Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1991 Nov;10(11):3263–3272. doi: 10.1002/j.1460-2075.1991.tb04890.x

Energetically distinct early and late stages of HlyB/HlyD-dependent secretion across both Escherichia coli membranes.

V Koronakis 1, C Hughes 1, E Koronakis 1
PMCID: PMC453051  PMID: 1915293

Abstract

The alternative secretion pathway which exports hemolysin across both Escherichia coli membranes into the surrounding medium is directed by an uncleaved C-terminal targeting signal and the membrane translocator proteins HlyD and HlyB. In order to identify stages and intermediates in this unconventional secretion process we have examined the effect of inhibition of the total proton motive force (delta P) and its components during the in vivo HlyB/HlyD-dependent export of a 22.4 kDa secretion competent HlyA C-terminal peptide (Actp). Secretion of Actp was severely inhibited by the proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which collapses simultaneously membrane potential delta psi and the proton gradient delta pH, and also by valinomycin/K+, a potassium ionophore which disrupts delta psi. The inhibition of secretion by valinomycin/K+ was ameliorated by imposition of a pH gradient, the second component of the delta P, and selective depletion of delta pH by nigericin also blocked secretion. This indicates that, as in the secretion of beta-lactamase to the periplasm, HlyB/D-directed secretion requires delta P itself and not specifically one of its components. However, inhibition of HlyB/D-dependent secretion was only marked when CCCP, valinomycin/K+ or nigericin were present during the early stage of Actp secretion; at a later stage the secretion was not significantly inhibited. HlyB/D-dependent secretion appears therefore to share with conventional secretion across the cytoplasmic membrane an early requirement for delta P, but comprises in addition a late stage which does not require delta P, delta psi or delta pH. The translocation intermediate identified in the delta P-independent late stage of secretion was associated with the membrane fraction. Analysis of the protease accessibility of this intermediate in whole cells and spheroplasts showed that it was not in the periplasm, nor was it exposed on the cell surface or on the periplasmic faces of either the inner or outer membranes. This may reflect its close association with the inner membrane or a membrane translocation complex.

Full text

PDF
3263

Images in this article

3264Image
on p.3264
3264Image
on p.3264
3265Image
on p.3265
3266Image
on p.3266
3266Image
on p.3266
3267Image
on p.3267
3268Image
on p.3268
3268Image
on p.3268
3269Image
on p.3269
3271Image
on p.3271

Selected References

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

  1. Baker K. P., Schaniel A., Vestweber D., Schatz G. A yeast mitochondrial outer membrane protein essential for protein import and cell viability. Nature. 1990 Dec 13;348(6302):605–609. doi: 10.1038/348605a0. [DOI] [PubMed] [Google Scholar]
  2. Bakker E. P., Randall L. L. The requirement for energy during export of beta-lactamase in Escherichia coli is fulfilled by the total protonmotive force. EMBO J. 1984 Apr;3(4):895–900. doi: 10.1002/j.1460-2075.1984.tb01902.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bishop L., Agbayani R., Jr, Ambudkar S. V., Maloney P. C., Ames G. F. Reconstitution of a bacterial periplasmic permease in proteoliposomes and demonstration of ATP hydrolysis concomitant with transport. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6953–6957. doi: 10.1073/pnas.86.18.6953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Castrillo J. L., Carrasco L. Adenovirus late protein synthesis is resistant to the inhibition of translation induced by poliovirus. J Biol Chem. 1987 May 25;262(15):7328–7334. [PubMed] [Google Scholar]
  5. 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]
  6. Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
  7. Date T., Goodman J. M., Wickner W. T. Procoat, the precursor of M13 coat protein, requires an electrochemical potential for membrane insertion. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4669–4673. doi: 10.1073/pnas.77.8.4669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davidson A. L., Nikaido H. Overproduction, solubilization, and reconstitution of the maltose transport system from Escherichia coli. J Biol Chem. 1990 Mar 15;265(8):4254–4260. [PubMed] [Google Scholar]
  9. Dean D. A., Fikes J. D., Gehring K., Bassford P. J., Jr, Nikaido H. Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein. J Bacteriol. 1989 Jan;171(1):503–510. doi: 10.1128/jb.171.1.503-510.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Economou A., Hamilton W. D., Johnston A. W., Downie J. A. The Rhizobium nodulation gene nodO encodes a Ca2(+)-binding protein that is exported without N-terminal cleavage and is homologous to haemolysin and related proteins. EMBO J. 1990 Feb;9(2):349–354. doi: 10.1002/j.1460-2075.1990.tb08117.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Enequist H. G., Hirst T. R., Harayama S., Hardy S. J., Randall L. L. Energy is required for maturation of exported proteins in Escherichia coli. Eur J Biochem. 1981 May 15;116(2):227–233. doi: 10.1111/j.1432-1033.1981.tb05323.x. [DOI] [PubMed] [Google Scholar]
  13. Felmlee T., Pellett S., Lee E. Y., Welch R. A. Escherichia coli hemolysin is released extracellularly without cleavage of a signal peptide. J Bacteriol. 1985 Jul;163(1):88–93. doi: 10.1128/jb.163.1.88-93.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geller B. L. Electrochemical potential releases a membrane-bound secretion intermediate of maltose-binding protein in Escherichia coli. J Bacteriol. 1990 Sep;172(9):4870–4876. doi: 10.1128/jb.172.9.4870-4876.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Geller B. L., Green H. M. Translocation of pro-OmpA across inner membrane vesicles of Escherichia coli occurs in two consecutive energetically distinct steps. J Biol Chem. 1989 Oct 5;264(28):16465–16469. [PubMed] [Google Scholar]
  16. 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]
  17. Gerlach J. H., Endicott J. A., Juranka P. F., Henderson G., Sarangi F., Deuchars K. L., Ling V. Homology between P-glycoprotein and a bacterial haemolysin transport protein suggests a model for multidrug resistance. Nature. 1986 Dec 4;324(6096):485–489. doi: 10.1038/324485a0. [DOI] [PubMed] [Google Scholar]
  18. Glaser P., Sakamoto H., Bellalou J., Ullmann A., Danchin A. Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis. EMBO J. 1988 Dec 1;7(12):3997–4004. doi: 10.1002/j.1460-2075.1988.tb03288.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gygi D., Nicolet J., Frey J., Cross M., Koronakis V., Hughes C. Isolation of the Actinobacillus pleuropneumoniae haemolysin gene and the activation and secretion of the prohaemolysin by the HlyC, HlyB and HlyD proteins of Escherichia coli. Mol Microbiol. 1990 Jan;4(1):123–128. doi: 10.1111/j.1365-2958.1990.tb02021.x. [DOI] [PubMed] [Google Scholar]
  20. Hartl F. U., Neupert W. Protein sorting to mitochondria: evolutionary conservations of folding and assembly. Science. 1990 Feb 23;247(4945):930–938. doi: 10.1126/science.2406905. [DOI] [PubMed] [Google Scholar]
  21. Hess J., Gentschev I., Goebel W., Jarchau T. Analysis of the haemolysin secretion system by PhoA-HlyA fusion proteins. Mol Gen Genet. 1990 Nov;224(2):201–208. doi: 10.1007/BF00271553. [DOI] [PubMed] [Google Scholar]
  22. Hirst T. R., Holmgren J. Transient entry of enterotoxin subunits into the periplasm occurs during their secretion from Vibrio cholerae. J Bacteriol. 1987 Mar;169(3):1037–1045. doi: 10.1128/jb.169.3.1037-1045.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hyde S. C., Emsley P., Hartshorn M. J., Mimmack M. M., Gileadi U., Pearce S. R., Gallagher M. P., Gill D. R., Hubbard R. E., Higgins C. F. Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature. 1990 Jul 26;346(6282):362–365. doi: 10.1038/346362a0. [DOI] [PubMed] [Google Scholar]
  24. Härtlein M., Schiessl S., Wagner W., Rdest U., Kreft J., Goebel W. Transport of hemolysin by Escherichia coli. J Cell Biochem. 1983;22(2):87–97. doi: 10.1002/jcb.240220203. [DOI] [PubMed] [Google Scholar]
  25. Issartel J. P., Koronakis V., Hughes C. Activation of Escherichia coli prohaemolysin to the mature toxin by acyl carrier protein-dependent fatty acylation. Nature. 1991 Jun 27;351(6329):759–761. doi: 10.1038/351759a0. [DOI] [PubMed] [Google Scholar]
  26. Koronakis V., Cross M., Senior B., Koronakis E., Hughes C. The secreted hemolysins of Proteus mirabilis, Proteus vulgaris, and Morganella morganii are genetically related to each other and to the alpha-hemolysin of Escherichia coli. J Bacteriol. 1987 Apr;169(4):1509–1515. doi: 10.1128/jb.169.4.1509-1515.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Koronakis V., Koronakis E., Hughes C. Comparison of the haemolysin secretion protein HlyB from Proteus vulgaris and Escherichia coli; site-directed mutagenesis causing impairment of export function. Mol Gen Genet. 1988 Aug;213(2-3):551–555. doi: 10.1007/BF00339631. [DOI] [PubMed] [Google Scholar]
  28. Koronakis V., Koronakis E., Hughes C. Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes. EMBO J. 1989 Feb;8(2):595–605. doi: 10.1002/j.1460-2075.1989.tb03414.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Létoffé S., Delepelaire P., Wandersman C. Protease secretion by Erwinia chrysanthemi: the specific secretion functions are analogous to those of Escherichia coli alpha-haemolysin. EMBO J. 1990 May;9(5):1375–1382. doi: 10.1002/j.1460-2075.1990.tb08252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mackman N., Baker K., Gray L., Haigh R., Nicaud J. M., Holland I. B. Release of a chimeric protein into the medium from Escherichia coli using the C-terminal secretion signal of haemolysin. EMBO J. 1987 Sep;6(9):2835–2841. doi: 10.1002/j.1460-2075.1987.tb02580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Michaelis S., Beckwith J. Mechanism of incorporation of cell envelope proteins in Escherichia coli. Annu Rev Microbiol. 1982;36:435–465. doi: 10.1146/annurev.mi.36.100182.002251. [DOI] [PubMed] [Google Scholar]
  32. Monaco J. J., Cho S., Attaya M. Transport protein genes in the murine MHC: possible implications for antigen processing. Science. 1990 Dec 21;250(4988):1723–1726. doi: 10.1126/science.2270487. [DOI] [PubMed] [Google Scholar]
  33. Murén E. M., Randall L. L. Export of alpha-amylase by Bacillus amyloliquefaciens requires proton motive force. J Bacteriol. 1985 Nov;164(2):712–716. doi: 10.1128/jb.164.2.712-716.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nakahama K., Yoshimura K., Marumoto R., Kikuchi M., Lee I. S., Hase T., Matsubara H. Cloning and sequencing of Serratia protease gene. Nucleic Acids Res. 1986 Jul 25;14(14):5843–5855. doi: 10.1093/nar/14.14.5843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pages J. M., Lazdunski C. Maturation of exported proteins in Escherichia coli. Requirement for energy, site and kinetics of processing. Eur J Biochem. 1982 Jun;124(3):561–566. [PubMed] [Google Scholar]
  36. Pfanner N., Neupert W. Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier. EMBO J. 1985 Nov;4(11):2819–2825. doi: 10.1002/j.1460-2075.1985.tb04009.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Pohlner J., Halter R., Beyreuther K., Meyer T. F. Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease. 1987 Jan 29-Feb 4Nature. 325(6103):458–462. doi: 10.1038/325458a0. [DOI] [PubMed] [Google Scholar]
  39. Prossnitz E., Gee A., Ames G. F. Reconstitution of the histidine periplasmic transport system in membrane vesicles. Energy coupling and interaction between the binding protein and the membrane complex. J Biol Chem. 1989 Mar 25;264(9):5006–5014. [PubMed] [Google Scholar]
  40. 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]
  41. Rommens J. M., Iannuzzi M. C., Kerem B., Drumm M. L., Melmer G., Dean M., Rozmahel R., Cole J. L., Kennedy D., Hidaka N. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science. 1989 Sep 8;245(4922):1059–1065. doi: 10.1126/science.2772657. [DOI] [PubMed] [Google Scholar]
  42. Schiebel E., Driessen A. J., Hartl F. U., Wickner W. Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase. Cell. 1991 Mar 8;64(5):927–939. doi: 10.1016/0092-8674(91)90317-r. [DOI] [PubMed] [Google Scholar]
  43. Schleyer M., Neupert W. Transport of proteins into mitochondria: translocational intermediates spanning contact sites between outer and inner membranes. Cell. 1985 Nov;43(1):339–350. doi: 10.1016/0092-8674(85)90039-x. [DOI] [PubMed] [Google Scholar]
  44. Schleyer M., Schmidt B., Neupert W. Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria. Eur J Biochem. 1982 Jun 15;125(1):109–116. doi: 10.1111/j.1432-1033.1982.tb06657.x. [DOI] [PubMed] [Google Scholar]
  45. Springer W., Goebel W. Synthesis and secretion of hemolysin by Escherichia coli. J Bacteriol. 1980 Oct;144(1):53–59. doi: 10.1128/jb.144.1.53-59.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Strathdee C. A., Lo R. Y. Extensive homology between the leukotoxin of Pasteurella haemolytica A1 and the alpha-hemolysin of Escherichia coli. Infect Immun. 1987 Dec;55(12):3233–3236. doi: 10.1128/iai.55.12.3233-3236.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  48. Thom J. R., Randall L. L. Role of the leader peptide of maltose-binding protein in two steps of the export process. J Bacteriol. 1988 Dec;170(12):5654–5661. doi: 10.1128/jb.170.12.5654-5661.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wagner W., Vogel M., Goebel W. Transport of hemolysin across the outer membrane of Escherichia coli requires two functions. J Bacteriol. 1983 Apr;154(1):200–210. doi: 10.1128/jb.154.1.200-210.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. Wong K. R., Buckley J. T. Proton motive force involved in protein transport across the outer membrane of Aeromonas salmonicida. Science. 1989 Nov 3;246(4930):654–656. doi: 10.1126/science.2814486. [DOI] [PubMed] [Google Scholar]
  53. Yamada H., Tokuda H., Mizushima S. Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli. J Biol Chem. 1989 Jan 25;264(3):1723–1728. [PubMed] [Google Scholar]
  54. 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 The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES