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Analysis of Tat Targeting Function and Twin-Arginine Signal Peptide Activity in Escherichia coli

  • Tracy Palmer
  • Ben C. Berks
  • Frank Sargent
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 619)

Abstract

The Tat system is a protein export system dedicated to the transport of folded proteins across the prokaryotic cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Proteins are targeted for export by the Tat system via N-terminal signal peptides harbouring an S-R-R-x-F-L-K ‘twin-arginine’ motif. In this chapter qualitative and quantitative assays for native Tat substrates in the model organism Escherichia coli are described. Genetic screening methods designed to allow the rapid positive selection of Tat signal peptide activity and the first positive selection for mutations that inactivate the Tat pathway are also presented. Finally isothermal titration calorimetry (ITC) methods for measuring the affinity of twin-arginine signal peptide–chaperone interactions are discussed.

Key words

Tat system twin-arginine signal peptide TMAO reductase hydrogenase chaperone protein–protein interaction 

Notes

Acknowledgments

We would particularly like to thank Prof. Gary Sawers (Halle-Wittenberg) for his help with developing some of the early methods for analysis of the E. coli Tat system, and all the members of our laboratories past and present. Work in our laboratories is or has been funded by the BBSRC, the MRC, the Wellcome Trust, the European Union and the Royal Society.

References

  1. 1.
    Theg, S. M., Cline, K., Finazzi, G., and Wollman, F. A. (2005) The energetics of the chloroplast Tat protein transport pathway revisited Trends Plant Sci 10, 153–154.CrossRefPubMedGoogle Scholar
  2. 2.
    Berks, B. C., Palmer, T., and Sargent, F. (2003) The Tat protein translocation pathway and its role in microbial physiology Adv Microb Physiol 47, 187–254.CrossRefPubMedGoogle Scholar
  3. 3.
    Berks, B. C., Palmer, T., and Sargent, F. (2005) Protein targeting by the bacterial twin–arginine translocation (Tat) pathway Curr Opin Microbiol 8, 174–181.CrossRefPubMedGoogle Scholar
  4. 4.
    Tullman-Ercek, D., DeLisa, M. P., Kawarasaki, Y., Iranpour, P., Ribnicky, B., Palmer, T., and Georgiou, G. (2007) Export pathway selectivity of Escherichia coli twin arginine translocation signal peptides J Biol Chem 282, 8309–8316.CrossRefPubMedGoogle Scholar
  5. 5.
    Weiner, J. H., Bilous, P. T., Shaw, G. M., Lubitz, S. P., Frost, L., Thomas, G. H., Cole, J. A., and Turner, R. J. (1998) A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins Cell 93, 93–101.CrossRefPubMedGoogle Scholar
  6. 6.
    Sargent, F., Bogsch, E. G., Stanley, N. R., Wexler, M., Robinson, C., Berks, B. C., and Palmer, T. (1998) Overlapping functions of components of a bacterial Sec-independent protein export pathway EMBO J 17, 3640–3650.CrossRefPubMedGoogle Scholar
  7. 7.
    Rodrigue, A., Chanal, A., Beck, K., Muller, M., and Wu, L. F. (1999) Co-translocation of a periplasmic enzyme complex by a hitchhiker mechanism through the bacterial tat pathway J Biol Chem 274, 13223–13228.CrossRefPubMedGoogle Scholar
  8. 8.
    Jack, R. L., Buchanan, G., Dubini, A., Hatzixanthis, K., Palmer, T., and Sargent, F. (2004) Coordinating assembly and export of complex bacterial proteins EMBO J 23, 3962–3972.CrossRefPubMedGoogle Scholar
  9. 9.
    Ize, B., Stanley, N. R., Buchanan, G., and Palmer, T. (2003) Role of the Escherichia coli Tat pathway in outer membrane integrity Mol Microbiol 48, 1183–1193.CrossRefPubMedGoogle Scholar
  10. 10.
    Ize, B., Coulthurst, S. J., Buchanan, G., Hatzixanthis, K., Caldelari, I., Barclay, E. C., Richardson, D. J., Palmer, T., and Sargent, F. (2009) Remnant signal peptides on non-exported enzymes: implications for the evolution of prokaryotic respiratory chains Microbiology 155, 3992–4004.Google Scholar
  11. 11.
    Thomas, J. D., Daniel, R. A., Errington, J., and Robinson, C. (2001) Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli Mol Microbiol 39, 47–53.CrossRefPubMedGoogle Scholar
  12. 12.
    Santini, C. L., Bernadac, A., Zhang, M., Chanal, A., Ize, B., Blanco, C., and Wu, L. F. (2001) Translocation of jellyfish green fluorescent protein via the Tat system of Escherichia coli and change of its periplasmic localization in response to osmotic up-shock J Biol Chem 276, 8159–8164.CrossRefPubMedGoogle Scholar
  13. 13.
    Blaudeck, N., Kreutzenbeck, P., Freudl, R., and Sprenger, G. A. (2003) Genetic analysis of pathway specificity during posttranslational protein translocation across the Escherichia coli plasma membrane J Bacteriol 185, 2811–2819.CrossRefPubMedGoogle Scholar
  14. 14.
    Hicks, M. G., Lee, P. A., Georgiou, G., Berks, B. C., and Palmer, T. (2005) Positive selection for loss-of-function tat mutations identifies critical residues required for TatA activity J Bacteriol 187, 2920–2925.CrossRefPubMedGoogle Scholar
  15. 15.
    Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, New York.Google Scholar
  16. 16.
    Cohen, G. N., and Rickenberg, H. V. (1956) Concentration specifique reversible des amino acides chez Escherichia coli. Ann Inst Pasteur (Paris) 91, 693–720.Google Scholar
  17. 17.
    McEwan, A. G., Jackson, J. B., and Ferguson, S. J. (1984) Rationalization of properties of nitrate reductases in Rhodopseudomonas capsulata Arch Microbiol 137, 344–349CrossRefGoogle Scholar
  18. 18.
    Osborn, M. J., Gander, J. E., and Parisi, E. (1972) Mechanism of assembly of the outer membrane of Salmonella typhimurium. Site of synthesis of lipopolysaccharide J Biol Chem 247, 3973–3986.PubMedGoogle Scholar
  19. 19.
    Silvestro, A., Pommier, J., and Giordano, G. (1988) The inducible trimethylamine-N-oxide reductase of Escherichia coli K12: biochemical and immunological studies Biochim Biophys Acta 954, 1–13.PubMedGoogle Scholar
  20. 20.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent J Biol Chem 193, 265–275.PubMedGoogle Scholar
  21. 21.
    Lester, R. L., and DeMoss, J. A. (1971) Effects of molybdate and selenite on formate and nitrate metabolism in Escherichia coli J Bacteriol 105, 1006–1014.PubMedGoogle Scholar
  22. 22.
    Ballantine, S. P., and Boxer, D. H. (1985) Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12 J Bacteriol 163, 454–459.PubMedGoogle Scholar
  23. 23.
    Tabor, S., and Richardson, C. C. (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes Proc Natl Acad Sci USA 82, 1074–1078.CrossRefPubMedGoogle Scholar
  24. 24.
    Stanley, N. R., Palmer, T., and Berks, B. C. (2000) The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli J Biol Chem 275, 11591–11596.CrossRefPubMedGoogle Scholar
  25. 25.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685.CrossRefPubMedGoogle Scholar
  26. 26.
    Maillard, J., Spronk, C. A., Buchanan, G., Lyall, V., Richardson, D. J., Palmer, T., Vuister, G. W., and Sargent, F. (2007) Structural diversity in twin-arginine signal peptide-binding proteins Proc Natl Acad Sci USA 104, 15641–15646.CrossRefPubMedGoogle Scholar
  27. 27.
    Li, H., Lin, X. M., Wang, S. Y., and Peng, X. X. (2007) Identification and antibody-therapeutic targeting of chloramphenicol-resistant outer membrane proteins in Escherichia coli J Proteome Res 6, 3628–3636.CrossRefPubMedGoogle Scholar
  28. 28.
    Caldelari, I., Palmer, T., and Sargent, F. (2008) Escherichia coli tat mutant strains are able to transport maltose in the absence of an active malE gene Arch Microbiol.Google Scholar
  29. 29.
    Pascal, M. C., Burini, J. F., and Chippaux, M. (1984) Regulation of the trimethylamine N-oxide (TMAO) reductase in Escherichia coli: analysis of tor::Mud1 operon fusion Mol Gen Genet 195, 351–355.CrossRefPubMedGoogle Scholar
  30. 30.
    Berg, B. L., and Stewart, V. (1990) Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12 Genetics 125, 691–702.PubMedGoogle Scholar
  31. 31.
    Bilous, P. T., Cole, S. T., Anderson, W. F., and Weiner, J. H. (1988) Nucleotide sequence of the dmsABC operon encoding the anaerobic dimethylsulphoxide reductase of Escherichia coli Mol Microbiol 2, 785–795.CrossRefPubMedGoogle Scholar
  32. 32.
    Cotter, P. A., and Gunsalus, R. P. (1989) Oxygen, nitrate, and molybdenum regulation of dmsABC gene expression in Escherichia coli J Bacteriol 171, 3817–3823.PubMedGoogle Scholar
  33. 33.
    Richard, D. J., Sawers, G., Sargent, F., McWalter, L., and Boxer, D. H. (1999) Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [NiFe] hydrogenases 1 and 2 of Escherichia coli Microbiology 145, 2903–2912.PubMedGoogle Scholar
  34. 34.
    Atlung, T., Nielsen, A., and Hansen, F. G. (1989) Isolation, characterization, and nucleotide sequence of appY, a regulatory gene for growth-phase-dependent gene expression in Escherichia coli J Bacteriol 171, 1683–1691.PubMedGoogle Scholar
  35. 35.
    Turnbull, W. B., and Daranas, A. H. (2003) On the value of c: can low affinity systems be studied by isothermal titration calorimetry? J Am Chem Soc 125, 14859–14866.CrossRefPubMedGoogle Scholar
  36. 36.
    Jack, R. L., Sargent, F., Berks, B. C., Sawers, G., and Palmer, T. (2001) Constitutive expression of Escherichia coli tat genes indicates an important role for the twin-arginine translocase during aerobic and anaerobic growth J Bacteriol 183, 1801–1804.CrossRefPubMedGoogle Scholar
  37. 37.
    Sargent, F., Stanley, N. R., Berks, B. C., and 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.CrossRefPubMedGoogle Scholar
  38. 38.
    Casadaban, M. J., and Cohen, S. N. (1979) 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 USA 76, 4530–4533.CrossRefPubMedGoogle Scholar
  39. 39.
    Bogsch, E. G., Sargent, F., Stanley, N. R., Berks, B. C., Robinson, C., and 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.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Tracy Palmer
    • 1
  • Ben C. Berks
    • 2
  • Frank Sargent
    • 1
  1. 1.Division of Molecular Microbiology, College of Life SciencesUniversity of DundeeDundeeScotland
  2. 2.Department of BiochemistryOxford UniversityOxfordUK

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