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. 1998 Feb 16;17(4):936–944. doi: 10.1093/emboj/17.4.936

The SecB chaperone is involved in the secretion of the Serratia marcescens HasA protein through an ABC transporter.

P Delepelaire 1, C Wandersman 1
PMCID: PMC1170443  PMID: 9463372

Abstract

The secretion pathways of the heme-binding protein HasA from Serratia marcescens and of the metalloproteases A, B, C and G from Erwinia chrysanthemi have been reconstituted in Escherichia coli. They are secreted in a single step from the cytoplasm across both membranes of the Gram-negative envelope, after recognition of their specific C-terminal secretion signal by their cognate ABC transporter. We report strong evidence that both HasA and the metalloproteases bind the SecB chaperone involved in the export of several envelope proteins via the Sec pathway. We also show that the secretion of the HasA protein is strongly dependent upon SecB in the reconstituted system, whereas that of the proteases is not. HasA secretion in the original host is strongly inhibited by a protein known to interfere with E.coli SecB function. We propose that the proteins secreted by the ABC pathway may have to be unfolded for efficient secretion.

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Selected References

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

  1. Akatsuka H., Binet R., Kawai E., Wandersman C., Omori K. Lipase secretion by bacterial hybrid ATP-binding cassette exporters: molecular recognition of the LipBCD, PrtDEF, and HasDEF exporters. J Bacteriol. 1997 Aug;179(15):4754–4760. doi: 10.1128/jb.179.15.4754-4760.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Binet R., Wandersman C. Cloning of the Serratia marcescens hasF gene encoding the Has ABC exporter outer membrane component: a TolC analogue. Mol Microbiol. 1996 Oct;22(2):265–273. doi: 10.1046/j.1365-2958.1996.00103.x. [DOI] [PubMed] [Google Scholar]
  3. Binet R., Wandersman C. Protein secretion by hybrid bacterial ABC-transporters: specific functions of the membrane ATPase and the membrane fusion protein. EMBO J. 1995 May 15;14(10):2298–2306. doi: 10.1002/j.1460-2075.1995.tb07224.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blight M. A., Holland I. B. Heterologous protein secretion and the versatile Escherichia coli haemolysin translocator. Trends Biotechnol. 1994 Nov;12(11):450–455. doi: 10.1016/0167-7799(94)90020-5. [DOI] [PubMed] [Google Scholar]
  5. Collier D. N., Bankaitis V. A., Weiss J. B., Bassford P. J., Jr The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein. Cell. 1988 Apr 22;53(2):273–283. doi: 10.1016/0092-8674(88)90389-3. [DOI] [PubMed] [Google Scholar]
  6. Collier D. N. SecB: a molecular chaperone of Escherichia coli protein secretion pathway. Adv Protein Chem. 1993;44:151–193. doi: 10.1016/s0065-3233(08)60567-7. [DOI] [PubMed] [Google Scholar]
  7. Delepelaire P. PrtD, the integral membrane ATP-binding cassette component of the Erwinia chrysanthemi metalloprotease secretion system, exhibits a secretion signal-regulated ATPase activity. J Biol Chem. 1994 Nov 11;269(45):27952–27957. [PubMed] [Google Scholar]
  8. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Fikes J. D., Bassford P. J., Jr Export of unprocessed precursor maltose-binding protein to the periplasm of Escherichia coli cells. J Bacteriol. 1987 Jun;169(6):2352–2359. doi: 10.1128/jb.169.6.2352-2359.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ghigo J. M., Létoffé S., Wandersman C. A new type of hemophore-dependent heme acquisition system of Serratia marcescens reconstituted in Escherichia coli. J Bacteriol. 1997 Jun;179(11):3572–3579. doi: 10.1128/jb.179.11.3572-3579.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gilson L., Mahanty H. K., Kolter R. Genetic analysis of an MDR-like export system: the secretion of colicin V. EMBO J. 1990 Dec;9(12):3875–3884. doi: 10.1002/j.1460-2075.1990.tb07606.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goebel W., Hedgpeth J. Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli. J Bacteriol. 1982 Sep;151(3):1290–1298. doi: 10.1128/jb.151.3.1290-1298.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gonsky R., Lebendiker M. A., Harary R., Banai Y., Kaempfer R. Binding of ATP to eukaryotic initiation factor 2. Differential modulation of mRNA-binding activity and GTP-dependent binding of methionyl-tRNAMetf. J Biol Chem. 1990 Jun 5;265(16):9083–9089. [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. Holland I. B., Kenny B., Blight M. Haemolysin secretion from E coli. Biochimie. 1990 Feb-Mar;72(2-3):131–141. doi: 10.1016/0300-9084(90)90138-7. [DOI] [PubMed] [Google Scholar]
  17. Hwang J., Zhong X., Tai P. C. Interactions of dedicated export membrane proteins of the colicin V secretion system: CvaA, a member of the membrane fusion protein family, interacts with CvaB and TolC. J Bacteriol. 1997 Oct;179(20):6264–6270. doi: 10.1128/jb.179.20.6264-6270.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Khisty V. J., Munske G. R., Randall L. L. Mapping of the binding frame for the chaperone SecB within a natural ligand, galactose-binding protein. J Biol Chem. 1995 Oct 27;270(43):25920–25927. doi: 10.1074/jbc.270.43.25920. [DOI] [PubMed] [Google Scholar]
  19. Kimsey H. H., Dagarag M. D., Kumamoto C. A. Diverse effects of mutation on the activity of the Escherichia coli export chaperone SecB. J Biol Chem. 1995 Sep 29;270(39):22831–22835. doi: 10.1074/jbc.270.39.22831. [DOI] [PubMed] [Google Scholar]
  20. Kumamoto C. A., Beckwith J. Evidence for specificity at an early step in protein export in Escherichia coli. J Bacteriol. 1985 Jul;163(1):267–274. doi: 10.1128/jb.163.1.267-274.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kumamoto C. A. Escherichia coli SecB protein associates with exported protein precursors in vivo. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5320–5324. doi: 10.1073/pnas.86.14.5320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kumamoto C. A., Gannon P. M. Effects of Escherichia coli secB mutations on pre-maltose binding protein conformation and export kinetics. J Biol Chem. 1988 Aug 15;263(23):11554–11558. [PubMed] [Google Scholar]
  23. Kumamoto C. A., Nault A. K. Characterization of the Escherichia coli protein-export gene secB. Gene. 1989 Jan 30;75(1):167–175. doi: 10.1016/0378-1119(89)90393-4. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Létoffé S., Delepelaire P., Wandersman C. Protein secretion in gram-negative bacteria: assembly of the three components of ABC protein-mediated exporters is ordered and promoted by substrate binding. EMBO J. 1996 Nov 1;15(21):5804–5811. [PMC free article] [PubMed] [Google Scholar]
  26. Létoffé S., Ghigo J. M., Wandersman C. Identification of two components of the Serratia marcescens metalloprotease transporter: protease SM secretion in Escherichia coli is TolC dependent. J Bacteriol. 1993 Nov;175(22):7321–7328. doi: 10.1128/jb.175.22.7321-7328.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Létoffé S., Ghigo J. M., Wandersman C. Iron acquisition from heme and hemoglobin by a Serratia marcescens extracellular protein. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9876–9880. doi: 10.1073/pnas.91.21.9876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Létoffé S., Ghigo J. M., Wandersman C. Secretion of the Serratia marcescens HasA protein by an ABC transporter. J Bacteriol. 1994 Sep;176(17):5372–5377. doi: 10.1128/jb.176.17.5372-5377.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Létoffé S., Wandersman C. Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B. J Bacteriol. 1992 Aug;174(15):4920–4927. doi: 10.1128/jb.174.15.4920-4927.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Michiels T., Cornelis G. R. Secretion of hybrid proteins by the Yersinia Yop export system. J Bacteriol. 1991 Mar;173(5):1677–1685. doi: 10.1128/jb.173.5.1677-1685.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Randall L. L. Peptide binding by chaperone SecB: implications for recognition of nonnative structure. Science. 1992 Jul 10;257(5067):241–245. doi: 10.1126/science.1631545. [DOI] [PubMed] [Google Scholar]
  33. Sandkvist M., Bagdasarian M. Suppression of temperature-sensitive assembly mutants of heat-labile enterotoxin B subunits. Mol Microbiol. 1993 Nov;10(3):635–645. doi: 10.1111/j.1365-2958.1993.tb00935.x. [DOI] [PubMed] [Google Scholar]
  34. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  35. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  36. Spratt B. G., Hedge P. J., te Heesen S., Edelman A., Broome-Smith J. K. Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene. 1986;41(2-3):337–342. doi: 10.1016/0378-1119(86)90117-4. [DOI] [PubMed] [Google Scholar]
  37. Stanley P. L., Diaz P., Bailey M. J., Gygi D., Juarez A., Hughes C. Loss of activity in the secreted form of Escherichia coli haemolysin caused by an rfaP lesion in core lipopolysaccharide assembly. Mol Microbiol. 1993 Nov;10(4):781–787. doi: 10.1111/j.1365-2958.1993.tb00948.x. [DOI] [PubMed] [Google Scholar]
  38. Topping T. B., Randall L. L. Chaperone SecB from Escherichia coli mediates kinetic partitioning via a dynamic equilibrium with its ligands. J Biol Chem. 1997 Aug 1;272(31):19314–19318. doi: 10.1074/jbc.272.31.19314. [DOI] [PubMed] [Google Scholar]
  39. Topping T. B., Randall L. L. Determination of the binding frame within a physiological ligand for the chaperone SecB. Protein Sci. 1994 May;3(5):730–736. doi: 10.1002/pro.5560030502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wandersman C., Delepelaire P., Letoffe S., Schwartz M. Characterization of Erwinia chrysanthemi extracellular proteases: cloning and expression of the protease genes in Escherichia coli. J Bacteriol. 1987 Nov;169(11):5046–5053. doi: 10.1128/jb.169.11.5046-5053.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wandersman C. Secretion across the bacterial outer membrane. Trends Genet. 1992 Sep;8(9):317–322. doi: 10.1016/0168-9525(92)90264-5. [DOI] [PubMed] [Google Scholar]
  42. Weiss J. B., Bassford P. J., Jr The folding properties of the Escherichia coli maltose-binding protein influence its interaction with SecB in vitro. J Bacteriol. 1990 Jun;172(6):3023–3029. doi: 10.1128/jb.172.6.3023-3029.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wild J., Walter W. A., Gross C. A., Altman E. Accumulation of secretory protein precursors in Escherichia coli induces the heat shock response. J Bacteriol. 1993 Jul;175(13):3992–3997. doi: 10.1128/jb.175.13.3992-3997.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Woestyn S., Sory M. P., Boland A., Lequenne O., Cornelis G. R. The cytosolic SycE and SycH chaperones of Yersinia protect the region of YopE and YopH involved in translocation across eukaryotic cell membranes. Mol Microbiol. 1996 Jun;20(6):1261–1271. doi: 10.1111/j.1365-2958.1996.tb02645.x. [DOI] [PubMed] [Google Scholar]
  45. Wolff N., Delepelaire P., Ghigo J. M., Delepierre M. Spectroscopic studies of the C-terminal secretion signal of the Serratia marcescens haem acquisition protein (HasA) in various membrane-mimetic environments. Eur J Biochem. 1997 Jan 15;243(1-2):400–407. doi: 10.1111/j.1432-1033.1997.0400a.x. [DOI] [PubMed] [Google Scholar]
  46. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  47. de Cock H., Tommassen J. Conservation of components of the Escherichia coli export machinery in prokaryotes. FEMS Microbiol Lett. 1991 May 15;64(2-3):195–199. doi: 10.1016/0378-1097(91)90594-z. [DOI] [PubMed] [Google Scholar]

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