Archives of Microbiology

, Volume 191, Issue 12, pp 919–925 | Cite as

Proteolytic processing of Escherichia coli twin-arginine signal peptides by LepB

  • Iris Lüke
  • Jennifer I. Handford
  • Tracy Palmer
  • Frank SargentEmail author
Short Communication


The twin-arginine translocation (Tat) apparatus is a protein targeting system found in the cytoplasmic membranes of many prokaryotes. Substrate proteins of the Tat pathway are synthesised with signal peptides bearing SRRxFLK ‘twin-arginine’ amino acid motifs. All Tat signal peptides have a common tripartite structure comprising a polar N-terminal region, followed by a hydrophobic region of variable length and a polar C-terminal region. In Escherichia coli, Tat signal peptides are proteolytically cleaved after translocation. The signal peptide C-terminal regions contain conserved AxA motifs, which are possible recognition sequences for leader peptidase I (LepB). In this work, the role of LepB in Tat signal peptide processing was addressed directly. Deliberate repression of lepB expression prevented processing of all Tat substrates tested, including SufI, AmiC, and a TorA-23K reporter protein. In addition, electron microscopy revealed gross defects in cell architecture and membrane integrity following depletion of cellular LepB protein levels.


Escherichia coli Tat protein transport pathway Signal peptidase I LepB protein 



We thank K. Hatzixanthis, K. Moore and M. G. Hicks (Norwich) for preliminary work on this project, and J.-W. de Gier (Stockholm) for providing an anti-LepB serum. This research was funded by the BBSRC through award BB/C006844/2 (to F.S.) and a postgraduate studentship (to J.I.H.). T.P. is an MRC senior non-clinical research fellow.


  1. Berks BC (1996) A common export pathway for proteins binding complex redox cofactors? Mol Microbiol 22:393–404CrossRefPubMedGoogle Scholar
  2. Bernadac A, Gavioli M, Lazzaroni JC, Raina S, Lloubes R (1998) Escherichia coli tol-pal mutants form outer membrane vesicles. J Bacteriol 180:4872–4878PubMedGoogle Scholar
  3. Cristobal S, de Gier JW, Nielsen H, von Heijne G (1999) Competition between Sec- and Tat-dependent protein translocation in Escherichia coli. EMBO J 18:2982–2990CrossRefPubMedGoogle Scholar
  4. Dalbey RE, Wickner W (1985) Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane. J Biol Chem 260:15925–15931PubMedGoogle Scholar
  5. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645CrossRefPubMedGoogle Scholar
  6. Dunn SD (1986) Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal Biochem 157:144–153CrossRefPubMedGoogle Scholar
  7. Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter. J Bacteriol 177:4121–4130PubMedGoogle Scholar
  8. Hatzixanthis K, Palmer T, Sargent F (2003) A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase. Mol Microbiol 49:1377–1390CrossRefPubMedGoogle Scholar
  9. Hayden JD, Ades SE (2008) The extracytoplasmic stress factor, sigmaE, is required to maintain cell envelope integrity in Escherichia coli. PLoS One 3:e1573CrossRefPubMedGoogle Scholar
  10. Ize B et al (2002) In vivo dissection of the Tat translocation pathway in Escherichia coli. J Mol Biol 317:327–335CrossRefPubMedGoogle Scholar
  11. Ize B, Stanley NR, Buchanan G, Palmer T (2003) Role of the Escherichia coli Tat pathway in outer membrane integrity. Mol Microbiol 48:1183–1193CrossRefPubMedGoogle Scholar
  12. Katsui N, Tsuchido T, Hiramatsu R, Fujikawa S, Takano M, Shibasaki I (1982) Heat-induced blebbing and vesiculation of the outer membrane of Escherichia coli. J Bacteriol 151:1523–1531PubMedGoogle Scholar
  13. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  14. Mejean V, Iobbi-Nivol C, Lepelletier M, Giordano G, Chippaux M, Pascal MC (1994) TMAO anaerobic respiration in Escherichia coli: involvement of the tor operon. Mol Microbiol 11:1169–1179CrossRefPubMedGoogle Scholar
  15. Natale P, Bruser T, Driessen AJ (2008) Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-distinct translocases and mechanisms. Biochim Biophys Acta 1778:1735–1756CrossRefPubMedGoogle Scholar
  16. Ng SY, Chaban B, VanDyke DJ, Jarrell KF (2007) Archaeal signal peptidases. Microbiology 153:305–314CrossRefPubMedGoogle Scholar
  17. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Syst 8:581–599CrossRefPubMedGoogle Scholar
  18. Paetzel M, Karla A, Strynadka NC, Dalbey RE (2002) Signal peptidases. Chem Rev 102:4549–4580CrossRefPubMedGoogle Scholar
  19. Sambasivarao D, Turner RJ, Simala-Grant JL, Shaw G, Hu J, Weiner JH (2000) Multiple roles for the twin arginine leader sequence of dimethyl sulfoxide reductase of Escherichia coli. J Biol Chem 275:22526–22531CrossRefPubMedGoogle Scholar
  20. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  21. Sargent F (2007) Constructing the wonders of the bacterial world: biosynthesis of complex enzymes. Microbiology 153:633–651CrossRefPubMedGoogle Scholar
  22. Sargent F et al (1998) Overlapping functions of components of a bacterial Sec-independent protein export pathway. EMBO J 17:3640–3650CrossRefPubMedGoogle Scholar
  23. Sargent F, Berks BC, Palmer T (2006) Pathfinders and trailblazers: a prokaryotic targeting system for transport of folded proteins. FEMS Microbiol Lett 254:198–207PubMedCrossRefGoogle Scholar
  24. Simons RW, Houman F, Kleckner N (1987) Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53:85–96CrossRefPubMedGoogle Scholar
  25. Stanley NR, Palmer T, Berks BC (2000) The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli. J Biol Chem 275:11591–11596CrossRefPubMedGoogle Scholar
  26. Stanley NR, Findlay K, Berks BC, Palmer T (2001) Escherichia coli strains blocked in Tat-dependent protein export exhibit pleiotropic defects in the cell envelope. J Bacteriol 183:139–144CrossRefPubMedGoogle Scholar
  27. Tabor S, Richardson CC (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82:1074–1078CrossRefPubMedGoogle Scholar
  28. Tarry M et al (2009) The Escherichia coli cell division protein and model Tat substrate SufI (FtsP) localizes to the septal ring and has a multicopper oxidase-like structure. J Mol Biol 386:504–519CrossRefPubMedGoogle Scholar
  29. Thornton J, Blakey D, Scanlon E, Merrick M (2006) The ammonia channel protein AmtB from Escherichia coli is a polytopic membrane protein with a cleavable signal peptide. FEMS Microbiol Lett 258:114–120CrossRefPubMedGoogle Scholar
  30. 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 USA 76:4350–4354CrossRefPubMedGoogle Scholar
  31. Tullman-Ercek D et al (2007) Export pathway selectivity of Escherichia coli twin arginine translocation signal peptides. J Biol Chem 282:8309–8316CrossRefPubMedGoogle Scholar
  32. Woldringh CL (2002) The role of co-transcriptional translation and protein translocation (transertion) in bacterial chromosome segregation. Mol Microbiol 45:17–29CrossRefPubMedGoogle Scholar
  33. Yahr TL, Wickner WT (2001) Functional reconstitution of bacterial Tat translocation in vitro. EMBO J 20:2472–2479CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Iris Lüke
    • 1
  • Jennifer I. Handford
    • 2
  • Tracy Palmer
    • 1
  • Frank Sargent
    • 1
    Email author
  1. 1.Division of Molecular Microbiology, College of Life SciencesUniversity of DundeeDundeeUK
  2. 2.Department of Molecular MicrobiologyJohn Innes CentreNorwichUK

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