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. 1994 Sep 1;13(17):3973–3982. doi: 10.1002/j.1460-2075.1994.tb06713.x

Systematic probing of the environment of a translocating secretory protein during translocation through the ER membrane.

W Mothes 1, S Prehn 1, T A Rapoport 1
PMCID: PMC395317  PMID: 8076593

Abstract

We have extended a previously developed photo-crosslinking approach to systematically probe the protein environment of the secretory protein preprolactin, trapped during its transfer through the endoplasmic reticulum membrane. Single photoreactive groups were placed at various positions of nascent polypeptide chains of various length, corresponding to different stages of the transport process, and photo-crosslinks to membrane proteins were analyzed. In all cases, the polypeptide segment extending from the ribosome was found to be located in a membrane environment that is formed almost exclusively from Sec61 alpha, the multi-spanning subunit of the Sec61p complex that is essential for translocation. At early stages of the translocation process, before cleavage of the signal sequence, almost the entire nascent chain emerged from the ribosome contacts Sec61 alpha. The 'translocating chain-associating membrane' protein interacts mainly with the region of the signal sequence preceding its hydrophobic core. Our results suggest that the nascent chain is transferred directly from the ribosome into a protein-conducting channel, the major constituent of which is Sec61 alpha.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blobel G., Sabatini D. D. Controlled proteolysis of nascent polypeptides in rat liver cell fractions. I. Location of the polypeptides within ribosomes. J Cell Biol. 1970 Apr;45(1):130–145. doi: 10.1083/jcb.45.1.130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brunner J., Richards F. M. Analysis of membranes photolabeled with lipid analogues. Reaction of phospholipids containing a disulfide group and a nitrene or carbene precursor with lipids and with gramicidin A. J Biol Chem. 1980 Apr 25;255(8):3319–3329. [PubMed] [Google Scholar]
  4. 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. 1993 Jun 18;73(6):1101–1115. doi: 10.1016/0092-8674(93)90640-c. [DOI] [PubMed] [Google Scholar]
  5. Görlich D., Hartmann E., Prehn S., Rapoport T. A. A protein of the endoplasmic reticulum involved early in polypeptide translocation. Nature. 1992 May 7;357(6373):47–52. doi: 10.1038/357047a0. [DOI] [PubMed] [Google Scholar]
  6. Görlich D., Kurzchalia T. V., Wiedmann M., Rapoport T. A. Probing the molecular environment of translocating polypeptide chains by cross-linking. Methods Cell Biol. 1991;34:241–262. doi: 10.1016/s0091-679x(08)61684-2. [DOI] [PubMed] [Google Scholar]
  7. Görlich D., Prehn S., Hartmann E., Herz J., Otto A., Kraft R., Wiedmann M., Knespel S., Dobberstein B., Rapoport T. A. The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents. J Cell Biol. 1990 Dec;111(6 Pt 1):2283–2294. doi: 10.1083/jcb.111.6.2283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Görlich D., Prehn S., Hartmann E., Kalies K. U., Rapoport T. A. A mammalian homolog of SEC61p and SECYp is associated with ribosomes and nascent polypeptides during translocation. Cell. 1992 Oct 30;71(3):489–503. doi: 10.1016/0092-8674(92)90517-g. [DOI] [PubMed] [Google Scholar]
  9. Görlich D., Rapoport T. A. Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell. 1993 Nov 19;75(4):615–630. doi: 10.1016/0092-8674(93)90483-7. [DOI] [PubMed] [Google Scholar]
  10. Hartmann E., Görlich D., Kostka S., Otto A., Kraft R., Knespel S., Bürger E., Rapoport T. A., Prehn S. A tetrameric complex of membrane proteins in the endoplasmic reticulum. Eur J Biochem. 1993 Jun 1;214(2):375–381. doi: 10.1111/j.1432-1033.1993.tb17933.x. [DOI] [PubMed] [Google Scholar]
  11. High S., Andersen S. S., Görlich D., Hartmann E., Prehn S., Rapoport T. A., Dobberstein B. Sec61p is adjacent to nascent type I and type II signal-anchor proteins during their membrane insertion. J Cell Biol. 1993 May;121(4):743–750. doi: 10.1083/jcb.121.4.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. High S., Martoglio B., Görlich D., Andersen S. S., Ashford A. J., Giner A., Hartmann E., Prehn S., Rapoport T. A., Dobberstein B. Site-specific photocross-linking reveals that Sec61p and TRAM contact different regions of a membrane-inserted signal sequence. J Biol Chem. 1993 Dec 15;268(35):26745–26751. [PubMed] [Google Scholar]
  13. Joly J. C., Wickner W. The SecA and SecY subunits of translocase are the nearest neighbors of a translocating preprotein, shielding it from phospholipids. EMBO J. 1993 Jan;12(1):255–263. doi: 10.1002/j.1460-2075.1993.tb05651.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kellaris K. V., Bowen S., Gilmore R. ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein. J Cell Biol. 1991 Jul;114(1):21–33. doi: 10.1083/jcb.114.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krieg U. C., Walter P., Johnson A. E. Photocrosslinking of the signal sequence of nascent preprolactin to the 54-kilodalton polypeptide of the signal recognition particle. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8604–8608. doi: 10.1073/pnas.83.22.8604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuroiwa T., Sakaguchi M., Mihara K., Omura T. Detection of a novel 9-kDa endoplasmic reticulum membrane protein in mammalian cells by chemical cross-linking with translocating nascent peptides. J Biochem. 1993 Oct;114(4):541–546. doi: 10.1093/oxfordjournals.jbchem.a124213. [DOI] [PubMed] [Google Scholar]
  17. Kurzchalia T. V., Wiedmann M., Girshovich A. S., Bochkareva E. S., Bielka H., Rapoport T. A. The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle. Nature. 1986 Apr 17;320(6063):634–636. doi: 10.1038/320634a0. [DOI] [PubMed] [Google Scholar]
  18. Malkin L. I., Rich A. Partial resistance of nascent polypeptide chains to proteolytic digestion due to ribosomal shielding. J Mol Biol. 1967 Jun 14;26(2):329–346. doi: 10.1016/0022-2836(67)90301-4. [DOI] [PubMed] [Google Scholar]
  19. Müsch A., Wiedmann M., Rapoport T. A. Yeast Sec proteins interact with polypeptides traversing the endoplasmic reticulum membrane. Cell. 1992 Apr 17;69(2):343–352. doi: 10.1016/0092-8674(92)90414-8. [DOI] [PubMed] [Google Scholar]
  20. Rapoport T. A. Transport of proteins across the endoplasmic reticulum membrane. Science. 1992 Nov 6;258(5084):931–936. doi: 10.1126/science.1332192. [DOI] [PubMed] [Google Scholar]
  21. Sanders S. L., Whitfield K. M., Vogel J. P., Rose M. D., Schekman R. W. Sec61p and BiP directly facilitate polypeptide translocation into the ER. Cell. 1992 Apr 17;69(2):353–365. doi: 10.1016/0092-8674(92)90415-9. [DOI] [PubMed] [Google Scholar]
  22. Shaw A. S., Rottier P. J., Rose J. K. Evidence for the loop model of signal-sequence insertion into the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7592–7596. doi: 10.1073/pnas.85.20.7592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Simon S. M., Blobel G. A protein-conducting channel in the endoplasmic reticulum. Cell. 1991 May 3;65(3):371–380. doi: 10.1016/0092-8674(91)90455-8. [DOI] [PubMed] [Google Scholar]
  24. Tajima S., Lauffer L., Rath V. L., Walter P. The signal recognition particle receptor is a complex that contains two distinct polypeptide chains. J Cell Biol. 1986 Oct;103(4):1167–1178. doi: 10.1083/jcb.103.4.1167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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 translation. J Cell Biol. 1991 Mar;112(5):809–821. doi: 10.1083/jcb.112.5.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Walter P., Ibrahimi I., Blobel G. Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol. 1981 Nov;91(2 Pt 1):545–550. doi: 10.1083/jcb.91.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wiedmann M., Goerlich D., Hartmann E., Kurzchalia T. V., Rapoport T. A. Photocrosslinking demonstrates proximity of a 34 kDa membrane protein to different portions of preprolactin during translocation through the endoplasmic reticulum. FEBS Lett. 1989 Nov 6;257(2):263–268. doi: 10.1016/0014-5793(89)81549-2. [DOI] [PubMed] [Google Scholar]
  28. Wiedmann M., Kurzchalia T. V., Bielka H., Rapoport T. A. Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking. J Cell Biol. 1987 Feb;104(2):201–208. doi: 10.1083/jcb.104.2.201. [DOI] [PMC free article] [PubMed] [Google Scholar]

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