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:

Sequential triage of transmembrane segments by Sec61α during biogenesis of a native multispanning membrane protein

Nature Structural & Molecular Biology volume 12, pages 870–878 (2005)Cite this article

Abstract

During polytopic protein biogenesis, the Sec61 translocon must rapidly orient and integrate multiple transmembrane segments (TMs) into the endoplasmic reticulum membrane. To understand this process, we examined interactions between Sec61α and all six TMs of the aquaporin-4 (AQP4) water channel at defined stages of synthesis using incorporated photo-cross-linking probes. Each TM interacted with and moved through the translocon in a highly ordered and sequential fashion. Strong asymmetric Sec61α cross-linking was observed for only one helix at a time, suggesting the presence of a single primary binding site. However, up to four TMs simultaneously contacted Sec61α from different molecular environments. Thus, AQP4 integration by Sec61α involves sequential triage of TMs from their initial portal of entry into multiple secondary sites within the translocon. This mechanism provides a means to facilitate early folding events before release into the lipid bilayer.

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: AQP4 biogenesis is unaffected by εANB-Lys.
Figure 2: Cross-linking to AQP4 integration intermediates.
Figure 3: Capturing transient TM2-translocon interactions.
Figure 4: Quantification of Sec61α-AQP4 cross-linking.
Figure 5: AQP4 TMs have unique Sec61α cross-linking profiles.
Figure 6: Sec61α simultaneously contacts multiple AQP4 TMs.
Figure 7: Model of AQP4 progression through Sec61α.

Similar content being viewed by others

References

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

    Article  Google Scholar 

  2. Rapoport, T., Goder, V., Heinrich, S. & Matlack, K. Membrane-protein integration and the role of the translocation channel. Trends Cell Biol. 14, 568–575 (2004).

    Article  CAS  Google Scholar 

  3. Menetret, J.-F. et al. Architecture of the ribosome-channel complex derived from native membranes. J. Mol. Biol. 348, 445–457 (2005).

    Article  CAS  Google Scholar 

  4. Görlich, D. & Rapoport, T. Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell 75, 615–630 (1993).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. High, S. et al. Site-specific photocrosslinking reveals that Sec61P and TRAM contact different regions of a membrane inserted signal sequence. J. Biol. Chem. 268, 26745–26751 (1993).

    CAS  PubMed  Google Scholar 

  8. Thrift, R.N., Andrews, D.W., Walter, P. & Johnson, A.E. A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translocation. J. Cell Biol. 112, 809–821 (1991).

    Article  CAS  Google Scholar 

  9. Mothes, W., Prehn, S. & Rapoport, T. 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 

  10. McCormick, P., Miao, Y., Shao, Y., Lin, J. & Johnson, A. Cotranslational protein integration into the ER membrane is mediated by the binding of nascent chains to translocon proteins. Mol. Cell 12, 329–341 (2003).

    Article  CAS  Google Scholar 

  11. Liao, S., Lin, J., Do, H. & Johnson, A. Both lumenal and cytosolic gating of the aqueous translocon pore are regulated from inside the ribosome during membrane protein integration. Cell 90, 31–42 (1997).

    Article  CAS  Google Scholar 

  12. Woolhead, C., McCormick, P. & Johnson, A. Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell 116, 725–736 (2004).

    Article  CAS  Google Scholar 

  13. High, S. et al. Sec61p is adjacent to nascent type I and type II signal-anchor proteins during their membrane insertion. J. Cell Biol. 121, 743–750 (1993).

    Article  CAS  Google Scholar 

  14. Martoglio, B., Hofmann, M., Brunner, J. & Dobberstein, B. The protein-conducting channel in the membrane of the endoplasmic reticulum is open laterally toward the lipid bilayer. Cell 81, 207–214 (1995).

    Article  CAS  Google Scholar 

  15. Do, H., Falcone, D., Lin, J., Andrews, D. & Johnson, A. The cotranslational integration of membrane proteins into the phospholipid bilayer is a multistep process. Cell 85, 369–378 (1996).

    Article  CAS  Google Scholar 

  16. Mothes, W. et al. Molecular mechanism of membrane protein integration into the endoplasmic reticulum. Cell 89, 523–533 (1997).

    Article  CAS  Google Scholar 

  17. Heinrich, S.U., Mothes, W., Brunner, J. & Rapoport, T. The Sec61p complex mediates the integration of a membrane protein by allowing lipid partitioning of the transmembrane domain. Cell 102, 233–244 (2000).

    Article  CAS  Google Scholar 

  18. Meacock, S., Lecomte, F., Crawshaw, S. & High, S. Different transmembrane domains associate with distinct endoplasmic reticulum components during membrane integration of a polytopic protein. Mol. Biol. Cell 13, 4114–4129 (2002).

    Article  CAS  Google Scholar 

  19. Heinrich, S. & Rapoport, T. Cooperation of transmembrane segments during integration of a double-spanning protein into the ER membrane. EMBO J. 22, 3654–3663 (2003).

    Article  CAS  Google Scholar 

  20. Higy, M., Junne, T. & Spiess, M. Topogenesis of membrane proteins at the endoplasmic reticulum. Biochemistry 43, 12716–12722 (2004).

    Article  CAS  Google Scholar 

  21. Alder, N. & Johnson, A. Cotranslational membrane protein biogenesis at the endoplasmic reticulum. J. Biol. Chem. 279, 22787–22790 (2004).

    Article  CAS  Google Scholar 

  22. Sadlish, H. & Skach, W. Biogenesis of CFTR and other polytopic membrane proteins; new roles for the ribosome-translocon complex. J. Membr. Biol. 202, 115–126 (2004).

    Article  CAS  Google Scholar 

  23. Skach, W. et al. Biogenesis and transmembrane topology of the CHIP28 water channel in the endoplasmic reticulum. J. Cell Biol. 125, 803–815 (1994).

    Article  CAS  Google Scholar 

  24. Skach, W. & Lingappa, V. Amino terminus 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 

  25. Lin, J. & Addison, R. A novel integration signal that is composed of two transmembrane segments is required to integrate the neorospora plasma membrane H+-ATPase into microsomes. J. Biol. Chem. 270, 6935–6941 (1995).

    Article  CAS  Google Scholar 

  26. Borel, A. & Simon, S. Biogenesis of polytopic membrane proteins: membrane segments of P-glycoprotein sequentially translocate to span the ER membrane. Biochemistry 35, 10587–10594 (1996).

    Article  CAS  Google Scholar 

  27. Hanein, D. et al. Oligomeric rings of the Sec61p complex induced by ligands required for protein translocation. Cell 87, 721–732 (1996).

    Article  CAS  Google Scholar 

  28. Beckmann, R. et al. Architecture of the protein-conducting channel associated with the translating 80S ribosome. Cell 107, 361–372 (2001).

    Article  CAS  Google Scholar 

  29. Morgan, D., Menetret, J., Neuhof, A., Rapoport, T. & Akey, C. Structure of the mammalian ribosome-channel complex at 17A resolution. J. Mol. Biol. 324, 871–886 (2002).

    Article  CAS  Google Scholar 

  30. Hegde, R. & Lingappa, V. Membrane protein biogenesis: regulated complexity at the endoplasmic reticulum. Cell 91, 575–582 (1997).

    Article  CAS  Google Scholar 

  31. Bibi, E. The role of the ribosome-translocon complex in translation and assembly of polytopic membrane proteins. Trends Biochem. Sci. 23, 51–55 (1998).

    Article  CAS  Google Scholar 

  32. Verkman, A. & Mitra, A. Structure and function of aquaporin water channels. Am. J. Physiol. Renal Physiol. 278, F13–F28 (2000).

    Article  CAS  Google Scholar 

  33. Agre, P. et al. Aquaporin water channels - from atomic structure to clinical medicine. J. Physiol. (Lond.) 542, 3–16 (2002).

    Article  CAS  Google Scholar 

  34. Fujiyoshi, Y. et al. Structure and function of water channels. Curr. Opin. Struct. Biol. 12, 509–515 (2002).

    Article  CAS  Google Scholar 

  35. Sui, H., Han, B.-G., Lee, J., Walian, P. & Jap, B. Structural basis of water specific transport through the AQP1 water channel. Nature 414, 872–878 (2001).

    Article  CAS  Google Scholar 

  36. Fu, D. et al. Structure of a glycerol-conducting channel and the basis for its selectivity. Science 290, 481–486 (2000).

    Article  CAS  Google Scholar 

  37. Shi, L.-B., Skach, W., Ma, T. & Verkman, A. Distinct biogenesis mechanisms for water channels MIWC and CHIP28 at the endoplasmic reticulum. Biochemistry 34, 8250–8256 (1995).

    Article  CAS  Google Scholar 

  38. Foster, W. et al. Identification of sequence determinants that direct different intracellular folding pathways for AQP1 and AQP4. J. Biol. Chem. 275, 34157–34165 (2000).

    Article  CAS  Google Scholar 

  39. Nilsson, I. et al. Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex. J. Cell Biol. 161, 715–725 (2003).

    Article  CAS  Google Scholar 

  40. Fons, R., Bogert, B. & Hegde, R. Substrate-specific function of the translocon-associated protein complex during translocation across the ER membrane. J. Cell Biol. 160, 529–539 (2003).

    Article  CAS  Google Scholar 

  41. Wilson, C. et al. Ribophorin I associates with a subset of membrane proteins after their integration at the Sec61 translocon. J. Biol. Chem. 280, 4195–4206 (2005).

    Article  CAS  Google Scholar 

  42. Snapp, E., Reinhart, G., Bogert, B., Lippencott-Schwartz, J. & Hegde, R. The organization of engaged and quiescent translocons in the endoplasmic reticulum of mammalian cells. J. Cell Biol. 164, 997–1007 (2004).

    Article  CAS  Google Scholar 

  43. Breyton, C., Haase, W., Rapoport, T., Kuehlbrandt, W. & Collinson, I. Three-dimensional structure of the bacterial protein-translocation complex SecYEG. Nature 418, 662–665 (2002).

    Article  CAS  Google Scholar 

  44. Manting, E., van der Does, C., Remigy, H., Engel, A. & Driessen, A. SecYEG assembles into a tetramer to form the active protein translocation channel. EMBO J. 19, 852–861 (2000).

    Article  CAS  Google Scholar 

  45. Hamman, B., Chen, J.-C., Johnson, E. & Johnson, A. The aqueous pore through the translocon has a diameter of 40–60Å during cotranslational protein translocation at the ER membrane. Cell 89, 535–544 (1997).

    Article  CAS  Google Scholar 

  46. Moss, K., Helm, A., Lu, Y., Bragin, A. & Skach, W. Coupled translocation events generate topologic heterogeneity at the endoplasmic reticulum membrane. Mol. Biol. Cell 9, 2681–2697 (1998).

    Article  CAS  Google Scholar 

  47. Carveth, K., Buck, T., Anthony, V. & Skach, W. Cooperativity and flexibility of cytsic fibrosis transmembrane conductance regulator transmembrane segments participate in membrane localization of a charged residue. J. Biol. Chem. 277, 39507–39514 (2002).

    Article  CAS  Google Scholar 

  48. Goder, V., Bieri, C. & Spiess, M. Glycosylation can influence topogenesis of membrane proteins and reveals dynamic reorientation of nascent polypeptides within the translocon. J. Cell Biol. 147, 257–266 (1999).

    Article  CAS  Google Scholar 

  49. Lu, Y. et al. Reorientation of Aquaporin-1 topology during maturation in the endoplasmic reticulum. Mol. Biol. Cell 11, 2973–2985 (2000).

    Article  CAS  Google Scholar 

  50. Plath, K., Mothes, W., Wilkinson, B., Stirling, C. & Rapoport, T. Signal sequence recognition in posttranslational protein transport across the yeast ER membrane. Cell 94, 795–807 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank C. Daniel and J. Knowles for their excellent technical assistance and P. McCormick for advice and suggestions. This work was supported by US National Institutes of Health grants DK51818 and GM53457 to W.R.S. and GM26494 to A.E.J., by the American Heart Association (H.S. and W.R.S.) and by the Robert A. Welch Foundation (A.E.J.).

Author information

Author notes

  1. Heather Sadlish and David Pitonzo: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Oregon Health & Sciences University, Portland, 97239, Oregon, USA

    Heather Sadlish, David Pitonzo & William R Skach

  2. Department of Medical Biochemistry and Genetics, Texas A&M University System, Health Science Center, College Station, 77843-1114, Texas, USA

    Arthur E Johnson

  3. Departments of Chemistry and of Biochemistry and Biophysics, Texas A&M University, College Station, 77843-1114, Texas, USA

    Arthur E Johnson

Authors
  1. Heather Sadlish
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Pitonzo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Arthur E Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. William R Skach
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to William R Skach.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Normalization of AQP4 translation and read-through products (PDF 379 kb)

Supplementary Fig. 2

AQP4-TRAM and Sec61 crosslinking (PDF 542 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sadlish, H., Pitonzo, D., Johnson, A. et al. Sequential triage of transmembrane segments by Sec61α during biogenesis of a native multispanning membrane protein. Nat Struct Mol Biol 12, 870–878 (2005). https://doi.org/10.1038/nsmb994

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb994

This article is cited by

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