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. 1998 Feb 2;17(3):696–705. doi: 10.1093/emboj/17.3.696

Sec-dependent membrane protein biogenesis: SecYEG, preprotein hydrophobicity and translocation kinetics control the stop-transfer function.

F Duong 1, W Wickner 1
PMCID: PMC1170419  PMID: 9450995

Abstract

Preprotein translocase catalyzes membrane protein integration as well as complete translocation. Membrane proteins must interrupt their translocation and be laterally released from the translocase into the lipid bilayer. We have analyzed the translocation arrest and lateral release activities of Escherichia coli preprotein translocase with an in vitro reaction and the preprotein proOmpA carrying a synthetic stop-transfer sequence. Membrane protein integration is catalytic, occurs with kinetics similar to those of proOmpA itself and only requires the functions of SecYEG and SecA. Though a strongly hydrophobic segment will direct the protein to leave the translocase and enter the lipid bilayer, a protein with a segment of intermediate hydrophobicity partitions equally between the translocated and membrane-integrated states. Analysis of the effects of PMF, varied ATP concentrations or synthetic translocation arrest show that the stop-translocation efficiency of a mildly hydrophobic segment depends on the translocation kinetics. In contrast, the lateral partitioning from translocase to lipids depends solely on temperature and does not require SecA ATP hydrolysis or SecA membrane cycling. Thus translocation arrest is controlled by the SecYEG translocase activity while lateral release and membrane integration are directed by the hydrophobicity of the segment itself. Our results suggest that a greater hydrophobicity is required for efficient translocation arrest than for lateral release into the membrane.

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Selected References

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

  1. Adams G. A., Rose J. K. Structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport. Cell. 1985 Jul;41(3):1007–1015. doi: 10.1016/s0092-8674(85)80081-7. [DOI] [PubMed] [Google Scholar]
  2. Akiyama Y., Ogura T., Ito K. Involvement of FtsH in protein assembly into and through the membrane. I. Mutations that reduce retention efficiency of a cytoplasmic reporter. J Biol Chem. 1994 Feb 18;269(7):5218–5224. [PubMed] [Google Scholar]
  3. Akiyama Y., Shirai Y., Ito K. Involvement of FtsH in protein assembly into and through the membrane. II. Dominant mutations affecting FtsH functions. J Biol Chem. 1994 Feb 18;269(7):5225–5229. [PubMed] [Google Scholar]
  4. Andersson H., von Heijne G. Membrane protein topology: effects of delta mu H+ on the translocation of charged residues explain the 'positive inside' rule. EMBO J. 1994 May 15;13(10):2267–2272. doi: 10.1002/j.1460-2075.1994.tb06508.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bassilana M., Wickner W. Purified Escherichia coli preprotein translocase catalyzes multiple cycles of precursor protein translocation. Biochemistry. 1993 Mar 16;32(10):2626–2630. doi: 10.1021/bi00061a021. [DOI] [PubMed] [Google Scholar]
  6. Borel A. C., Simon S. M. Biogenesis of polytopic membrane proteins: membrane segments assemble within translocation channels prior to membrane integration. Cell. 1996 May 3;85(3):379–389. doi: 10.1016/s0092-8674(00)81116-2. [DOI] [PubMed] [Google Scholar]
  7. Brundage L., Hendrick J. P., Schiebel E., Driessen A. J., Wickner W. The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell. 1990 Aug 24;62(4):649–657. doi: 10.1016/0092-8674(90)90111-q. [DOI] [PubMed] [Google Scholar]
  8. Chen H., Kendall D. A. Artificial transmembrane segments. Requirements for stop transfer and polypeptide orientation. J Biol Chem. 1995 Jun 9;270(23):14115–14122. doi: 10.1074/jbc.270.23.14115. [DOI] [PubMed] [Google Scholar]
  9. Chen L., Tai P. C. ATP is essential for protein translocation into Escherichia coli membrane vesicles. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4384–4388. doi: 10.1073/pnas.82.13.4384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chuck S. L., Lingappa V. R. Analysis of a pause transfer sequence from apolipoprotein B. J Biol Chem. 1993 Oct 25;268(30):22794–22801. [PubMed] [Google Scholar]
  11. Claros M. G., von Heijne G. TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci. 1994 Dec;10(6):685–686. doi: 10.1093/bioinformatics/10.6.685. [DOI] [PubMed] [Google Scholar]
  12. Crooke E., Brundage L., Rice M., Wickner W. ProOmpA spontaneously folds in a membrane assembly competent state which trigger factor stabilizes. EMBO J. 1988 Jun;7(6):1831–1835. doi: 10.1002/j.1460-2075.1988.tb03015.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cunningham K., Lill R., Crooke E., Rice M., Moore K., Wickner W., Oliver D. SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA. EMBO J. 1989 Mar;8(3):955–959. doi: 10.1002/j.1460-2075.1989.tb03457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Davis N. G., Boeke J. D., Model P. Fine structure of a membrane anchor domain. J Mol Biol. 1985 Jan 5;181(1):111–121. doi: 10.1016/0022-2836(85)90329-8. [DOI] [PubMed] [Google Scholar]
  15. Davis N. G., Model P. An artificial anchor domain: hydrophobicity suffices to stop transfer. Cell. 1985 Jun;41(2):607–614. doi: 10.1016/s0092-8674(85)80033-7. [DOI] [PubMed] [Google Scholar]
  16. Douville K., Price A., Eichler J., Economou A., Wickner W. SecYEG and SecA are the stoichiometric components of preprotein translocase. J Biol Chem. 1995 Aug 25;270(34):20106–20111. doi: 10.1074/jbc.270.34.20106. [DOI] [PubMed] [Google Scholar]
  17. Driessen A. J. Precursor protein translocation by the Escherichia coli translocase is directed by the protonmotive force. EMBO J. 1992 Mar;11(3):847–853. doi: 10.1002/j.1460-2075.1992.tb05122.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Duong F., Wickner W. Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme. EMBO J. 1997 May 15;16(10):2756–2768. doi: 10.1093/emboj/16.10.2756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Duong F., Wickner W. The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling. EMBO J. 1997 Aug 15;16(16):4871–4879. doi: 10.1093/emboj/16.16.4871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Economou A., Wickner W. SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell. 1994 Sep 9;78(5):835–843. doi: 10.1016/s0092-8674(94)90582-7. [DOI] [PubMed] [Google Scholar]
  21. Eichler J., Brunner J., Wickner W. The protease-protected 30 kDa domain of SecA is largely inaccessible to the membrane lipid phase. EMBO J. 1997 May 1;16(9):2188–2196. doi: 10.1093/emboj/16.9.2188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Engelman D. M., Steitz T. A., Goldman A. Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu Rev Biophys Biophys Chem. 1986;15:321–353. doi: 10.1146/annurev.bb.15.060186.001541. [DOI] [PubMed] [Google Scholar]
  23. Geller B. L., Movva N. R., Wickner W. Both ATP and the electrochemical potential are required for optimal assembly of pro-OmpA into Escherichia coli inner membrane vesicles. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4219–4222. doi: 10.1073/pnas.83.12.4219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hahne K., Haucke V., Ramage L., Schatz G. Incomplete arrest in the outer membrane sorts NADH-cytochrome b5 reductase to two different submitochondrial compartments. Cell. 1994 Dec 2;79(5):829–839. doi: 10.1016/0092-8674(94)90072-8. [DOI] [PubMed] [Google Scholar]
  25. Hanada M., Nishiyama K. I., Mizushima S., Tokuda H. Reconstitution of an efficient protein translocation machinery comprising SecA and the three membrane proteins, SecY, SecE, and SecG (p12). J Biol Chem. 1994 Sep 23;269(38):23625–23631. [PubMed] [Google Scholar]
  26. Ito K. The major pathways of protein translocation across membranes. Genes Cells. 1996 Apr;1(4):337–346. doi: 10.1046/j.1365-2443.1996.34034.x. [DOI] [PubMed] [Google Scholar]
  27. Kuroiwa T., Sakaguchi M., Mihara K., Omura T. Structural requirements for interruption of protein translocation across rough endoplasmic reticulum membrane. J Biochem. 1990 Nov;108(5):829–834. doi: 10.1093/oxfordjournals.jbchem.a123288. [DOI] [PubMed] [Google Scholar]
  28. Kuroiwa T., Sakaguchi M., Mihara K., Omura T. Systematic analysis of stop-transfer sequence for microsomal membrane. J Biol Chem. 1991 May 15;266(14):9251–9255. [PubMed] [Google Scholar]
  29. Lee E., Manoil C. Mutations eliminating the protein export function of a membrane-spanning sequence. J Biol Chem. 1994 Nov 18;269(46):28822–28828. [PubMed] [Google Scholar]
  30. MacIntyre S., Freudl R., Eschbach M. L., Henning U. An artificial hydrophobic sequence functions as either an anchor or a signal sequence at only one of two positions within the Escherichia coli outer membrane protein OmpA. J Biol Chem. 1988 Dec 15;263(35):19053–19059. [PubMed] [Google Scholar]
  31. Martoglio B., Hofmann M. W., Brunner J., Dobberstein B. The protein-conducting channel in the membrane of the endoplasmic reticulum is open laterally toward the lipid bilayer. Cell. 1995 Apr 21;81(2):207–214. doi: 10.1016/0092-8674(95)90330-5. [DOI] [PubMed] [Google Scholar]
  32. Mothes W., Heinrich S. U., Graf R., Nilsson I., von Heijne G., Brunner J., Rapoport T. A. Molecular mechanism of membrane protein integration into the endoplasmic reticulum. Cell. 1997 May 16;89(4):523–533. doi: 10.1016/s0092-8674(00)80234-2. [DOI] [PubMed] [Google Scholar]
  33. 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. 1994 Mar 11;269(10):7617–7622. [PubMed] [Google Scholar]
  34. Nicchitta C. V., Blobel G. Assembly of translocation-competent proteoliposomes from detergent-solubilized rough microsomes. Cell. 1990 Jan 26;60(2):259–269. doi: 10.1016/0092-8674(90)90741-v. [DOI] [PubMed] [Google Scholar]
  35. Persson B., Argos P. Prediction of transmembrane segments in proteins utilising multiple sequence alignments. J Mol Biol. 1994 Mar 25;237(2):182–192. doi: 10.1006/jmbi.1994.1220. [DOI] [PubMed] [Google Scholar]
  36. Sato K., Mori H., Yoshida M., Tagaya M., Mizushima S. In vitro analysis of the stop-transfer process during translocation across the cytoplasmic membrane of Escherichia coli. J Biol Chem. 1997 Aug 8;272(32):20082–20087. doi: 10.1074/jbc.272.32.20082. [DOI] [PubMed] [Google Scholar]
  37. Sato K., Mori H., Yoshida M., Tagaya M., Mizushima S. Short hydrophobic segments in the mature domain of ProOmpA determine its stepwise movement during translocation across the cytoplasmic membrane of Escherichia coli. J Biol Chem. 1997 Feb 28;272(9):5880–5886. doi: 10.1074/jbc.272.9.5880. [DOI] [PubMed] [Google Scholar]
  38. Schatz P. J., Beckwith J. Genetic analysis of protein export in Escherichia coli. Annu Rev Genet. 1990;24:215–248. doi: 10.1146/annurev.ge.24.120190.001243. [DOI] [PubMed] [Google Scholar]
  39. Schiebel E., Driessen A. J., Hartl F. U., Wickner W. Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase. Cell. 1991 Mar 8;64(5):927–939. doi: 10.1016/0092-8674(91)90317-r. [DOI] [PubMed] [Google Scholar]
  40. Shiozuka K., Tani K., Mizushima S., Tokuda H. The proton motive force lowers the level of ATP required for the in vitro translocation of a secretory protein in Escherichia coli. J Biol Chem. 1990 Nov 5;265(31):18843–18847. [PubMed] [Google Scholar]
  41. Spiess M., Handschin C., Baker K. P. Stop-transfer activity of hydrophobic sequences depends on the translation system. J Biol Chem. 1989 Nov 15;264(32):19117–19124. [PubMed] [Google Scholar]
  42. Weiss J. B., Ray P. H., Bassford P. J., Jr Purified secB protein of Escherichia coli retards folding and promotes membrane translocation of the maltose-binding protein in vitro. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8978–8982. doi: 10.1073/pnas.85.23.8978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wickner W., Leonard M. R. Escherichia coli preprotein translocase. J Biol Chem. 1996 Nov 22;271(47):29514–29516. doi: 10.1074/jbc.271.47.29514. [DOI] [PubMed] [Google Scholar]
  44. Wolfe P. B., Rice M., Wickner W. Effects of two sec genes on protein assembly into the plasma membrane of Escherichia coli. J Biol Chem. 1985 Feb 10;260(3):1836–1841. [PubMed] [Google Scholar]
  45. Yost C. S., Lopez C. D., Prusiner S. B., Myers R. M., Lingappa V. R. Non-hydrophobic extracytoplasmic determinant of stop transfer in the prion protein. Nature. 1990 Feb 15;343(6259):669–672. doi: 10.1038/343669a0. [DOI] [PubMed] [Google Scholar]
  46. Zerial M., Huylebroeck D., Garoff H. Foreign transmembrane peptides replacing the internal signal sequence of transferrin receptor allow its translocation and membrane binding. Cell. 1987 Jan 16;48(1):147–155. doi: 10.1016/0092-8674(87)90365-5. [DOI] [PubMed] [Google Scholar]
  47. van Klompenburg W., Nilsson I., von Heijne G., de Kruijff B. Anionic phospholipids are determinants of membrane protein topology. EMBO J. 1997 Jul 16;16(14):4261–4266. doi: 10.1093/emboj/16.14.4261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. von Heijne G., Gavel Y. Topogenic signals in integral membrane proteins. Eur J Biochem. 1988 Jul 1;174(4):671–678. doi: 10.1111/j.1432-1033.1988.tb14150.x. [DOI] [PubMed] [Google Scholar]
  49. von Heijne G. Getting greasy: how transmembrane polypeptide segments integrate into the lipid bilayer. Mol Microbiol. 1997 Apr;24(2):249–253. doi: 10.1046/j.1365-2958.1997.3351702.x. [DOI] [PubMed] [Google Scholar]
  50. von Heijne G. Membrane proteins: from sequence to structure. Annu Rev Biophys Biomol Struct. 1994;23:167–192. doi: 10.1146/annurev.bb.23.060194.001123. [DOI] [PubMed] [Google Scholar]
  51. von Heijne G. Towards a comparative anatomy of N-terminal topogenic protein sequences. J Mol Biol. 1986 May 5;189(1):239–242. doi: 10.1016/0022-2836(86)90394-3. [DOI] [PubMed] [Google Scholar]

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