Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1992 Sep;11(9):3165–3174. doi: 10.1002/j.1460-2075.1992.tb05393.x

Suppressor analysis suggests a multistep, cyclic mechanism for protein secretion in Escherichia coli.

K Bieker-Brady 1, T J Silhavy 1
PMCID: PMC1326449  PMID: 1387081

Abstract

The sec/prl gene products catalyze the translocation of precursor proteins from the cytoplasm of Escherichia coli. Recessive, conditionally lethal mutant alleles of these genes (sec mutations) cause a generalized defect in protein secretion; dominant suppressor mutant alleles (prl mutations) restore export of precursor proteins with altered signal sequences. In prl strains, a precursor protein with a defective signal sequence can be selectively targeted to the suppressor gene product. When a precursor LacZ hybrid protein is used, the targeted prl protein is inactivated by the large, toxic hybrid molecule, a result termed suppressor-directed inactivation (SDI). Using SDI, two different secretion-related complexes can be generated: a pretranslocation complex that contains a hybrid protein with an unprocessed signal sequence, and a translocation complex in which the hybrid protein is jammed in transmembrane orientation with the signal sequence cleaved. Additional Sec proteins that are contained within, and thus sequestered by, each of these complexes can be identified when their functional levels are lowered using the conditional lethal sec mutations. Results of this genetic analysis suggest a multistep pathway for protein secretion in which the translocation machinery assembles on demand.

Full text

PDF
3165

Images in this article

3167Image
on p.3167
3168Image
on p.3168

Selected References

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

  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. 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]
  3. Bassford P. J., Jr Export of the periplasmic maltose-binding protein of Escherichia coli. J Bioenerg Biomembr. 1990 Jun;22(3):401–439. doi: 10.1007/BF00763175. [DOI] [PubMed] [Google Scholar]
  4. Bieker K. L., Phillips G. J., Silhavy T. J. The sec and prl genes of Escherichia coli. J Bioenerg Biomembr. 1990 Jun;22(3):291–310. doi: 10.1007/BF00763169. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brickman E. R., Oliver D. B., Garwin J. L., Kumamoto C., Beckwith J. The use of extragenic suppressors to define genes involved in protein export in Escherichia coli. Mol Gen Genet. 1984;196(1):24–27. doi: 10.1007/BF00334087. [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. 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]
  12. Emr S. D., Silhavy T. J. Mutations affecting localization of an Escherichia coli outer membrane protein, the bacteriophage lambda receptor. J Mol Biol. 1980 Jul 25;141(1):63–90. doi: 10.1016/s0022-2836(80)80029-5. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Gardel C., Johnson K., Jacq A., Beckwith J. The secD locus of E.coli codes for two membrane proteins required for protein export. EMBO J. 1990 Oct;9(10):3209–3216. doi: 10.1002/j.1460-2075.1990.tb07519.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hartl F. U., Lecker S., Schiebel E., Hendrick J. P., Wickner W. The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane. Cell. 1990 Oct 19;63(2):269–279. doi: 10.1016/0092-8674(90)90160-g. [DOI] [PubMed] [Google Scholar]
  16. Kumamoto C. A. SecB protein: a cytosolic export factor that associates with nascent exported proteins. J Bioenerg Biomembr. 1990 Jun;22(3):337–351. doi: 10.1007/BF00763171. [DOI] [PubMed] [Google Scholar]
  17. Lee C., Li P., Inouye H., Brickman E. R., Beckwith J. Genetic studies on the inability of beta-galactosidase to be translocated across the Escherichia coli cytoplasmic membrane. J Bacteriol. 1989 Sep;171(9):4609–4616. doi: 10.1128/jb.171.9.4609-4616.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Mizushima S., Tokuda H. In vitro translocation of bacterial secretory proteins and energy requirements. J Bioenerg Biomembr. 1990 Jun;22(3):389–399. doi: 10.1007/BF00763174. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Oliver D. B., Cabelli R. J., Jarosik G. P. SecA protein: autoregulated initiator of secretory precursor protein translocation across the E. coli plasma membrane. J Bioenerg Biomembr. 1990 Jun;22(3):311–336. doi: 10.1007/BF00763170. [DOI] [PubMed] [Google Scholar]
  22. Phillips G. J., Silhavy T. J. Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid proteins in E. coli. Nature. 1990 Apr 26;344(6269):882–884. doi: 10.1038/344882a0. [DOI] [PubMed] [Google Scholar]
  23. Randall L. L., Hardy S. J., Thom J. R. Export of protein: a biochemical view. Annu Rev Microbiol. 1987;41:507–541. doi: 10.1146/annurev.mi.41.100187.002451. [DOI] [PubMed] [Google Scholar]
  24. Riggs P. D., Derman A. I., Beckwith J. A mutation affecting the regulation of a secA-lacZ fusion defines a new sec gene. Genetics. 1988 Apr;118(4):571–579. doi: 10.1093/genetics/118.4.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Schatz P. J., Bieker K. L., Ottemann K. M., Silhavy T. J., Beckwith J. One of three transmembrane stretches is sufficient for the functioning of the SecE protein, a membrane component of the E. coli secretion machinery. EMBO J. 1991 Jul;10(7):1749–1757. doi: 10.1002/j.1460-2075.1991.tb07699.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Stader J., Gansheroff L. J., Silhavy T. J. New suppressors of signal-sequence mutations, prlG, are linked tightly to the secE gene of Escherichia coli. Genes Dev. 1989 Jul;3(7):1045–1052. doi: 10.1101/gad.3.7.1045. [DOI] [PubMed] [Google Scholar]
  30. Stader J., Silhavy T. J. A progenitor of the outer membrane LamB trimer. J Bacteriol. 1988 Apr;170(4):1973–1974. doi: 10.1128/jb.170.4.1973-1974.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stoker N. G., Fairweather N. F., Spratt B. G. Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene. 1982 Jun;18(3):335–341. doi: 10.1016/0378-1119(82)90172-x. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Wickner W., Driessen A. J., Hartl F. U. The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu Rev Biochem. 1991;60:101–124. doi: 10.1146/annurev.bi.60.070191.000533. [DOI] [PubMed] [Google Scholar]

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

RESOURCES