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. 1988 Jan;170(1):126–131. doi: 10.1128/jb.170.1.126-131.1988

Requirement of heat-labile cytoplasmic protein factors for posttranslational translocation of OmpA protein precursors into Escherichia coli membrane vesicles.

Q P Weng 1, L L Chen 1, P C Tai 1
PMCID: PMC210615  PMID: 3275607

Abstract

The involvement of possible cytoplasmic factors in ATP-dependent postttranslational translocation of proteins into Escherichia coli membrane vesicles was examined. The precursor of OmpA protein was partially purified by DEAE-cellulose chromatography, and its translocation was found to require material from the soluble cytoplasmic fraction. The fractionated active cytoplasmic translocation factor (CTF) was protease sensitive, micrococcal nuclease insensitive, N-ethylmaleimide resistant, and heat labile. The heat sensitivity of the CTF allowed its specific and preferential inactivation in the crude-precursor synthesis mixture, which provided a simple and rapid assay procedure for the factor during purification. Two active fractions were detected upon further fractionation: the major one was about 8S in sucrose gradient centrifugation and 120 kilodaltons by Sephadex filtration, whereas the other was about 4S and 60 kilodaltons in sucrose gradient centrifugation and by Sephadex filtration, respectively. The active fractions could also be fractionated by DEAE-Sepharose chromatography. These CTFs are apparently different from the previously reported 12S export factor (M. Muller and G. Blobel, Proc. Natl. Acad. Sci. USA 81:7737-7741, 1984).

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Selected References

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

  1. Caulfield M. P., Furlong D., Tai P. C., Davis B. D. Secretory S complex of Bacillus subtilis forms a large, organized structure when released from ribosomes. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4031–4035. doi: 10.1073/pnas.82.12.4031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Caulfield M. P., Horiuchi S., Tai P. C., Davis B. D. The 64-kilodalton membrane protein of Bacillus subtilis is also present as a multiprotein complex on membrane-free ribosomes. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7772–7776. doi: 10.1073/pnas.81.24.7772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen L. L., Tai P. C. Evidence for the involvement of ATP in co-translational protein translocation. Nature. 1987 Jul 9;328(6126):164–166. doi: 10.1038/328164a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Chen L., Rhoads D., Tai P. C. Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli. J Bacteriol. 1985 Mar;161(3):973–980. doi: 10.1128/jb.161.3.973-980.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Crooke E., Wickner W. Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly-competent form. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5216–5220. doi: 10.1073/pnas.84.15.5216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eilers M., Schatz G. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature. 1986 Jul 17;322(6076):228–232. doi: 10.1038/322228a0. [DOI] [PubMed] [Google Scholar]
  9. Hall M. N., Gabay J., Schwartz M. Evidence for a coupling of synthesis and export of an outer membrane protein in Escherichia coli. EMBO J. 1983;2(1):15–19. doi: 10.1002/j.1460-2075.1983.tb01373.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Infante A. A., Baierlein R. Pressure-induced dissociation of sedimenting ribosomes: effect on sedimentation patterns. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1780–1785. doi: 10.1073/pnas.68.8.1780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Josefsson L. G., Randall L. L. Different exported proteins in E. coli show differences in the temporal mode of processing in vivo. Cell. 1981 Jul;25(1):151–157. doi: 10.1016/0092-8674(81)90239-7. [DOI] [PubMed] [Google Scholar]
  12. Lee C. A., Beckwith J. Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis. J Bacteriol. 1986 Jun;166(3):878–883. doi: 10.1128/jb.166.3.878-883.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Müller M., Blobel G. In vitro translocation of bacterial proteins across the plasma membrane of Escherichia coli. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7421–7425. doi: 10.1073/pnas.81.23.7421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Müller M., Blobel G. Protein export in Escherichia coli requires a soluble activity. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7737–7741. doi: 10.1073/pnas.81.24.7737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ohta S., Schatz G. A purified precursor polypeptide requires a cytosolic protein fraction for import into mitochondria. EMBO J. 1984 Mar;3(3):651–657. doi: 10.1002/j.1460-2075.1984.tb01862.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Oliver D. B., Beckwith J. E. coli mutant pleiotropically defective in the export of secreted proteins. Cell. 1981 Sep;25(3):765–772. doi: 10.1016/0092-8674(81)90184-7. [DOI] [PubMed] [Google Scholar]
  17. Oliver D. B., Beckwith J. Regulation of a membrane component required for protein secretion in Escherichia coli. Cell. 1982 Aug;30(1):311–319. doi: 10.1016/0092-8674(82)90037-x. [DOI] [PubMed] [Google Scholar]
  18. Randall L. L., Hardy S. J. Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell. 1986 Sep 12;46(6):921–928. doi: 10.1016/0092-8674(86)90074-7. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Spirin A. S. On the equilibrium of the association-dissociation reaction of ribosomal subparticles and on the existance of the so-called '60 S intermediate' ('swollen 70 S') during centrifugation of the equilibrium mixture. FEBS Lett. 1971 May 20;14(5):349–353. doi: 10.1016/0014-5793(71)80298-3. [DOI] [PubMed] [Google Scholar]
  21. Strauch K. L., Kumamoto C. A., Beckwith J. Does secA mediate coupling between secretion and translation in Escherichia coli? J Bacteriol. 1986 May;166(2):505–512. doi: 10.1128/jb.166.2.505-512.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tai P. C. Biochemical studies of bacterial protein export. Curr Top Microbiol Immunol. 1986;125:43–58. doi: 10.1007/978-3-642-71251-7_5. [DOI] [PubMed] [Google Scholar]
  23. Walter P., Blobel G. Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature. 1982 Oct 21;299(5885):691–698. doi: 10.1038/299691a0. [DOI] [PubMed] [Google Scholar]
  24. 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. 1981 Nov;91(2 Pt 1):557–561. doi: 10.1083/jcb.91.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Waters M. G., Chirico W. J., Blobel G. Protein translocation across the yeast microsomal membrane is stimulated by a soluble factor. J Cell Biol. 1986 Dec;103(6 Pt 2):2629–2636. doi: 10.1083/jcb.103.6.2629. [DOI] [PMC free article] [PubMed] [Google Scholar]

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