Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1988 Jun;7(6):1831–1835. doi: 10.1002/j.1460-2075.1988.tb03015.x

ProOmpA spontaneously folds in a membrane assembly competent state which trigger factor stabilizes.

E Crooke 1, L Brundage 1, M Rice 1, W Wickner 1
PMCID: PMC457175  PMID: 3049077

Abstract

The precursor protein proOmpA can translocate across purified Escherichia coli inner membrane vesicles in the absence of any other soluble proteins. ProOmpA, purified 2000-fold in the presence of 8 M urea, is competent for translocation following rapid renaturation via dilution. ATP, the transmembrane electrochemical potential, and functional secY protein are essential for the translocation of proOmpA renatured by dilution. The kinetics of its translocation and the level of translocation at each concentration of ATP are indistinguishable from that of proOmpA renatured by dialysis with trigger factor. After dilution, the proOmpA rapidly loses its competence for membrane assembly. However, this competence is stabilized by trigger factor. Assembly-competent proOmpA is in a protease-sensitive conformation, whereas proOmpA which has lost this competence is more resistant to degradation. This suggests that the primary role for trigger factor in in vitro protein translocation is to maintain precursor proteins in a translocation-competent conformation. We propose that a properly folded precursor protein and ATP are the only soluble components which are essential for bacterial protein translocation.

Full text

PDF
1831

Images in this article

1832Image
on p.1832
1832Image
on p.1832
1832Image
on p.1832
1832Image
on p.1832
1833Image
on p.1833
1833Image
on p.1833

Selected References

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

  1. Bacallao R., Crooke E., Shiba K., Wickner W., Ito K. The secY protein can act post-translationally to promote bacterial protein export. J Biol Chem. 1986 Sep 25;261(27):12907–12910. [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. Eilers M., Oppliger W., Schatz G. Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria. EMBO J. 1987 Apr;6(4):1073–1077. doi: 10.1002/j.1460-2075.1987.tb04860.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Folz R. J., Gordon J. I. The effects of deleting the propeptide from human preproapolipoprotein A-I on co-translational translocation and signal peptidase processing. J Biol Chem. 1987 Dec 15;262(35):17221–17230. [PubMed] [Google Scholar]
  9. 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]
  10. Hansen W., Garcia P. D., Walter P. In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor. Cell. 1986 May 9;45(3):397–406. doi: 10.1016/0092-8674(86)90325-9. [DOI] [PubMed] [Google Scholar]
  11. Ito K., Date T., Wickner W. Synthesis, assembly into the cytoplasmic membrane, and proteolytic processing of the precursor of coliphage M13 coat protein. J Biol Chem. 1980 Mar 10;255(5):2123–2130. [PubMed] [Google Scholar]
  12. 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]
  13. Newmeyer D. D., Lucocq J. M., Bürglin T. R., De Robertis E. M. Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation. EMBO J. 1986 Mar;5(3):501–510. doi: 10.1002/j.1460-2075.1986.tb04239.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Oliver D. B. Identification of five new essential genes involved in the synthesis of a secreted protein in Escherichia coli. J Bacteriol. 1985 Jan;161(1):285–291. doi: 10.1128/jb.161.1.285-291.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Oliver D. B., Liss L. R. prlA-mediated suppression of signal sequence mutations is modulated by the secA gene product of Escherichia coli K-12. J Bacteriol. 1985 Feb;161(2):817–819. doi: 10.1128/jb.161.2.817-819.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pain D., Blobel G. Protein import into chloroplasts requires a chloroplast ATPase. Proc Natl Acad Sci U S A. 1987 May;84(10):3288–3292. doi: 10.1073/pnas.84.10.3288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Perara E., Rothman R. E., Lingappa V. R. Uncoupling translocation from translation: implications for transport of proteins across membranes. Science. 1986 Apr 18;232(4748):348–352. doi: 10.1126/science.3961485. [DOI] [PubMed] [Google Scholar]
  20. Pfanner N., Tropschug M., Neupert W. Mitochondrial protein import: nucleoside triphosphates are involved in conferring import-competence to precursors. Cell. 1987 Jun 19;49(6):815–823. doi: 10.1016/0092-8674(87)90619-2. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Rothblatt J. A., Meyer D. I. Secretion in yeast: translocation and glycosylation of prepro-alpha-factor in vitro can occur via an ATP-dependent post-translational mechanism. EMBO J. 1986 May;5(5):1031–1036. doi: 10.1002/j.1460-2075.1986.tb04318.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rothblatt J. A., Webb J. R., Ammerer G., Meyer D. I. Secretion in yeast: structural features influencing the post-translational translocation of prepro-alpha-factor in vitro. EMBO J. 1987 Nov;6(11):3455–3463. doi: 10.1002/j.1460-2075.1987.tb02669.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Verner K., Schatz G. Import of an incompletely folded precursor protein into isolated mitochondria requires an energized inner membrane, but no added ATP. EMBO J. 1987 Aug;6(8):2449–2456. doi: 10.1002/j.1460-2075.1987.tb02524.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Waters M. G., Blobel G. Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis. J Cell Biol. 1986 May;102(5):1543–1550. doi: 10.1083/jcb.102.5.1543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Weng Q. P., Chen L. L., Tai P. C. Requirement of heat-labile cytoplasmic protein factors for posttranslational translocation of OmpA protein precursors into Escherichia coli membrane vesicles. J Bacteriol. 1988 Jan;170(1):126–131. doi: 10.1128/jb.170.1.126-131.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wickner W. The assembly of proteins into biological membranes: The membrane trigger hypothesis. Annu Rev Biochem. 1979;48:23–45. doi: 10.1146/annurev.bi.48.070179.000323. [DOI] [PubMed] [Google Scholar]
  30. Wiech H., Sagstetter M., Müller G., Zimmermann R. The ATP requiring step in assembly of M13 procoat protein into microsomes is related to preservation of transport competence of the precursor protein. EMBO J. 1987 Apr;6(4):1011–1016. doi: 10.1002/j.1460-2075.1987.tb04853.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Wolfe P. B., Wickner W. Bacterial leader peptidase, a membrane protein without a leader peptide, uses the same export pathway as pre-secretory proteins. Cell. 1984 Apr;36(4):1067–1072. doi: 10.1016/0092-8674(84)90056-4. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES