Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1985 Dec 1;4(12):3351–3356. doi: 10.1002/j.1460-2075.1985.tb04088.x

The SecY membrane component of the bacterial protein export machinery: analysis by new electrophoretic methods for integral membrane proteins.

Y Akiyama, K Ito
PMCID: PMC554665  PMID: 3004955

Abstract

The product of the secY (prlA) gene (the SecY protein) involved in protein export in Escherichia coli was overproduced and localized in the cytoplasmic (inner) membrane. Because of its strong interaction with a non-ionic detergent (NP40), it partitioned into the detergent layer during electroblotting through a NP40-containing gel (detergent blotting), and it formed a horizontal streak in the O'Farrell two-dimensional gel electrophoretic system. Consequently, we developed an alternative two-dimensional gel procedure, which proved useful for analysis of integral membrane proteins, especially in combination with detergent blotting. SDS-gel electrophoresis was carried out successively through gels of lower (first dimension) and higher (second dimension) sieving effects. Many membrane proteins, unlike soluble proteins, formed spots off and above the diagonal line, and all of these spots partitioned exclusively into the detergent layer. A characteristic pattern of integral membrane proteins of E. coli was thus obtained and the spot of the SecY protein in the cytoplasmic membrane was identified even when it was not overproduced. These results show that the gene secY specifies an integral membrane component of the protein export machinery.

Full text

PDF
3355

Images in this article

3352Fig. 1.
on p.3352
3352Fig. 2.
on p.3352
3353Fig. 3.
on p.3353
3353Fig. 4.
on p.3353
3354Fig. 5.
on p.3354
3355Fig. 6.
on p.3355
3355Fig. 7.
on p.3355

Selected References

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

  1. Ames G. F., Nikaido K. Two-dimensional gel electrophoresis of membrane proteins. Biochemistry. 1976 Feb 10;15(3):616–623. doi: 10.1021/bi00648a026. [DOI] [PubMed] [Google Scholar]
  2. Bankaitis V. A., Bassford P. J., Jr Proper interaction between at least two components is required for efficient export of proteins to the Escherichia coli cell envelope. J Bacteriol. 1985 Jan;161(1):169–178. doi: 10.1128/jb.161.1.169-178.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beyreuther K., Bieseler B., Ehring R., Griesser H. W., Mieschendahl M., Müller-Hill B., Triesch I. Investigation of structure and function of lactose permease of Escherichia coli. Biochem Soc Trans. 1980 Dec;8(6):675–676. doi: 10.1042/bst0080675. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Emr S. D., Bassford P. J., Jr Localization and processing of outer membrane and periplasmic proteins in Escherichia coli strains harboring export-specific suppressor mutations. J Biol Chem. 1982 May 25;257(10):5852–5860. [PubMed] [Google Scholar]
  8. 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]
  9. Ferro-Novick S., Honma M., Beckwith J. The product of gene secC is involved in the synthesis of exported proteins in E. coli. Cell. 1984 Aug;38(1):211–217. doi: 10.1016/0092-8674(84)90542-7. [DOI] [PubMed] [Google Scholar]
  10. Gilmore R., Walter P., Blobel G. Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor. J Cell Biol. 1982 Nov;95(2 Pt 1):470–477. doi: 10.1083/jcb.95.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Innis M. A., Tokunaga M., Williams M. E., Loranger J. M., Chang S. Y., Chang S., Wu H. C. Nucleotide sequence of the Escherichia coli prolipoprotein signal peptidase (lsp) gene. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3708–3712. doi: 10.1073/pnas.81.12.3708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ito K. Identification of the secY (prlA) gene product involved in protein export in Escherichia coli. Mol Gen Genet. 1984;197(2):204–208. doi: 10.1007/BF00330964. [DOI] [PubMed] [Google Scholar]
  13. Ito K., Sato T., Yura T. Synthesis and assembly of the membrane proteins in E. coli. Cell. 1977 Jul;11(3):551–559. doi: 10.1016/0092-8674(77)90073-3. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kiino D. R., Silhavy T. J. Mutation prlF1 relieves the lethality associated with export of beta-galactosidase hybrid proteins in Escherichia coli. J Bacteriol. 1984 Jun;158(3):878–883. doi: 10.1128/jb.158.3.878-883.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kreibich G., Ulrich B. L., Sabatini D. D. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes. J Cell Biol. 1978 May;77(2):464–487. doi: 10.1083/jcb.77.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kumamoto C. A., Beckwith J. Mutations in a new gene, secB, cause defective protein localization in Escherichia coli. J Bacteriol. 1983 Apr;154(1):253–260. doi: 10.1128/jb.154.1.253-260.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Lively M. O., Walsh K. A. Hen oviduct signal peptidase is an integral membrane protein. J Biol Chem. 1983 Aug 10;258(15):9488–9495. [PubMed] [Google Scholar]
  21. Meyer D. I., Krause E., Dobberstein B. Secretory protein translocation across membranes-the role of the "docking protein'. Nature. 1982 Jun 24;297(5868):647–650. doi: 10.1038/297647a0. [DOI] [PubMed] [Google Scholar]
  22. Nomura M., Gourse R., Baughman G. Regulation of the synthesis of ribosomes and ribosomal components. Annu Rev Biochem. 1984;53:75–117. doi: 10.1146/annurev.bi.53.070184.000451. [DOI] [PubMed] [Google Scholar]
  23. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Osborn M. J., Munson R. Separation of the inner (cytoplasmic) and outer membranes of Gram-negative bacteria. Methods Enzymol. 1974;31:642–653. doi: 10.1016/0076-6879(74)31070-1. [DOI] [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. Shultz J., Silhavy T. J., Berman M. L., Fiil N., Emr S. D. A previously unidentified gene in the spc operon of Escherichia coli K12 specifies a component of the protein export machinery. Cell. 1982 Nov;31(1):227–235. doi: 10.1016/0092-8674(82)90422-6. [DOI] [PubMed] [Google Scholar]
  29. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  30. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  31. Walter P., Gilmore R., Blobel G. Protein translocation across the endoplasmic reticulum. Cell. 1984 Aug;38(1):5–8. doi: 10.1016/0092-8674(84)90520-8. [DOI] [PubMed] [Google Scholar]
  32. Wolfe P. B., Wickner W., Goodman J. M. Sequence of the leader peptidase gene of Escherichia coli and the orientation of leader peptidase in the bacterial envelope. J Biol Chem. 1983 Oct 10;258(19):12073–12080. [PubMed] [Google Scholar]
  33. Yu F., Yamada H., Daishima K., Mizushima S. Nucleotide sequence of the lspA gene, the structural gene for lipoprotein signal peptidase of Escherichia coli. FEBS Lett. 1984 Jul 23;173(1):264–268. doi: 10.1016/0014-5793(84)81060-1. [DOI] [PubMed] [Google Scholar]

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

RESOURCES