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. 1998 Jan 2;17(1):101–112. doi: 10.1093/emboj/17.1.101

A novel sec-independent periplasmic protein translocation pathway in Escherichia coli.

C L Santini 1, B Ize 1, A Chanal 1, M Müller 1, G Giordano 1, L F Wu 1
PMCID: PMC1170362  PMID: 9427745

Abstract

The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a soluble periplasmic molybdoenzyme. The precursor of this enzyme possesses a cleavable N-terminal signal sequence which contains a twin-arginine motif. By using various moa, mob and mod mutants defective in different steps of molybdocofactor biosynthesis, we demonstrate that acquisition of the molybdocofactor in the cytoplasm is a prerequisite for the translocation of the TMAO reductase. The activation and translocation of the TMAO reductase precursor are post-translational processes, and activation is dissociable from translocation. The export of the TMAO reductase is driven mainly by the proton motive force, whereas sodium azide exhibits a limited effect on the export. The most intriguing observation is that translocation of the TMAO reductase across the cytoplasmic membrane is independent of the SecY, SecE, SecA and SecB proteins. Depletion of Ffh, a core component of the signal recognition particle of E. coli, appears to have a slight effect on the export of the TMAO reductase. These results strongly suggest that the translocation of the molybdoenzyme TMAO reductase into the periplasm uses a mechanism fundamentally different from general protein translocation.

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Selected References

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

  1. Ames G. F., Prody C., Kustu S. Simple, rapid, and quantitative release of periplasmic proteins by chloroform. J Bacteriol. 1984 Dec;160(3):1181–1183. doi: 10.1128/jb.160.3.1181-1183.1984. [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. Barrett E. L., Kwan H. S. Bacterial reduction of trimethylamine oxide. Annu Rev Microbiol. 1985;39:131–149. doi: 10.1146/annurev.mi.39.100185.001023. [DOI] [PubMed] [Google Scholar]
  4. Bassilana M., Gwizdek C. In vivo membrane assembly of the E.coli polytopic protein, melibiose permease, occurs via a Sec-independent process which requires the protonmotive force. EMBO J. 1996 Oct 1;15(19):5202–5208. [PMC free article] [PubMed] [Google Scholar]
  5. Berks B. C. A common export pathway for proteins binding complex redox cofactors? Mol Microbiol. 1996 Nov;22(3):393–404. doi: 10.1046/j.1365-2958.1996.00114.x. [DOI] [PubMed] [Google Scholar]
  6. Bogsch E., Brink S., Robinson C. Pathway specificity for a delta pH-dependent precursor thylakoid lumen protein is governed by a 'Sec-avoidance' motif in the transfer peptide and a 'Sec-incompatible' mature protein. EMBO J. 1997 Jul 1;16(13):3851–3859. doi: 10.1093/emboj/16.13.3851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boyington J. C., Gladyshev V. N., Khangulov S. V., Stadtman T. C., Sun P. D. Crystal structure of formate dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4S4 cluster. Science. 1997 Feb 28;275(5304):1305–1308. doi: 10.1126/science.275.5304.1305. [DOI] [PubMed] [Google Scholar]
  8. Campbell A. M., del Campillo-Campbell A., Villaret D. B. Molybdate reduction by Escherichia coli K-12 and its chl mutants. Proc Natl Acad Sci U S A. 1985 Jan;82(1):227–231. doi: 10.1073/pnas.82.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Chaddock A. M., Mant A., Karnauchov I., Brink S., Herrmann R. G., Klösgen R. B., Robinson C. A new type of signal peptide: central role of a twin-arginine motif in transfer signals for the delta pH-dependent thylakoidal protein translocase. EMBO J. 1995 Jun 15;14(12):2715–2722. doi: 10.1002/j.1460-2075.1995.tb07272.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chan M. K., Mukund S., Kletzin A., Adams M. W., Rees D. C. Structure of a hyperthermophilic tungstopterin enzyme, aldehyde ferredoxin oxidoreductase. Science. 1995 Mar 10;267(5203):1463–1469. doi: 10.1126/science.7878465. [DOI] [PubMed] [Google Scholar]
  12. Creighton A. M., Hulford A., Mant A., Robinson D., Robinson C. A monomeric, tightly folded stromal intermediate on the delta pH-dependent thylakoidal protein transport pathway. J Biol Chem. 1995 Jan 27;270(4):1663–1669. doi: 10.1074/jbc.270.4.1663. [DOI] [PubMed] [Google Scholar]
  13. Guzman L. M., Belin D., Carson M. J., Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121–4130. doi: 10.1128/jb.177.14.4121-4130.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Iobbi-Nivol C., Pommier J., Simala-Grant J., Méjean V., Giordano G. High substrate specificity and induction characteristics of trimethylamine-N-oxide reductase of Escherichia coli. Biochim Biophys Acta. 1996 May 2;1294(1):77–82. doi: 10.1016/0167-4838(95)00271-5. [DOI] [PubMed] [Google Scholar]
  15. Joshi M. S., Johnson J. L., Rajagopalan K. V. Molybdenum cofactor biosynthesis in Escherichia coli mod and mog mutants. J Bacteriol. 1996 Jul;178(14):4310–4312. doi: 10.1128/jb.178.14.4310-4312.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jourlin C., Ansaldi M., Méjean V. Transphosphorylation of the TorR response regulator requires the three phosphorylation sites of the TorS unorthodox sensor in Escherichia coli. J Mol Biol. 1997 Apr 11;267(4):770–777. doi: 10.1006/jmbi.1997.0919. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Luirink J., ten Hagen-Jongman C. M., van der Weijden C. C., Oudega B., High S., Dobberstein B., Kusters R. An alternative protein targeting pathway in Escherichia coli: studies on the role of FtsY. EMBO J. 1994 May 15;13(10):2289–2296. doi: 10.1002/j.1460-2075.1994.tb06511.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Macfarlane J., Müller M. The functional integration of a polytopic membrane protein of Escherichia coli is dependent on the bacterial signal-recognition particle. Eur J Biochem. 1995 Nov 1;233(3):766–771. doi: 10.1111/j.1432-1033.1995.766_3.x. [DOI] [PubMed] [Google Scholar]
  21. Maupin-Furlow J. A., Rosentel J. K., Lee J. H., Deppenmeier U., Gunsalus R. P., Shanmugam K. T. Genetic analysis of the modABCD (molybdate transport) operon of Escherichia coli. J Bacteriol. 1995 Sep;177(17):4851–4856. doi: 10.1128/jb.177.17.4851-4856.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Murphy C. K., Beckwith J. Residues essential for the function of SecE, a membrane component of the Escherichia coli secretion apparatus, are located in a conserved cytoplasmic region. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2557–2561. doi: 10.1073/pnas.91.7.2557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Méjean V., Iobbi-Nivol C., Lepelletier M., Giordano G., Chippaux M., Pascal M. C. TMAO anaerobic respiration in Escherichia coli: involvement of the tor operon. Mol Microbiol. 1994 Mar;11(6):1169–1179. doi: 10.1111/j.1365-2958.1994.tb00393.x. [DOI] [PubMed] [Google Scholar]
  24. Nivière V., Wong S. L., Voordouw G. Site-directed mutagenesis of the hydrogenase signal peptide consensus box prevents export of a beta-lactamase fusion protein. J Gen Microbiol. 1992 Oct;138(10):2173–2183. doi: 10.1099/00221287-138-10-2173. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Osborn M. J., Gander J. E., Parisi E. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Site of synthesis of lipopolysaccharide. J Biol Chem. 1972 Jun 25;247(12):3973–3986. [PubMed] [Google Scholar]
  27. Phillips G. J., Silhavy T. J. The E. coli ffh gene is necessary for viability and efficient protein export. Nature. 1992 Oct 22;359(6397):744–746. doi: 10.1038/359744a0. [DOI] [PubMed] [Google Scholar]
  28. Przybyla A. E., Robbins J., Menon N., Peck H. D., Jr Structure-function relationships among the nickel-containing hydrogenases. FEMS Microbiol Rev. 1992 Feb;8(2):109–135. doi: 10.1111/j.1574-6968.1992.tb04960.x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Rapoport T. A., Jungnickel B., Kutay U. Protein transport across the eukaryotic endoplasmic reticulum and bacterial inner membranes. Annu Rev Biochem. 1996;65:271–303. doi: 10.1146/annurev.bi.65.070196.001415. [DOI] [PubMed] [Google Scholar]
  32. Rech S., Wolin C., Gunsalus R. P. Properties of the periplasmic ModA molybdate-binding protein of Escherichia coli. J Biol Chem. 1996 Feb 2;271(5):2557–2562. doi: 10.1074/jbc.271.5.2557. [DOI] [PubMed] [Google Scholar]
  33. Robinson C., Klösgen R. B. Targeting of proteins into and across the thylakoid membrane--a multitude of mechanisms. Plant Mol Biol. 1994 Oct;26(1):15–24. doi: 10.1007/BF00039516. [DOI] [PubMed] [Google Scholar]
  34. Rodrigue A., Boxer D. H., Mandrand-Berthelot M. A., Wu L. F. Requirement for nickel of the transmembrane translocation of NiFe-hydrogenase 2 in Escherichia coli. FEBS Lett. 1996 Aug 26;392(2):81–86. doi: 10.1016/0014-5793(96)00788-0. [DOI] [PubMed] [Google Scholar]
  35. Rosentel J. K., Healy F., Maupin-Furlow J. A., Lee J. H., Shanmugam K. T. Molybdate and regulation of mod (molybdate transport), fdhF, and hyc (formate hydrogenlyase) operons in Escherichia coli. J Bacteriol. 1995 Sep;177(17):4857–4864. doi: 10.1128/jb.177.17.4857-4864.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schatz G., Dobberstein B. Common principles of protein translocation across membranes. Science. 1996 Mar 15;271(5255):1519–1526. doi: 10.1126/science.271.5255.1519. [DOI] [PubMed] [Google Scholar]
  37. Schatz P. J., Riggs P. D., Jacq A., Fath M. J., Beckwith J. The secE gene encodes an integral membrane protein required for protein export in Escherichia coli. Genes Dev. 1989 Jul;3(7):1035–1044. doi: 10.1101/gad.3.7.1035. [DOI] [PubMed] [Google Scholar]
  38. Schindelin H., Kisker C., Hilton J., Rajagopalan K. V., Rees D. C. Crystal structure of DMSO reductase: redox-linked changes in molybdopterin coordination. Science. 1996 Jun 14;272(5268):1615–1621. doi: 10.1126/science.272.5268.1615. [DOI] [PubMed] [Google Scholar]
  39. Schneider F., Löwe J., Huber R., Schindelin H., Kisker C., Knäblein J. Crystal structure of dimethyl sulfoxide reductase from Rhodobacter capsulatus at 1.88 A resolution. J Mol Biol. 1996 Oct 18;263(1):53–69. doi: 10.1006/jmbi.1996.0555. [DOI] [PubMed] [Google Scholar]
  40. Scott D., Amy N. K. Molybdenum accumulation in chlD mutants of Escherichia coli. J Bacteriol. 1989 Mar;171(3):1284–1287. doi: 10.1128/jb.171.3.1284-1287.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Seluanov A., Bibi E. FtsY, the prokaryotic signal recognition particle receptor homologue, is essential for biogenesis of membrane proteins. J Biol Chem. 1997 Jan 24;272(4):2053–2055. doi: 10.1074/jbc.272.4.2053. [DOI] [PubMed] [Google Scholar]
  42. Silvestro A., Pommier J., Pascal M. C., Giordano G. The inducible trimethylamine N-oxide reductase of Escherichia coli K12: its localization and inducers. Biochim Biophys Acta. 1989 Nov 30;999(2):208–216. doi: 10.1016/0167-4838(89)90220-3. [DOI] [PubMed] [Google Scholar]
  43. Stewart V., MacGregor C. H. Nitrate reductase in Escherichia coli K-12: involvement of chlC, chlE, and chlG loci. J Bacteriol. 1982 Aug;151(2):788–799. doi: 10.1128/jb.151.2.788-799.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Subramani S. Convergence of model systems for peroxisome biogenesis. Curr Opin Cell Biol. 1996 Aug;8(4):513–518. doi: 10.1016/s0955-0674(96)80029-9. [DOI] [PubMed] [Google Scholar]
  45. Subramani S. Protein import into peroxisomes and biogenesis of the organelle. Annu Rev Cell Biol. 1993;9:445–478. doi: 10.1146/annurev.cb.09.110193.002305. [DOI] [PubMed] [Google Scholar]
  46. Takagi M., Tsuchiya T., Ishimoto M. Proton translocation coupled to trimethylamine N-oxide reduction in anaerobically grown Escherichia coli. J Bacteriol. 1981 Dec;148(3):762–768. doi: 10.1128/jb.148.3.762-768.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Traxler B., Murphy C. Insertion of the polytopic membrane protein MalF is dependent on the bacterial secretion machinery. J Biol Chem. 1996 May 24;271(21):12394–12400. doi: 10.1074/jbc.271.21.12394. [DOI] [PubMed] [Google Scholar]
  48. Ulbrandt N. D., Newitt J. A., Bernstein H. D. The E. coli signal recognition particle is required for the insertion of a subset of inner membrane proteins. Cell. 1997 Jan 24;88(2):187–196. doi: 10.1016/s0092-8674(00)81839-5. [DOI] [PubMed] [Google Scholar]
  49. Werner P. K., Saier M. H., Jr, Müller M. Membrane insertion of the mannitol permease of Escherichia coli occurs under conditions of impaired SecA function. J Biol Chem. 1992 Dec 5;267(34):24523–24532. [PubMed] [Google Scholar]
  50. Wolfe P. B., Rice M., Wickner W. Effects of two sec genes on protein assembly into the plasma membrane of Escherichia coli. J Biol Chem. 1985 Feb 10;260(3):1836–1841. [PubMed] [Google Scholar]
  51. Wu L. F., Mandrand M. A. Microbial hydrogenases: primary structure, classification, signatures and phylogeny. FEMS Microbiol Rev. 1993 Apr;10(3-4):243–269. doi: 10.1111/j.1574-6968.1993.tb05870.x. [DOI] [PubMed] [Google Scholar]
  52. de Gier J. W., Mansournia P., Valent Q. A., Phillips G. J., Luirink J., von Heijne G. Assembly of a cytoplasmic membrane protein in Escherichia coli is dependent on the signal recognition particle. FEBS Lett. 1996 Dec 16;399(3):307–309. doi: 10.1016/s0014-5793(96)01354-3. [DOI] [PubMed] [Google Scholar]

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