Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Nov;86(22):8673–8677. doi: 10.1073/pnas.86.22.8673

Detergent disruption of bacterial inner membranes and recovery of protein translocation activity.

K Cunningham 1, W T Wickner 1
PMCID: PMC298350  PMID: 2554324

Abstract

Isolation of the integral membrane components of protein translocation requires methods for fractionation and functional reconstitution. We treated inner-membrane vesicles of Escherichia coli with mixtures of octyl beta-D-glucoside, phospholipids, and an integral membrane carrier protein under conditions that extract most of the membrane proteins into micellar solution. Upon dialysis, proteoliposomes were reconstituted that supported translocation of radiochemically pure [35S]pro-OmpA (the precursor of outer membrane protein A). Translocation into these proteoliposomes required ATP hydrolysis and membrane proteins, indicating that the reaction is that of the inner membrane. The suspension of membranes in detergent was separated into supernatant and pellet fractions by ultracentrifugation. After reconstitution, translocation activity was observed in both fractions, but processing by leader peptidase of translocated pro-OmpA to OmpA was not detectable in the reconstituted pellet fraction. Processing activity was restored by addition of pure leader peptidase as long as this enzyme was added before detergent removal, indicating that the translocation activity is not associated with detergent-resistant membrane vesicles. These results show that protein translocation activity can be recovered from detergent-disrupted membrane vesicles, providing a first step towards the goal of isolating the solubilized components.

Full text

PDF
8676

Images in this article

8674Image
on p.8674
8675Image
on p.8675
8675Image
on p.8675
8675Image
on p.8675
8675Image
on p.8675
8675Image
on p.8675
8676Image
on p.8676
8676Image
on p.8676

Selected References

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

  1. Akiyama Y., Ito K. The SecY membrane component of the bacterial protein export machinery: analysis by new electrophoretic methods for integral membrane proteins. EMBO J. 1985 Dec 1;4(12):3351–3356. doi: 10.1002/j.1460-2075.1985.tb04088.x. [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. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Bochkareva E. S., Lissin N. M., Girshovich A. S. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. Nature. 1988 Nov 17;336(6196):254–257. doi: 10.1038/336254a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Chang C. N., Model P., Blobel G. Membrane biogenesis: cotranslational integration of the bacteriophage f1 coat protein into an Escherichia coli membrane fraction. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1251–1255. doi: 10.1073/pnas.76.3.1251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Chen L., Tai P. C. Effects of nucleotides on ATP-dependent protein translocation into Escherichia coli membrane vesicles. J Bacteriol. 1986 Nov;168(2):828–832. doi: 10.1128/jb.168.2.828-832.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Crooke E., Brundage L., Rice M., Wickner W. ProOmpA spontaneously folds in a membrane assembly competent state which trigger factor stabilizes. EMBO J. 1988 Jun;7(6):1831–1835. doi: 10.1002/j.1460-2075.1988.tb03015.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Crooke E., Guthrie B., Lecker S., Lill R., Wickner W. ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle. Cell. 1988 Sep 23;54(7):1003–1011. doi: 10.1016/0092-8674(88)90115-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Cunningham K., Wickner W. Specific recognition of the leader region of precursor proteins is required for the activation of translocation ATPase of Escherichia coli. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8630–8634. doi: 10.1073/pnas.86.22.8630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Fikes J. D., Bassford P. J., Jr Novel secA alleles improve export of maltose-binding protein synthesized with a defective signal peptide. J Bacteriol. 1989 Jan;171(1):402–409. doi: 10.1128/jb.171.1.402-409.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gardel C., Benson S., Hunt J., Michaelis S., Beckwith J. secD, a new gene involved in protein export in Escherichia coli. J Bacteriol. 1987 Mar;169(3):1286–1290. doi: 10.1128/jb.169.3.1286-1290.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Green N., Alexander H., Olson A., Alexander S., Shinnick T. M., Sutcliffe J. G., Lerner R. A. Immunogenic structure of the influenza virus hemagglutinin. Cell. 1982 Mar;28(3):477–487. doi: 10.1016/0092-8674(82)90202-1. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Lecker S., Lill R., Ziegelhoffer T., Georgopoulos C., Bassford P. J., Jr, Kumamoto C. A., Wickner W. Three pure chaperone proteins of Escherichia coli--SecB, trigger factor and GroEL--form soluble complexes with precursor proteins in vitro. EMBO J. 1989 Sep;8(9):2703–2709. doi: 10.1002/j.1460-2075.1989.tb08411.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lill R., Crooke E., Guthrie B., Wickner W. The "trigger factor cycle" includes ribosomes, presecretory proteins, and the plasma membrane. Cell. 1988 Sep 23;54(7):1013–1018. doi: 10.1016/0092-8674(88)90116-x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Makino S., Woolford J. L., Jr, Tanford C., Webster R. E. Interaction of deoxycholate and of detergents with the coat protein of bacteriophage f1. J Biol Chem. 1975 Jun 10;250(11):4327–4332. [PubMed] [Google Scholar]
  28. Moore K. E., Dalbey R. E., Wickner W. In vitro insertion of leader peptidase into Escherichia coli membrane vesicles. J Bacteriol. 1988 Sep;170(9):4395–4398. doi: 10.1128/jb.170.9.4395-4398.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Newman M. J., Foster D. L., Wilson T. H., Kaback H. R. Purification and reconstitution of functional lactose carrier from Escherichia coli. J Biol Chem. 1981 Nov 25;256(22):11804–11808. [PubMed] [Google Scholar]
  31. Newman M. J., Wilson T. H. Solubilization and reconstitution of the lactose transport system from Escherichia coli. J Biol Chem. 1980 Nov 25;255(22):10583–10586. [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. Weiss J. B., Ray P. H., Bassford P. J., Jr Purified secB protein of Escherichia coli retards folding and promotes membrane translocation of the maltose-binding protein in vitro. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8978–8982. doi: 10.1073/pnas.85.23.8978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wickner W. T., Lodish H. F. Multiple mechanisms of protein insertion into and across membranes. Science. 1985 Oct 25;230(4724):400–407. doi: 10.1126/science.4048938. [DOI] [PubMed] [Google Scholar]
  38. Wolfe P. B., Silver P., Wickner W. The isolation of homogeneous leader peptidase from a strain of Escherichia coli which overproduces the enzyme. J Biol Chem. 1982 Jul 10;257(13):7898–7902. [PubMed] [Google Scholar]
  39. Zimmermann R., Wickner W. Energetics and intermediates of the assembly of Protein OmpA into the outer membrane of Escherichia coli. J Biol Chem. 1983 Mar 25;258(6):3920–3925. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES