pancreatic microsomes in both orientations. Furthermore, changing the conserved charged residues at the amino terminus did not alter the ratio of the two forms obtained 36. These results and other exceptions to the positive-inside rule 37suggest that orientation is established by an as-yet unidentified balance of properties, and that the distribution of positive charges surrounding the TM domain is only one of the important variables. The translocon component(s) that presumably mediates the orientation of a TM sequence is unknown. Integration Is a regulated, multi-atep process. Thus, co-translational integration is not simply a one-step insertion of the TM sequence into the hydrophobic bilayer. Instead, several choices must be made at and by the translocon during the processing of polypeptides for integration. Experimental evidence for the multistep nature of this process is provided by photo-crosslinking data, which reveal that the TM domain passes sequentially through at least three different proteinaceous environments at the translocon, and that the TM sequence does not leave the translocon until translation terminates u. The movement of a TM sequence from the aqueous 368
TIBS 21 -
pore into the translocon and its ultimate release into the bilayer therefore appears to be highly regulated, presumably to ensure proper recognition, orientation and insertion of TM sequences, especially in multi-spanning membrane proteins. Moreover, current data suggest that TRAM plays a prominent role in such integration processing n.
Retrotranslocation Recent evidence suggests that rapid ER-associated protein degradation is mediated by proteasomes following movement of the protein from the ER lumen to the cytoplasm38. This movement, which we have termed retrotranslocation, must occur without destroying the permeability barrier between the cytoplasm and the ER lumen. As the translocon transports polypeptides without compromising the ER membrane during translocation, it is reasonable to wonder whether the translocon might also mediate retrotranslocation. Although there is no direct evidence implicating the translocon in this type of protein movement, calnexin is required for the retrotranslocation and degradation of nonglycosylated forms of proalpha factor in a yeast in vitro system 38. Calnexin has also been shown to be involved in the folding of polypeptides in the ER lumen before their translation has been completed TM. Calnexin is therefore a likely component of the extended translocon, and this proximity is consistent with a role for the translocon in retrotranslocation. Translocated MHC class I polypeptides can ratum to the cytoplasm. Newly-synthesized MHC class I molecules are selectively released into the cytoplasm and degraded in cells synthesizing the cytomegalovirus protein US11 (Ref. 40). Full length MHC class I molecules are recovered in an $100 fraction from USllproducing cells when cytoplasmic proteasomes are chemically inactivated, suggesting the surprising possibility that even the transmembrane domain of the MHC class I protein is transported back to the cytoplasm. Much of the new molecule has traversed the translocon before retrotranslocation, as demonstrated by the presence of glycosylated and signal sequence-cleaved MHC class I molecules in cytosoP~ As the only viral protein required to trigger rapid degradation of MHC class I is US11 (itself an integral ER membrane protein) and as extraction of an integrated TM
OCTOBER 1996
sequence would be thermodynamically difficult, it is unlikely that US11 creates an MHC class I specific translocation site that pulls the MHC class I polypeptide out of the lipid bilayer. Instead, we speculate that US11 targets new MHC class I proteins to a translocondependent pathway that is used to return other rapidly degraded molecules to the cytoplasm before integration. Consistent with this possibility, the movement of a polypeptide through the translocon is not necessarily continuous, nor unidirectional, as noted above. The release of exotoxin A fusion proteins, while still attached to the ribosome, is consistent with an export function for the translocon 28. Hence, the translocon might play a role in quality control by preventing the release to the ER lumen or to the lipid bilayer of proteins destined for degradation. The characteristics of some other rapidly degraded integral membrane and lumenal proteins are also consistent with retrotranslocation to the degradative pathway by the translocon. In cells synthesizing apolipoprotein B, rapid degradation has been shown to occur before the molecule is fully translocated across the ER membrane 41. Degradation of mutant cystic fibrosis transmembrane conductance regulator (CFTR) molecules has also been shown to require both cytoplasmic proteasomes and ubiquitination, possibly by Ubc6, a protein that can contact the transiocon 42-44. However, the site of CFTR degradation has not been rigorously examined. Finally, sequences within the transmembrane domains of incorrectly or incompletely assembled oligomeric proteins have been shown to target the polypeptides for rapid degradation 45. These observations, coupled with recent data suggesting that membrane integration is tightly regulated at the translocon 11, are consistent with rapid degradation resulting from retrotranslocation from the translocon to the degradative machinery in the cytoplasm. Concluding remarks A primary function of the translocon in the ER membrane of eukaryotes is to facilitate protein transit from the cytoplasm to the ER lumen. It is also clear that the translocon plays an active role in the selection, orientation and insertion of TM sequences into the bilayer. Furthermore, evidence is accumulating that the translocon participates in processes not conventionally associated
TIBS 21 -
TALKINGPOINT
OCTOBER 1996
with the vectorial cytoplasm-to-lumen transport of nascent polypeptides. The range of activities demonstrated or presumed to occur in the immediate vicinity of the translocon suggests that the translocon is an important regulatory site, and that further surprises are likely to emerge as our understanding of the topography and dynamics of the translocon increases,
Acknowledgements We thank V. Lingappa and H. Ploegh for providing manuscripts before publication, Work in the authors' laboratories was supported by a grant and a scientist award from the Medical Research Council of Canada (D. W. A.), by NIH grant R01 GM 26494 (A. E. J.), and by the Robert A. Welch Foundation (A. E. J.). We apologize to those whose work was not cited because of the limitation in the number of references permitted in this article.
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Pete Jeffs is a freelancer working in Paris, France 369