1887

Abstract

Protein translocation via the Tat machinery in thylakoids and bacteria occurs through a cooperation between the TatA, TatB and TatC subunits, of which the TatC protein forms the initial Tat substrate-binding site. The Tat machinery lacks TatB and comprises two separate TatAC complexes with distinct substrate specificities: PhoD is secreted by the TatAdCd complex, whereas YwbN is secreted by the TatAyCy complex. To study the role of the Gram-positive TatC proteins in Tat-dependent protein secretion efficiency, we applied several genetic engineering approaches to modify and analyse the TatCd and TatCy proteins. Cytoplasmic and transmembrane domain exchange between TatCd and TatCy resulted in stable chimeric proteins that were unable to secrete both known substrates of the Tat system. Site-directed mutagenesis of conserved residues in the N-terminal part of both TatC proteins revealed significant differences in the degree of importance of these residues between TatCd, TatCy and TatC. In addition, two small C-terminal deletions in TatCy completely abolished YwbN translocation, indicating that this terminus is essential for Tat translocation activity. Important differences from previous observations for TatC and implications for substrate binding and translocation are discussed.

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2009-06-01
2024-12-02
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References

  1. Alami M., Lüke I., Deitermann S., Eisner G., Koch H. G., Brunner J., Müller M. 2003; Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli . Mol Cell 12:937–946
    [Google Scholar]
  2. Allen S. C., Barrett C. M., Ray N., Robinson C. 2002; Essential cytoplasmic domains in the Escherichia coli TatC protein. J Biol Chem 277:10362–10366
    [Google Scholar]
  3. Barnett J. P., Eijlander R. T., Kuipers O. P., Robinson C. 2008; A minimal Tat system from a gram-positive organism: a bifunctional TatA subunit participates in discrete TatAC and TatA complexes. J Biol Chem 283:2534–2542
    [Google Scholar]
  4. Barrett C. M., Mangels D., Robinson C. 2005; Mutations in subunits of the Escherichia coli twin-arginine translocase block function via differing effects on translocation activity or Tat complex structure. J Mol Biol 347:453–463
    [Google Scholar]
  5. Behrendt J., Standar K., Lindenstrauss U., Brüser T. 2004; Topological studies on the twin-arginine translocase component TatC. FEMS Microbiol Lett 234:303–308
    [Google Scholar]
  6. Bogsch E. G., Sargent F., Stanley N. R., Berks B. C., Robinson C., Palmer T. 1998; An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria. J Biol Chem 273:18003–18006
    [Google Scholar]
  7. Bron S., Venema G. 1972; Ultraviolet interaction and excision repair in Bacillus subtilis . I. Construction and characterization of a transformable eightfold auxotrophic strain and two ultraviolet-sensitive derivatives. Mutat Res 15:1–10
    [Google Scholar]
  8. Bron S., Bolhuis A., Tjalsma H., Holsappel S., Venema G., van Dijl J. M. 1998; Protein secretion and possible roles for multiple signal peptidases for precursor processing in bacilli. J Biotechnol 64:3–13
    [Google Scholar]
  9. Buchanan G., de Leeuw E., Stanley N. R., Wexler M., Berks B. C., Sargent F., Palmer T. 2002; Functional complexity of the twin-arginine translocase TatC component revealed by site-directed mutagenesis. Mol Microbiol 43:1457–1470
    [Google Scholar]
  10. Cline K., Mori H. 2001; Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport. J Cell Biol 154:719–729
    [Google Scholar]
  11. Eder S., Liu W., Hulett F. M. 1999; Mutational analysis of the phoD promoter in Bacillus subtilis : implications for PhoP binding and promoter activation of Pho regulon promoters. J Bacteriol 181:2017–2025
    [Google Scholar]
  12. Eijlander R. T., Jongbloed J. D., Kuipers O. P. 2009; Relaxed specificity of the Bacillus subtilis TatAdCd translocase in Tat-dependent protein secretion. J Bacteriol 191:196–202
    [Google Scholar]
  13. Gérard F., Cline K. 2006; Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site. J Biol Chem 281:6130–6135
    [Google Scholar]
  14. Gérard F., Cline K. 2007; The thylakoid proton gradient promotes an advanced stage of signal peptide binding deep within the Tat pathway receptor complex. J Biol Chem 282:5263–5272
    [Google Scholar]
  15. Gohlke U., Pullan L., McDevitt C. A., Porcelli I., de Leeuw E., Palmer T., Saibil H. R., Berks B. C. 2005; The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter. Proc Natl Acad Sci U S A 102:10482–10486
    [Google Scholar]
  16. Härtl B., Wehrl W., Wiegert T., Homuth G., Schumann W. 2001; Development of a new integration site within the Bacillus subtilis chromosome and construction of compatible expression cassettes. J Bacteriol 183:2696–2699
    [Google Scholar]
  17. Holzapfel E., Eisner G., Alami M., Barrett C. M. L., Buchanan G., Lüke I., Betton J. M., Robinson C., Palmer T. other authors 2007; The entire N-terminal half of TatC is involved in twin-arginine precursor binding. Biochemistry 46:2892–2898
    [Google Scholar]
  18. Jongbloed J. D., Martin U., Antelmann H., Hecker M., Tjalsma H., Venema G., Bron S., van Dijl J. M., Müller J. 2000; TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway. J Biol Chem 275:41350–41357
    [Google Scholar]
  19. Jongbloed J. D., Grieger U., Antelmann H., Hecker M., Nijland R., Bron S., van Dijl J. M. 2004; Two minimal Tat translocases in Bacillus . Mol Microbiol 54:1319–1325
    [Google Scholar]
  20. Kreutzenbeck P., Kröger C., Lausberg F., Blaudeck N., Sprenger G. A., Freudl R. 2007; Escherichia coli twin-arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities. J Biol Chem 282:7903–7911
    [Google Scholar]
  21. Kunst F., Ogasawara N., Moszer I., Albertini A. M., Alloni G., Azevedo V., Bertero M. G., Bessières P., Bolotin A. other authors 1997; The complete genome sequence of the gram-positive bacterium Bacillus subtilis . Nature 390:249–256
    [Google Scholar]
  22. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  23. Lange C., Müller S. D., Walther T. H., Bürck J., Ulrich A. S. 2007; Structure analysis of the protein translocating channel TatA in membranes using a multi-construct approach. Biochim Biophys Acta 17682627–2634
    [Google Scholar]
  24. Lee P. A., Tullman-Ercek D., Georgiou G. 2006; The bacterial twin-arginine translocation pathway. Annu Rev Microbiol 60:373–395
    [Google Scholar]
  25. Mangels D., Mathers J., Bolhuis A., Robinson C. 2005; The core TatABC complex of the twin-arginine translocase in Escherichia coli : TatC drives assembly whereas TatA is essential for stability. J Mol Biol 345:415–423
    [Google Scholar]
  26. McDevitt C. A., Buchanan G., Sargent F., Palmer T., Berks B. C. 2006; Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels. FEBS J 273:5656–5668
    [Google Scholar]
  27. Mendel S., McCarthy A., Barnett J. P., Eijlander R. T., Kuipers O. P., Robinson C. 2008; The Escherichia coli TatABC system and a Bacillus subtilis TatAC-type system recognise three distinct targeting determinants in twin-arginine signal peptides. J Mol Biol 375:661–672
    [Google Scholar]
  28. Punginelli C., Maldonado B., Grahl S., Jack R., Alami M., Schröder J., Berks B. C., Palmer T. 2007; Cysteine-scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC component. J Bacteriol 189:5482–5494
    [Google Scholar]
  29. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  30. Sargent F., Bogsch E. G., Stanley N. R., Wexler M., Robinson C., Berks B. C., Palmer T. 1998; Overlapping functions of components of a bacterial Sec-independent protein export pathway. EMBO J 17:3640–3650
    [Google Scholar]
  31. Sargent F., Stanley N. R., Berks B. C., Palmer T. 1999; Sec-independent protein translocation in Escherichia coli . A distinct and pivotal role for the TatB protein. J Biol Chem 274:36073–36082
    [Google Scholar]
  32. Sargent F., Berks B. C., Palmer T. 2006; Pathfinders and trailblazers: a prokaryotic targeting system for transport of folded proteins. FEMS Microbiol Lett 254:198–207
    [Google Scholar]
  33. Strauch E. M., Georgiou G. 2007; Escherichia coli tatC mutations that suppress defective twin-arginine transporter signal peptides. J Mol Biol 374:283–291
    [Google Scholar]
  34. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354
    [Google Scholar]
  35. van der Vossen J. M. B. M., van der Lelie D., Venema G. 1987; Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl Environ Microbiol 53:2452–2457
    [Google Scholar]
  36. van Dijl J. M., de Jong A., Venema G., Bron S. 1995; Identification of the potential active site of the signal peptidase SipS of Bacillus subtilis . Structural and functional similarities with LexA-like proteases. J Biol Chem 270:3611–3618
    [Google Scholar]
  37. Wertman K. F., Wyman A. R., Botstein D. 1986; Host/vector interactions which affect the viability of recombinant phage lambda clones. Gene 49:253–262
    [Google Scholar]
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