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. 1997 Dec;179(23):7386–7393. doi: 10.1128/jb.179.23.7386-7393.1997

Differential translocation of protein precursors across SecY-deficient membranes of Escherichia coli: SecY is not obligatorily required for translocation of certain secretory proteins in vitro.

Y B Yang 1, J Lian 1, P C Tai 1
PMCID: PMC179689  PMID: 9393703

Abstract

SecY, a component of the protein translocation system in Escherichia coli, was depleted at a nonpermissive temperature in a strain which had a temperature-sensitive polar effect on the expression of its secY. Membrane vesicles prepared from these cells, when grown at the nonpermissive temperature, contained about 5% SecY and similarly low levels of SecG. As expected, translocation of alkaline phosphatase precursors across these SecY-deficient membranes was severely impaired and appeared to be directly related to the decrease of SecY amounts. However, despite such a dramatic reduction in SecY and SecG levels, these membranes exhibited 50 to 70% of the wild-type translocation activity, including the processing of the signal peptide, of OmpA precursor (proOmpA). This translocation activity in SecY-deficient membranes was still SecA and ATP dependent and was not unique to proOmpA, as lipoprotein and lambda receptor protein precursors were also transported efficiently. Membranes that were reconstituted from these SecY-depleted membranes contained undetectable amounts of SecY yet were also shown to possess substantial translocation activity for proOmpA. These results indicate that the requirement of SecY for translocation is not obligatory for all secretory proteins and may depend on the nature of precursors. Consequently, it is unlikely that SecY is the essential core channel through which all precursors traverse across membranes; rather, SecY probably contributes to efficiency and specificity.

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Selected References

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  1. Akimaru J., Matsuyama S., Tokuda H., Mizushima S. Reconstitution of a protein translocation system containing purified SecY, SecE, and SecA from Escherichia coli. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6545–6549. doi: 10.1073/pnas.88.15.6545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akiyama Y., Ito K. Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli. EMBO J. 1987 Nov;6(11):3465–3470. doi: 10.1002/j.1460-2075.1987.tb02670.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arkowitz R. A., Wickner W. SecD and SecF are required for the proton electrochemical gradient stimulation of preprotein translocation. EMBO J. 1994 Feb 15;13(4):954–963. doi: 10.1002/j.1460-2075.1994.tb06340.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baba T., Jacq A., Brickman E., Beckwith J., Taura T., Ueguchi C., Akiyama Y., Ito K. Characterization of cold-sensitive secY mutants of Escherichia coli. J Bacteriol. 1990 Dec;172(12):7005–7010. doi: 10.1128/jb.172.12.7005-7010.1990. [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. Bieker K. L., Silhavy T. J. PrlA (SecY) and PrlG (SecE) interact directly and function sequentially during protein translocation in E. coli. Cell. 1990 Jun 1;61(5):833–842. doi: 10.1016/0092-8674(90)90193-i. [DOI] [PubMed] [Google Scholar]
  7. Bieker K. L., Silhavy T. J. PrlA is important for the translocation of exported proteins across the cytoplasmic membrane of Escherichia coli. Proc Natl Acad Sci U S A. 1989 Feb;86(3):968–972. doi: 10.1073/pnas.86.3.968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  9. Brundage L., Fimmel C. J., Mizushima S., Wickner W. SecY, SecE, and band 1 form the membrane-embedded domain of Escherichia coli preprotein translocase. J Biol Chem. 1992 Feb 25;267(6):4166–4170. [PubMed] [Google Scholar]
  10. 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]
  11. Cabelli R. J., Chen L., Tai P. C., Oliver D. B. SecA protein is required for secretory protein translocation into E. coli membrane vesicles. Cell. 1988 Nov 18;55(4):683–692. doi: 10.1016/0092-8674(88)90227-9. [DOI] [PubMed] [Google Scholar]
  12. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  13. Cerretti D. P., Dean D., Davis G. R., Bedwell D. M., Nomura M. The spc ribosomal protein operon of Escherichia coli: sequence and cotranscription of the ribosomal protein genes and a protein export gene. Nucleic Acids Res. 1983 May 11;11(9):2599–2616. doi: 10.1093/nar/11.9.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chen L. L., Tai P. C. Roles of H+-ATPase and proton motive force in ATP-dependent protein translocation in vitro. J Bacteriol. 1986 Jul;167(1):389–392. doi: 10.1128/jb.167.1.389-392.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Chen X., Xu H., Tai P. C. A significant fraction of functional SecA is permanently embedded in the membrane. SecA cycling on and off the membrane is not essential during protein translocation. J Biol Chem. 1996 Nov 22;271(47):29698–29706. doi: 10.1074/jbc.271.47.29698. [DOI] [PubMed] [Google Scholar]
  17. DAVIS B. D., MINGIOLI E. S. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. doi: 10.1128/jb.60.1.17-28.1950. [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. Economou A., Pogliano J. A., Beckwith J., Oliver D. B., Wickner W. SecA membrane cycling at SecYEG is driven by distinct ATP binding and hydrolysis events and is regulated by SecD and SecF. Cell. 1995 Dec 29;83(7):1171–1181. doi: 10.1016/0092-8674(95)90143-4. [DOI] [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. Emr S. D., Hanley-Way S., Silhavy T. J. Suppressor mutations that restore export of a protein with a defective signal sequence. Cell. 1981 Jan;23(1):79–88. doi: 10.1016/0092-8674(81)90272-5. [DOI] [PubMed] [Google Scholar]
  22. Fandl J. P., Cabelli R., Oliver D., Tai P. C. SecA suppresses the temperature-sensitive SecY24 defect in protein translocation in Escherichia coli membrane vesicles. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8953–8957. doi: 10.1073/pnas.85.23.8953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fandl J. P., Tai P. C. Biochemical evidence for the secY24 defect in Escherichia coli protein translocation and its suppression by soluble cytoplasmic factors. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7448–7452. doi: 10.1073/pnas.84.21.7448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Flower A. M., Doebele R. C., Silhavy T. J. PrlA and PrlG suppressors reduce the requirement for signal sequence recognition. J Bacteriol. 1994 Sep;176(18):5607–5614. doi: 10.1128/jb.176.18.5607-5614.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Flower A. M., Osborne R. S., Silhavy T. J. The allele-specific synthetic lethality of prlA-prlG double mutants predicts interactive domains of SecY and SecE. EMBO J. 1995 Mar 1;14(5):884–893. doi: 10.1002/j.1460-2075.1995.tb07070.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gesteland R. F. Isolation and characterization of ribonuclease I mutants of Escherichia coli. J Mol Biol. 1966 Mar;16(1):67–84. doi: 10.1016/s0022-2836(66)80263-2. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Hirata H., Altendorf K., Harold F. M. Energy coupling in membrane vesicles of Escherichia coli. I. Accumulation of metabolites in response to an electrical potential. J Biol Chem. 1974 May 10;249(9):2939–2945. [PubMed] [Google Scholar]
  29. Ito K., Cerretti D. P., Nashimoto H., Nomura M. Characterization of an amber mutation in the structural gene for ribosomal protein L15, which impairs the expression of the protein export gene, secY, in Escherichia coli. EMBO J. 1984 Oct;3(10):2319–2324. doi: 10.1002/j.1460-2075.1984.tb02133.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ito K., Wittekind M., Nomura M., Shiba K., Yura T., Miura A., Nashimoto H. A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins. Cell. 1983 Mar;32(3):789–797. doi: 10.1016/0092-8674(83)90065-x. [DOI] [PubMed] [Google Scholar]
  31. Kim Y. J., Rajapandi T., Oliver D. SecA protein is exposed to the periplasmic surface of the E. coli inner membrane in its active state. Cell. 1994 Sep 9;78(5):845–853. doi: 10.1016/s0092-8674(94)90602-5. [DOI] [PubMed] [Google Scholar]
  32. Kleerebezem M., Crielaard W., Tommassen J. Involvement of stress protein PspA (phage shock protein A) of Escherichia coli in maintenance of the protonmotive force under stress conditions. EMBO J. 1996 Jan 2;15(1):162–171. [PMC free article] [PubMed] [Google Scholar]
  33. Kumamoto C. A., Chen L., Fandl J., Tai P. C. Purification of the Escherichia coli secB gene product and demonstration of its activity in an in vitro protein translocation system. J Biol Chem. 1989 Feb 5;264(4):2242–2249. [PubMed] [Google Scholar]
  34. Kumamoto C. A. Molecular chaperones and protein translocation across the Escherichia coli inner membrane. Mol Microbiol. 1991 Jan;5(1):19–22. doi: 10.1111/j.1365-2958.1991.tb01821.x. [DOI] [PubMed] [Google Scholar]
  35. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  36. Lill R., Cunningham K., Brundage L. A., Ito K., Oliver D., Wickner W. SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli. EMBO J. 1989 Mar;8(3):961–966. doi: 10.1002/j.1460-2075.1989.tb03458.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Maloney P. C., Kashket E. R., Wilson T. H. A protonmotive force drives ATP synthesis in bacteria. Proc Natl Acad Sci U S A. 1974 Oct;71(10):3896–3900. doi: 10.1073/pnas.71.10.3896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Matsuyama S., Akimaru J., Mizushima S. SecE-dependent overproduction of SecY in Escherichia coli. Evidence for interaction between two components of the secretory machinery. FEBS Lett. 1990 Aug 20;269(1):96–100. doi: 10.1016/0014-5793(90)81128-b. [DOI] [PubMed] [Google Scholar]
  39. Matsuyama S., Fujita Y., Mizushima S. SecD is involved in the release of translocated secretory proteins from the cytoplasmic membrane of Escherichia coli. EMBO J. 1993 Jan;12(1):265–270. doi: 10.1002/j.1460-2075.1993.tb05652.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nishiyama K., Hanada M., Tokuda H. Disruption of the gene encoding p12 (SecG) reveals the direct involvement and important function of SecG in the protein translocation of Escherichia coli at low temperature. EMBO J. 1994 Jul 15;13(14):3272–3277. doi: 10.1002/j.1460-2075.1994.tb06628.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nishiyama K., Kabuyama Y., Akimaru J., Matsuyama S., Tokuda H., Mizushima S. SecY is an indispensable component of the protein secretory machinery of Escherichia coli. Biochim Biophys Acta. 1991 May 31;1065(1):89–97. doi: 10.1016/0005-2736(91)90015-z. [DOI] [PubMed] [Google Scholar]
  42. Nishiyama K., Mizushima S., Tokuda H. A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli. EMBO J. 1993 Sep;12(9):3409–3415. doi: 10.1002/j.1460-2075.1993.tb06015.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nishiyama K., Mizushima S., Tokuda H. Preferential interaction of Sec-G with Sec-E stabilizes an unstable Sec-E derivative in the Escherichia coli cytoplasmic membrane. Biochem Biophys Res Commun. 1995 Dec 5;217(1):217–223. doi: 10.1006/bbrc.1995.2766. [DOI] [PubMed] [Google Scholar]
  44. Nishiyama K., Mizushima S., Tokuda H. The carboxyl-terminal region of SecE interacts with SecY and is functional in the reconstitution of protein translocation activity in Escherichia coli. J Biol Chem. 1992 Apr 5;267(10):7170–7176. [PubMed] [Google Scholar]
  45. Nouwen N., de Kruijff B., Tommassen J. prlA suppressors in Escherichia coli relieve the proton electrochemical gradient dependency of translocation of wild-type precursors. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5953–5957. doi: 10.1073/pnas.93.12.5953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Osborne R. S., Silhavy T. J. PrlA suppressor mutations cluster in regions corresponding to three distinct topological domains. EMBO J. 1993 Sep;12(9):3391–3398. doi: 10.1002/j.1460-2075.1993.tb06013.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Powers T., Walter P. Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor. EMBO J. 1997 Aug 15;16(16):4880–4886. doi: 10.1093/emboj/16.16.4880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rensing S. A., Maier U. G. The SecY protein family: comparative analysis and phylogenetic relationships. Mol Phylogenet Evol. 1994 Sep;3(3):187–191. doi: 10.1006/mpev.1994.1021. [DOI] [PubMed] [Google Scholar]
  50. Rhoads D. B., Tai P. C., Davis B. D. Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into inner membrane vesicles. J Bacteriol. 1984 Jul;159(1):63–70. doi: 10.1128/jb.159.1.63-70.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sagara K., Matsuyama S., Mizushima S. SecF stabilizes SecD and SecY, components of the protein translocation machinery of the Escherichia coli cytoplasmic membrane. J Bacteriol. 1994 Jul;176(13):4111–4116. doi: 10.1128/jb.176.13.4111-4116.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schatz G., Dobberstein B. Common principles of protein translocation across membranes. Science. 1996 Mar 15;271(5255):1519–1526. doi: 10.1126/science.271.5255.1519. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Shiba K., Ito K., Yura T., Cerretti D. P. A defined mutation in the protein export gene within the spc ribosomal protein operon of Escherichia coli: isolation and characterization of a new temperature-sensitive secY mutant. EMBO J. 1984 Mar;3(3):631–635. doi: 10.1002/j.1460-2075.1984.tb01859.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tai P. C., Tian G., Xu H., Lian J. P., Yu J. N. In vitro protein translocation into Escherichia coli inverted membrane vesicles. Methods Cell Biol. 1991;34:167–187. [PubMed] [Google Scholar]
  56. Taura T., Akiyama Y., Ito K. Genetic analysis of SecY: additional export-defective mutations and factors affecting their phenotypes. Mol Gen Genet. 1994 May 10;243(3):261–269. doi: 10.1007/BF00301061. [DOI] [PubMed] [Google Scholar]
  57. Tian G., Wu H. C., Ray P. H., Tai P. C. Temperature-dependent insertion of prolipoprotein into Escherichia coli membrane vesicles and requirements for ATP, soluble factors, and functional SecY protein for the overall translocation process. J Bacteriol. 1989 Apr;171(4):1987–1997. doi: 10.1128/jb.171.4.1987-1997.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Tokuda H., Akimaru J., Matsuyama S., Nishiyama K., Mizushima S. Purification of SecE and reconstitution of SecE-dependent protein translocation activity. FEBS Lett. 1991 Feb 25;279(2):233–236. doi: 10.1016/0014-5793(91)80156-w. [DOI] [PubMed] [Google Scholar]
  59. Tschauder S., Driessen A. J., Freudl R. Cloning and molecular characterization of the secY genes from Bacillus licheniformis and Staphylococcus carnosus: comparative analysis of nine members of the SecY family. Mol Gen Genet. 1992 Oct;235(1):147–152. doi: 10.1007/BF00286192. [DOI] [PubMed] [Google Scholar]
  60. Ulbrandt N. D., Newitt J. A., Bernstein H. D. The E. coli signal recognition particle is required for the insertion of a subset of inner membrane proteins. Cell. 1997 Jan 24;88(2):187–196. doi: 10.1016/s0092-8674(00)81839-5. [DOI] [PubMed] [Google Scholar]
  61. Watanabe M., Blobel G. SecA protein is required for translocation of a model precursor protein into inverted vesicles of Escherichia coli plasma membrane. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9011–9015. doi: 10.1073/pnas.90.19.9011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Watanabe M., Nicchitta C. V., Blobel G. Reconstitution of protein translocation from detergent-solubilized Escherichia coli inverted vesicles: PrlA protein-deficient vesicles efficiently translocate precursor proteins. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1960–1964. doi: 10.1073/pnas.87.5.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. 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]
  64. Yang Y. B., Yu N., Tai P. C. SecE-depleted membranes of Escherichia coli are active. SecE is not obligatorily required for the in vitro translocation of certain protein precursors. J Biol Chem. 1997 May 23;272(21):13660–13665. doi: 10.1074/jbc.272.21.13660. [DOI] [PubMed] [Google Scholar]

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