A Facile Reporter System for the Experimental Identification of Twin-Arginine Translocation (Tat) Signal Peptides from All Kingdoms of Life

• Published in: Journal of molecular biology 2008-01, Vol.375 (3), p.595-603
• Main Authors: Widdick, David A., Eijlander, Robyn T., van Dijl, Jan Maarten, Kuipers, Oscar P., Palmer, Tracy
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Arginine - metabolism; Bacillus subtilis; Bacillus subtilis - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; Genes, Reporter; Glycoside Hydrolases - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; Plant Proteins - genetics; Plant Proteins - metabolism; protein export; Protein Sorting Signals - genetics; Protein Sorting Signals - physiology; Protein Transport; Pseudomonas syringae - metabolism; reporter system; Streptomyces; Streptomyces coelicolor; Streptomyces coelicolor - metabolism; Substrate Specificity; Tat system; twin-arginine signal peptide
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2007.11.002

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 semiquantitative 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.