Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Cotranslational folding inhibits translocation from within the ribosome–Sec61 translocon complex

Nature Structural & Molecular Biology volume 21, pages 228–235 (2014)Cite this article

Subjects

Abstract

Eukaryotic secretory proteins cross the endoplasmic reticulum (ER) membrane through a protein-conducting channel contained within the ribosome–Sec61translocon complex (RTC). Using a zinc-finger sequence as a folding switch, we show that cotranslational folding of a secretory passenger inhibits translocation in canine ER microsomes and in human cells. Folding occurs within a cytosolically inaccessible environment, after ER targeting but before initiation of translocation, and it is most effective when the folded domain is 15–54 residues beyond the signal sequence. Under these conditions, substrate is diverted into cytosol at the stage of synthesis in which unfolded substrate enters the ER lumen. Moreover, the translocation block is reversed by passenger unfolding even after cytosol emergence. These studies identify an enclosed compartment within the assembled RTC that allows a short span of nascent chain to reversibly abort translocation in a substrate-specific manner.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Zinc-induced folding blocks cotranslational pPL translocation in vitro.
Figure 2: Zinc-induced folding blocks translocation in cultured cells and in a native protein substrate.
Figure 3: Zinc-finger folding does not block ER targeting.
Figure 4: Zinc-induced folding blocks translocation at a post-ER targeting step.
Figure 5: Passenger folding inhibits translocation of ribosome-bound nascent chains.
Figure 6: Zinc-finger folding directs the nascent chain into cytosol at 162 aa.
Figure 7: Passenger-induced translocation block is reversible upon unfolding.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

Swiss-Prot

References

  1. Park, E. & Rapoport, T.A. Mechanisms of Sec61/SecY-mediated protein translocation across membranes. Annu. Rev. Biophys. 41, 21–40 (2012).

    Article  CAS  Google Scholar 

  2. Shao, S. & Hegde, R.S. Membrane protein insertion at the endoplasmic reticulum. Annu. Rev. Cell Dev. Biol. 27, 25–56 (2011).

    Article  CAS  Google Scholar 

  3. Skach, W.R. Cellular mechanisms of membrane protein folding. Nat. Struct. Mol. Biol. 16, 606–612 (2009).

    Article  CAS  Google Scholar 

  4. Johnson, A.E. The structural and functional coupling of two molecular machines, the ribosome and the translocon. J. Cell Biol. 185, 765–767 (2009).

    Article  CAS  Google Scholar 

  5. Song, W., Raden, D., Mandon, E.C. & Gilmore, R. Role of Sec61α in the regulated transfer of the ribosome-nascent chain complex from the signal recognition particle to the translocation channel. Cell 100, 333–343 (2000).

    Article  CAS  Google Scholar 

  6. Holtkamp, W. et al. Dynamic switch of the signal recognition particle from scanning to targeting. Nat. Struct. Mol. Biol. 19, 1332–1337 (2012).

    Article  CAS  Google Scholar 

  7. Van den Berg, B. et al. X-ray structure of a protein-conducting channel. Nature 427, 36–44 (2004).

    Article  CAS  Google Scholar 

  8. Becker, T. et al. Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome. Science 326, 1369–1373 (2009).

    Article  CAS  Google Scholar 

  9. Crowley, K.S., Liao, S., Worrell, V.E., Reinhart, G.D. & Johnson, A.E. Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore. Cell 78, 461–471 (1994).

    Article  CAS  Google Scholar 

  10. Devaraneni, P.K. et al. Stepwise insertion and inversion of a type II signal anchor sequence in the ribosome-Sec61 translocon complex. Cell 146, 134–147 (2011).

    Article  CAS  Google Scholar 

  11. Frauenfeld, J. et al. Cryo-EM structure of the ribosome–SecYE complex in the membrane environment. Nat. Struct. Mol. Biol. 18, 614–621 (2011).

    Article  CAS  Google Scholar 

  12. Johnson, A.E. & van Waes, M.A. The translocon: a dynamic gateway at the ER membrane. Annu. Rev. Cell Dev. Biol. 15, 799–842 (1999).

    Article  Google Scholar 

  13. Jungnickel, B. & Rapoport, T.A. A posttargeting signal sequence recognition event in the endoplasmic reticulum membrane. Cell 82, 261–270 (1995).

    Article  CAS  Google Scholar 

  14. Kim, S.J., Mitra, D., Salerno, J.R. & Hegde, R.S. Signal sequences control gating of the protein translocation channel in a substrate-specific manner. Dev. Cell 2, 207–217 (2002).

    Article  CAS  Google Scholar 

  15. Rutkowski, D.T., Lingappa, V.R. & Hegde, R.S. Substrate-specific regulation of the ribosome- translocon junction by N-terminal signal sequences. Proc. Natl. Acad. Sci. USA 98, 7823–7828 (2001).

    Article  CAS  Google Scholar 

  16. Lingappa, V.R., Chaidez, J., Yost, C.S. & Hedgpeth, J. Determinants for protein localization: β-lactamase signal sequence directs globin across microsomal membranes. Proc. Natl. Acad. Sci. USA 81, 456–460 (1984).

    Article  CAS  Google Scholar 

  17. Levine, C.G., Mitra, D., Sharma, A., Smith, C.L. & Hegde, R.S. The efficiency of protein compartmentalization into the secretory pathway. Mol. Biol. Cell 16, 279–291 (2005).

    Article  CAS  Google Scholar 

  18. Andrews, D.W., Perara, E., Lesser, C. & Lingappa, V.R. Sequences beyond the cleavage site influence signal peptide function. J. Biol. Chem. 263, 15791–15798 (1988).

    CAS  PubMed  Google Scholar 

  19. Kim, S.J., Rahbar, R. & Hegde, R.S. Combinatorial control of prion protein biogenesis by the signal sequence and transmembrane domain. J. Biol. Chem. 276, 26132–26140 (2001).

    Article  CAS  Google Scholar 

  20. Hegde, R.S. & Kang, S.W. The concept of translocational regulation. J. Cell Biol. 182, 225–232 (2008).

    Article  CAS  Google Scholar 

  21. Goder, V., Junne, T. & Spiess, M. Sec61p contributes to signal sequence orientation according to the positive-inside rule. Mol. Biol. Cell 15, 1470–1478 (2004).

    Article  CAS  Google Scholar 

  22. von Heijne, G. Analysis of the distribution of charged residues in the N-terminal region of signal sequences: implications for protein export in prokaryotic and eukaryotic cells. EMBO J. 3, 2315–2318 (1984).

    Article  CAS  Google Scholar 

  23. Crowley, K.S., Reinhart, G.D. & Johnson, A.E. The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation. Cell 73, 1101–1115 (1993).

    Article  CAS  Google Scholar 

  24. Arkowitz, R.A., Joly, J.C. & Wickner, W. Translocation can drive the unfolding of a preprotein domain. EMBO J. 12, 243–253 (1993).

    Article  CAS  Google Scholar 

  25. Bonardi, F. et al. Probing the SecYEG translocation pore size with preproteins conjugated with sizable rigid spherical molecules. Proc. Natl. Acad. Sci. USA 108, 7775–7780 (2011).

    Article  Google Scholar 

  26. Denzer, A.J., Nabholz, C.E. & Spiess, M. Transmembrane orientation of signal-anchor proteins is affected by the folding state but not the size of the N-terminal domain. EMBO J. 14, 6311–6317 (1995).

    Article  CAS  Google Scholar 

  27. Perara, E., Rothman, R.E. & Lingappa, V.R. Uncoupling translocation from translation: implications for transport of proteins across membranes. Science 232, 348–352 (1986).

    Article  CAS  Google Scholar 

  28. Kowarik, M., Kung, S., Martoglio, B. & Helenius, A. Protein folding during cotranslational translocation in the endoplasmic reticulum. Mol. Cell 10, 769–778 (2002).

    Article  CAS  Google Scholar 

  29. Cheng, Z. & Gilmore, R. Slow translocon gating causes cytosolic exposure of transmembrane and lumenal domains during membrane protein integration. Nat. Struct. Mol. Biol. 13, 930–936 (2006).

    Article  CAS  Google Scholar 

  30. Mandon, E.C., Trueman, S.F. & Gilmore, R. Translocation of proteins through the Sec61 and SecYEG channels. Curr. Opin. Cell Biol. 21, 501–507 (2009).

    Article  CAS  Google Scholar 

  31. Park, E. et al. Structure of the SecY channel during initiation of protein translocation. Nature, 10.1038/nature12720 (23 October 2013).

  32. Kosolapov, A. & Deutsch, C. Tertiary interactions within the ribosomal exit tunnel. Nat. Struct. Mol. Biol. 16, 405–411 (2009).

    Article  CAS  Google Scholar 

  33. O'Brien, E.P., Hsu, S.-T.D., Christodoulou, J., Vendruscolo, M. & Dobson, C.M. Transient tertiary structure formation within the ribosome exit port. J. Am. Chem. Soc. 132, 16928–16937 (2010).

    Article  CAS  Google Scholar 

  34. Tu, L., Khanna, P. & Deutsch, C. Transmembrane segments form tertiary hairpins in the folding vestibule of the ribosome. J. Mol. Biol. 426, 185–198 (2014).

    Article  CAS  Google Scholar 

  35. Párraga, G. et al. Zinc-dependent structure of a single-finger domain of yeast ADR1. Science 241, 1489–1492 (1988).

    Article  Google Scholar 

  36. Bowers, P.M., Schaufler, L.E. & Klevit, R.E. A folding transition and novel zinc finger accessory domain in the transcription factor ADR1. Nat. Struct. Biol. 6, 478–485 (1999).

    Article  CAS  Google Scholar 

  37. Buchsbaum, J.C. & Berg, J.M. Kinetics of metal binding by a zinc finger peptide. Inorganica Chim. Acta 297, 217–219 (2000).

    Article  CAS  Google Scholar 

  38. Petersen, T.N., Brunak, S., von Heijne, G. & Nielsen, H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8, 785–786 (2011).

    Article  CAS  Google Scholar 

  39. Lu, J. & Deutsch, C. Folding zones inside the ribosomal exit tunnel. Nat. Struct. Mol. Biol. 12, 1123–1129 (2005).

    Article  CAS  Google Scholar 

  40. Voss, N.R., Gerstein, M., Steitz, T.A. & Moore, P.B. The geometry of the ribosomal polypeptide exit tunnel. J. Mol. Biol. 360, 893–906 (2006).

    Article  CAS  Google Scholar 

  41. Lu, J. & Deutsch, C. Secondary structure formation of a transmembrane segment in Kv channels. Biochemistry 44, 8230–8243 (2005).

    Article  CAS  Google Scholar 

  42. Mothes, W., Prehn, S. & Rapoport, T.A. Systematic probing of the environment of a translocating secretory protein during translocation through the ER membrane. EMBO J. 13, 3973–3982 (1994).

    Article  CAS  Google Scholar 

  43. Heinz, U., Kiefer, M., Tholey, A. & Adolph, H.W. On the competition for available zinc. J. Biol. Chem. 280, 3197–3207 (2005).

    Article  CAS  Google Scholar 

  44. Connolly, T., Collins, P. & Gilmore, R. Access of proteinase K to partially translocated nascent polypeptides in intact and detergent-solubilized membranes. J. Cell Biol. 108, 299–307 (1989).

    Article  CAS  Google Scholar 

  45. Walter, P. & Blobel, G. Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J. Cell Biol. 91, 557–561 (1981).

    Article  CAS  Google Scholar 

  46. Clemons, W.M. Jr., Menetret, J.F., Akey, C.W. & Rapoport, T.A. Structural insight into the protein translocation channel. Curr. Opin. Struct. Biol. 14, 390–396 (2004).

    Article  CAS  Google Scholar 

  47. du Plessis, D.J., Berrelkamp, G., Nouwen, N. & Driessen, A.J. The lateral gate of SecYEG opens during protein translocation. J. Biol. Chem. 284, 15805–15814 (2009).

    Article  CAS  Google Scholar 

  48. Lycklama a Nijeholt, J.A., Wu, Z.C. & Driessen, A.J. Conformational dynamics of the plug domain of the SecYEG protein-conducting channel. J. Biol. Chem. 286, 43881–43890 (2011).

    Article  CAS  Google Scholar 

  49. Hegde, R.S. & Lingappa, V.R. Sequence-specific alteration of the ribosome-membrane junction exposes nascent secretory proteins to the cytosol. Cell 85, 217–228 (1996).

    Article  CAS  Google Scholar 

  50. Liao, S., Lin, J., Do, H. & Johnson, A.E. Both lumenal and cytosolic gating of the aqueous ER translocon pore are regulated from inside the ribosome during membrane protein integration. Cell 90, 31–41 (1997).

    Article  CAS  Google Scholar 

  51. Sadlish, H., Pitonzo, D., Johnson, A.E. & Skach, W.R. Sequential triage of transmembrane segments by Sec61α during biogenesis of a native multispanning membrane protein. Nat. Struct. Mol. Biol. 12, 870–878 (2005).

    Article  CAS  Google Scholar 

  52. Yost, C.S., Hedgpeth, J. & Lingappa, V.R. A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems. Cell 34, 759–766 (1983).

    Article  CAS  Google Scholar 

  53. Fujita, H., Yamagishi, M., Kida, Y. & Sakaguchi, M. Positive charges on the translocating polypeptide chain arrest movement through the translocon. J. Cell Sci. 124, 4184–4193 (2011).

    Article  CAS  Google Scholar 

  54. Chuck, S.L. & Lingappa, V.R. Pause transfer: a topogenic sequence in apolipoprotein B mediates stopping and restarting of translocation. Cell 68, 9–21 (1992).

    Article  CAS  Google Scholar 

  55. Nakahara, D.H., Lingappa, V.R. & Chuck, S.L. Translocational pausing is a common step in the biogenesis of unconventional integral membrane and secretory proteins. J. Biol. Chem. 269, 7617–7622 (1994).

    CAS  PubMed  Google Scholar 

  56. Mitchell, D.M. et al. Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence. Proc. Natl. Acad. Sci. USA 95, 14733–14738 (1998).

    Article  CAS  Google Scholar 

  57. Fisher, E.A. et al. The degradation of apolipoprotein B100 is mediated by the ubiquitin-proteasome pathway and involves heat shock protein 70. J. Biol. Chem. 272, 20427–20434 (1997).

    Article  CAS  Google Scholar 

  58. Cuchel, M. et al. Inhibition of microsomal triglyceride transfer protein in familial hypercholesterolemia. N. Engl. J. Med. 356, 148–156 (2007).

    Article  CAS  Google Scholar 

  59. Fukada, T., Yamasaki, S., Nishida, K., Murakami, M. & Hirano, T. Zinc homeostasis and signaling in health and diseases: zinc signaling. J. Biol. Inorg. Chem. 16, 1123–1134 (2011).

    Article  CAS  Google Scholar 

  60. Skach, W.R., Calayag, M.C. & Lingappa, V.R. Evidence for an alternate model of human P-glycoprotein structure and biogenesis. J. Biol. Chem. 268, 6903–6908 (1993).

    CAS  PubMed  Google Scholar 

  61. Skach, W.R. & Lingappa, V.R. Amino-terminal assembly of human P-glycoprotein at the endoplasmic reticulum is directed by cooperative actions of two internal sequences. J. Biol. Chem. 268, 23552–23561 (1993).

    CAS  PubMed  Google Scholar 

  62. Ota, T. et al. Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat. Genet. 36, 40–45 (2004).

    Article  Google Scholar 

  63. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

    Article  CAS  Google Scholar 

  64. Carlson, E., Bays, N., David, L. & Skach, W.R. Reticulocyte lysate as a model system to study endoplasmic reticulum membrane protein degradation. Methods Mol. Biol. 301, 185–205 (2005).

    CAS  PubMed  Google Scholar 

  65. Matsumura, Y., Rooney, L. & Skach, W.R. In vitro methods for CFTR biogenesis. Methods Mol. Biol. 741, 233–253 (2011).

    Article  CAS  Google Scholar 

  66. Lee, B.-C., Chu, T.K., Dill, K.A. & Zuckermann, R.N. Biomimetic nanostructures: creating a high-affinity zinc-binding site in a folded nonbiological polymer. J. Am. Chem. Soc. 130, 8847–8855 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank V. Hilser, P. Devaraneni and members of the Skach laboratory for valuable discussion. This work was supported by US National Institutes of Health grants GM53457 (W.R.S.), DK51818 (W.R.S.), F32 GM083568 (B.J.C.) and T32 HL083808 (B.J.C.), by the Cystic Fibrosis Foundation Therapeutics (W.R.S.) and by US National Science Foundation grant MCB0746589 (U.S.) and American Heart Association grant 12PRE11470005 (J.E.).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Oregon Health and Sciences University, Portland, Oregon, USA

    Brian J Conti, Johannes Elferich, Zhongying Yang, Ujwal Shinde & William R Skach

Authors
  1. Brian J Conti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Johannes Elferich
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhongying Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ujwal Shinde
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. William R Skach
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.J.C. conceived the project, designed and executed experiments, analyzed results and assisted in writing the manuscript. J.E. and U.S. carried out the bioinformatics analysis and assisted in writing the manuscript. Z.Y. designed and carried out molecular biology experiments. W.R.S. designed experiments, analyzed results and assisted in writing the manuscript.

Corresponding author

Correspondence to William R Skach.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Translocation effect of zinc-finger incorporation within the preprolactin passenger.

a) Schematic showing preprolactin (pPL) constructs and location of the signal sequence (gray), ADR1a zinc finger insertion sites (black), and the pPL passenger domain (white). The N-linked glycosylation consensus site location created by ADR1a insertion is shown by asterisk. b) Left panels show phosphorimage of in vitro synthesized [35S]Methionine-labeled pPL polypeptides translated in the presence and absence of CRMs and 0.5 mM Zn+2 as indicated. Unprocessed, signal cleaved, and glycosylated polypeptides are designated with filled circles, open circles and asterisks, respectively. Right panels show same products after proteinase K (PK) digestion.

Supplementary Figure 2 The entire SDS-PAGE gel lane(s) for each of the cropped gel images shown in Figures 1–5 of the main text are provided to improve transparency of the data.

The corresponding main figures and lane labels are specified at the top of each image. Description of symbols and experimental conditions for each image are included in the main figure legends. Approximate migration of MW standards is indicated on the left.

Supplementary Figure 3 PEGylation of pPL45-Zn Cys* RNCs in the presence of Zn+2.

RNCs containing pPL45-Zn synthesized in the presence of Zn+2 were isolated and subjected to pegylation with or without addition of 0.5M NaCl or 1% digitonin. Samples were treated with RNase prior to SDS-PAGE and phosphorimaging.

Supplementary Figure 4 The entire SDS-PAGE gel lane(s) for each of the cropped gel images shown in Figures 6 and 7 of the main text are provided for completeness.

The corresponding main figures and lane labels are specified at the top of each image. Symbols and experimental conditions for each image are described in the main figure legends. Approximate migration of MW standards is indicated on the left.

Supplementary Figure 5 Identification of pPL45-Zn Cys* peptide and peptidyl-tRNA PEGylated species.

The pPL45-Zn Cys* 162-mer was translated without Zn+2 or NYT and incubated with PEG-mal as described in methods. After pegylation, samples were analyzed directly by SDS-PAGE (left four lanes) or digested with RNase prior to SDS-PAGE (right four lanes). The labile peptide-tRNA bond is partially hydrolyzed during sample analysis giving rise to peptide alone (asterisk) and peptidyl-tRNA (open circle), respectively, each of which undergoes variable pegylation at the four potential Cys residues. This gives rise to a complex pattern of multiple pegylated species with overlapping mobility as indicated at the left side of the phosphorimage. The origin of pegylated bands are much better delineated following RNase treatment to remove peptidyl-tRNA species (shown on the right).

Supplementary Figure 6 Frequency and identity of N-terminal domains in secretory versus cytosolic and nuclear proteins.

a) Table showing percentage of cytosolic and nuclear or secretory proteins that contain structurally defined domains in the first 100 residues from the N-terminus or the first 100 residues beyond the predicted signal sequence based on structural classification via SCOP and CATH databases. Top row includes all domains, whereas bottom row includes only those domains shorter than 50 residues. Actual number of proteins with domains and total proteins are shown in parentheses. Although the number of proteins varies between the databases, both analyses show that discrete domains are more commonly found in N-terminal regions of secretory proteins. b) Identity of the 15 most frequent domains found based on the SCOP database is indicated and plotted as the number of cytosolic and nuclear or secretory proteins that contained the domain. Average domain length is shown in parenthesis. Similar results were obtained using the CATH database, but are not shown since CATH uses numerical identifiers. c) Of the domains located within the first 100 residues of the secretory cohort based on the SCOP database, 70.4% had annotated disulfide bridges in the Uniprot database.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6. (PDF 359 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Conti, B., Elferich, J., Yang, Z. et al. Cotranslational folding inhibits translocation from within the ribosome–Sec61 translocon complex. Nat Struct Mol Biol 21, 228–235 (2014). https://doi.org/10.1038/nsmb.2779

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2779

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing