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. 1998 Jul 1;17(13):3640–3650. doi: 10.1093/emboj/17.13.3640

Overlapping functions of components of a bacterial Sec-independent protein export pathway.

F Sargent 1, E G Bogsch 1, N R Stanley 1, M Wexler 1, C Robinson 1, B C Berks 1, T Palmer 1
PMCID: PMC1170700  PMID: 9649434

Abstract

We describe the identification of two Escherichia coli genes required for the export of cofactor-containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor-containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.

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Selected References

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  1. Andrews S. C., Berks B. C., McClay J., Ambler A., Quail M. A., Golby P., Guest J. R. A 12-cistron Escherichia coli operon (hyf) encoding a putative proton-translocating formate hydrogenlyase system. Microbiology. 1997 Nov;143(Pt 11):3633–3647. doi: 10.1099/00221287-143-11-3633. [DOI] [PubMed] [Google Scholar]
  2. Atlung T., Nielsen A., Hansen F. G. Isolation, characterization, and nucleotide sequence of appY, a regulatory gene for growth-phase-dependent gene expression in Escherichia coli. J Bacteriol. 1989 Mar;171(3):1683–1691. doi: 10.1128/jb.171.3.1683-1691.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bailey M. J., Hughes C., Koronakis V. RfaH and the ops element, components of a novel system controlling bacterial transcription elongation. Mol Microbiol. 1997 Dec;26(5):845–851. doi: 10.1046/j.1365-2958.1997.6432014.x. [DOI] [PubMed] [Google Scholar]
  4. Ballantine S. P., Boxer D. H. Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12. J Bacteriol. 1985 Aug;163(2):454–459. doi: 10.1128/jb.163.2.454-459.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Berg B. L., Li J., Heider J., Stewart V. Nitrate-inducible formate dehydrogenase in Escherichia coli K-12. I. Nucleotide sequence of the fdnGHI operon and evidence that opal (UGA) encodes selenocysteine. J Biol Chem. 1991 Nov 25;266(33):22380–22385. [PubMed] [Google Scholar]
  7. Berg B. L., Stewart V. Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12. Genetics. 1990 Aug;125(4):691–702. doi: 10.1093/genetics/125.4.691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Bernhard M., Benelli B., Hochkoeppler A., Zannoni D., Friedrich B. Functional and structural role of the cytochrome b subunit of the membrane-bound hydrogenase complex of Alcaligenes eutrophus H16. Eur J Biochem. 1997 Aug 15;248(1):179–186. doi: 10.1111/j.1432-1033.1997.00179.x. [DOI] [PubMed] [Google Scholar]
  10. Bilous P. T., Cole S. T., Anderson W. F., Weiner J. H. Nucleotide sequence of the dmsABC operon encoding the anaerobic dimethylsulphoxide reductase of Escherichia coli. Mol Microbiol. 1988 Nov;2(6):785–795. doi: 10.1111/j.1365-2958.1988.tb00090.x. [DOI] [PubMed] [Google Scholar]
  11. Bilous P. T., Weiner J. H. Dimethyl sulfoxide reductase activity by anaerobically grown Escherichia coli HB101. J Bacteriol. 1985 Jun;162(3):1151–1155. doi: 10.1128/jb.162.3.1151-1155.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Blattner F. R., Plunkett G., 3rd, Bloch C. A., Perna N. T., Burland V., Riley M., Collado-Vides J., Glasner J. D., Rode C. K., Mayhew G. F. The complete genome sequence of Escherichia coli K-12. Science. 1997 Sep 5;277(5331):1453–1462. doi: 10.1126/science.277.5331.1453. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Brøndsted L., Atlung T. Anaerobic regulation of the hydrogenase 1 (hya) operon of Escherichia coli. J Bacteriol. 1994 Sep;176(17):5423–5428. doi: 10.1128/jb.176.17.5423-5428.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. COHEN G. N., RICKENBERG H. V. Concentration spécifique réversible des amino acides chez Escherichia coli. Ann Inst Pasteur (Paris) 1956 Nov;91(5):693–720. [PubMed] [Google Scholar]
  16. Casadaban M. J., Cohen S. N. Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4530–4533. doi: 10.1073/pnas.76.9.4530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Clark S. A., Theg S. M. A folded protein can be transported across the chloroplast envelope and thylakoid membranes. Mol Biol Cell. 1997 May;8(5):923–934. doi: 10.1091/mbc.8.5.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Clausmeyer S., Klösgen R. B., Herrmann R. G. Protein import into chloroplasts. The hydrophilic lumenal proteins exhibit unexpected import and sorting specificities in spite of structurally conserved transit peptides. J Biol Chem. 1993 Jul 5;268(19):13869–13876. [PubMed] [Google Scholar]
  20. 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]
  21. Daniels D. L., Plunkett G., 3rd, Burland V., Blattner F. R. Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes. Science. 1992 Aug 7;257(5071):771–778. doi: 10.1126/science.1379743. [DOI] [PubMed] [Google Scholar]
  22. Dreusch A., Bürgisser D. M., Heizmann C. W., Zumft W. G. Lack of copper insertion into unprocessed cytoplasmic nitrous oxide reductase generated by an R20D substitution in the arginine consensus motif of the signal peptide. Biochim Biophys Acta. 1997 Apr 11;1319(2-3):311–318. doi: 10.1016/s0005-2728(96)00174-0. [DOI] [PubMed] [Google Scholar]
  23. Enoch H. G., Lester R. L. The purification and properties of formate dehydrogenase and nitrate reductase from Escherichia coli. J Biol Chem. 1975 Sep 10;250(17):6693–6705. [PubMed] [Google Scholar]
  24. Fincher V., McCaffery M., Cline K. Evidence for a loop mechanism of protein transport by the thylakoid Delta pH pathway. FEBS Lett. 1998 Feb 13;423(1):66–70. doi: 10.1016/s0014-5793(98)00066-0. [DOI] [PubMed] [Google Scholar]
  25. Glockner A. B., Zumft W. G. Sequence analysis of an internal 9.72-kb segment from the 30-kb denitrification gene cluster of Pseudomonas stutzeri. Biochim Biophys Acta. 1996 Nov 12;1277(1-2):6–12. doi: 10.1016/s0005-2728(96)00108-9. [DOI] [PubMed] [Google Scholar]
  26. Graham A. The organization of hydrogenase in the cytoplasmic membrane of Escherichia coli. Biochem J. 1981 Aug 1;197(2):283–291. doi: 10.1042/bj1970283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hamilton C. M., Aldea M., Washburn B. K., Babitzke P., Kushner S. R. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. doi: 10.1128/jb.171.9.4617-4622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Harboe N., Ingild A. Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand J Immunol Suppl. 1973;1:161–164. doi: 10.1111/j.1365-3083.1973.tb03798.x. [DOI] [PubMed] [Google Scholar]
  29. Izard J. W., Kendall D. A. Signal peptides: exquisitely designed transport promoters. Mol Microbiol. 1994 Sep;13(5):765–773. doi: 10.1111/j.1365-2958.1994.tb00469.x. [DOI] [PubMed] [Google Scholar]
  30. Jacobi A., Rossmann R., Böck A. The hyp operon gene products are required for the maturation of catalytically active hydrogenase isoenzymes in Escherichia coli. Arch Microbiol. 1992;158(6):444–451. doi: 10.1007/BF00276307. [DOI] [PubMed] [Google Scholar]
  31. Jones R. W., Garland P. B. Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane. Biochem J. 1977 Apr 15;164(1):199–211. doi: 10.1042/bj1640199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kalman L. V., Gunsalus R. P. Identification of a second gene involved in global regulation of fumarate reductase and other nitrate-controlled genes for anaerobic respiration in Escherichia coli. J Bacteriol. 1989 Jul;171(7):3810–3816. doi: 10.1128/jb.171.7.3810-3816.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  34. Lyons L. B., Zinder N. D. The genetic map of the filamentous bacteriophage f1. Virology. 1972 Jul;49(1):45–60. doi: 10.1016/s0042-6822(72)80006-0. [DOI] [PubMed] [Google Scholar]
  35. Macinga D. R., Cook G. M., Poole R. K., Rather P. N. Identification and characterization of aarF, a locus required for production of ubiquinone in Providencia stuartii and Escherichia coli and for expression of 2'-N-acetyltransferase in P. stuartii. J Bacteriol. 1998 Jan;180(1):128–135. doi: 10.1128/jb.180.1.128-135.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Menon N. K., Robbins J., Der Vartanian M., Patil D., Peck H. D., Jr, Menon A. L., Robson R. L., Przybyla A. E. Carboxy-terminal processing of the large subunit of [NiFe] hydrogenases. FEBS Lett. 1993 Sep 27;331(1-2):91–95. doi: 10.1016/0014-5793(93)80303-c. [DOI] [PubMed] [Google Scholar]
  37. Menon N. K., Robbins J., Peck H. D., Jr, Chatelus C. Y., Choi E. S., Przybyla A. E. Cloning and sequencing of a putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames. J Bacteriol. 1990 Apr;172(4):1969–1977. doi: 10.1128/jb.172.4.1969-1977.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Oliver D. B., Cabelli R. J., Dolan K. M., Jarosik G. P. Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8227–8231. doi: 10.1073/pnas.87.21.8227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Pascal M. C., Burini J. F., Chippaux M. Regulation of the trimethylamine N-oxide (TMAO) reductase in Escherichia coli: analysis of tor::Mud1 operon fusion. Mol Gen Genet. 1984;195(1-2):351–355. doi: 10.1007/BF00332770. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Roffey R. A., Theg S. M. Analysis of the Import of Carboxyl-Terminal Truncations of the 23-Kilodalton Subunit of the Oxygen-Evolving Complex Suggests That Its Structure Is an Important Determinant for Thylakoid Transport. Plant Physiol. 1996 Aug;111(4):1329–1338. doi: 10.1104/pp.111.4.1329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rossmann R., Maier T., Lottspeich F., Böck A. Characterisation of a protease from Escherichia coli involved in hydrogenase maturation. Eur J Biochem. 1995 Jan 15;227(1-2):545–550. doi: 10.1111/j.1432-1033.1995.tb20422.x. [DOI] [PubMed] [Google Scholar]
  47. Santini C. L., Ize B., Chanal A., Müller M., Giordano G., Wu L. F. A novel sec-independent periplasmic protein translocation pathway in Escherichia coli. EMBO J. 1998 Jan 2;17(1):101–112. doi: 10.1093/emboj/17.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sawers G. The hydrogenases and formate dehydrogenases of Escherichia coli. Antonie Van Leeuwenhoek. 1994;66(1-3):57–88. doi: 10.1007/BF00871633. [DOI] [PubMed] [Google Scholar]
  49. Sawers R. G., Ballantine S. P., Boxer D. H. Differential expression of hydrogenase isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme. J Bacteriol. 1985 Dec;164(3):1324–1331. doi: 10.1128/jb.164.3.1324-1331.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Settles A. M., Yonetani A., Baron A., Bush D. R., Cline K., Martienssen R. Sec-independent protein translocation by the maize Hcf106 protein. Science. 1997 Nov 21;278(5342):1467–1470. doi: 10.1126/science.278.5342.1467. [DOI] [PubMed] [Google Scholar]
  52. Silvestro A., Pommier J., Giordano G. The inducible trimethylamine-N-oxide reductase of Escherichia coli K12: biochemical and immunological studies. Biochim Biophys Acta. 1988 Apr 28;954(1):1–13. doi: 10.1016/0167-4838(88)90049-0. [DOI] [PubMed] [Google Scholar]
  53. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Voelker R., Barkan A. Two nuclear mutations disrupt distinct pathways for targeting proteins to the chloroplast thylakoid. EMBO J. 1995 Aug 15;14(16):3905–3914. doi: 10.1002/j.1460-2075.1995.tb00062.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weiner J. H., Bilous P. T., Shaw G. M., Lubitz S. P., Frost L., Thomas G. H., Cole J. A., Turner R. J. A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins. Cell. 1998 Apr 3;93(1):93–101. doi: 10.1016/s0092-8674(00)81149-6. [DOI] [PubMed] [Google Scholar]
  57. Weiner J. H., Rothery R. A., Sambasivarao D., Trieber C. A. Molecular analysis of dimethylsulfoxide reductase: a complex iron-sulfur molybdoenzyme of Escherichia coli. Biochim Biophys Acta. 1992 Aug 28;1102(1):1–18. doi: 10.1016/0005-2728(92)90059-b. [DOI] [PubMed] [Google Scholar]
  58. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

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