A facile reporter system for the experimental identification of twin-arginine translocation (Tat) signal peptides from all kingdoms of life

David A. Widdick, Robyn T. Eijlander, Jan Maarten van Dijl, Oscar P. Kuipers, Tracy Palmer*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

We have developed a reporter protein system for the experimental verification of twin-arginine signal peptides. This reporter system is based on the Streptomyces coelicolor agarase protein, which is secreted into the growth medium by the twin-arginine translocation (Tat) pathway and whose extracellular activity can be assayed colorimetrically in a serniquantitative manner. Replacement of the native agarase signal peptide with previously characterized twin-arginine signal peptides from other Gram-positive and Gram-negative bacteria resulted in efficient Tat-dependent export of agarase. Candidate twin-arginine signal peptides from archaeal proteins as well as plant thylakoid-targeting sequences were also demonstrated to mediate agarase translocation. A naturally occurring variant signal peptide with an arginine-glutamine motif instead of the consensus di-arginine was additionally recognized as a Tat-targeting sequence by Streptomyces. Application of the agarase assay to previously uncharacterized candidate Tat signal peptides from Bacillus subtilis identified two further probable Tat substrates in this organism. This is the first versatile reporter system for Tat signal peptide identification. (c) 2007 Elsevier Ltd. All rights reserved.

Original languageEnglish
Pages (from-to)595-603
Number of pages9
JournalJournal of Molecular Biology
Volume375
Issue number3
DOIs
Publication statusPublished - 18-Jan-2008

Keywords

  • protein export
  • Tat system
  • twin-arginine signal peptide
  • reporter system
  • Streptomyces
  • DEPENDENT PROTEIN-TRANSPORT
  • SEC-INDEPENDENT PROTEIN
  • RIESKE FE/S PROTEIN
  • AGARASE GENE DAGA
  • DELTA-PH PATHWAY
  • ESCHERICHIA-COLI
  • BACILLUS-SUBTILIS
  • EXPORT PATHWAY
  • STREPTOMYCES-COELICOLOR
  • PSEUDOMONAS-SYRINGAE

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