Expression of the bifunctional Bacillus subtilis TatAd protein in Escherichia coli reveals distinct TatA/B-family and Ta
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ORIGINAL PAPER
Expression of the bifunctional Bacillus subtilis TatAd protein in Escherichia coli reveals distinct TatA/B-family and TatB-specific domains James P. Barnett • Janna Lawrence Sharon Mendel • Colin Robinson
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Received: 3 February 2011 / Revised: 22 March 2011 / Accepted: 23 March 2011 / Published online: 9 April 2011 Ó Springer-Verlag 2011
Abstract In the Tat protein export pathway of Gramnegative bacteria, TatA and TatB are homologous proteins that carry out distinct and essential functions in separate sub-complexes. In contrast, Gram-positive Tat systems usually lack TatB and the TatA protein is bifunctional. We have used a mutagenesis approach to delineate TatA/Btype domains in the bifunctional TatAd protein from Bacillus subtilis. This involved expression of mutated TatAd variants in Escherichia coli and tests to determine whether the variants could function as TatA or TatB by complementing E. coli tatA and/or tatB mutants. We show that mutations in the C-terminal half of the transmembrane span and the subsequent FGP ‘hinge’ motif are critical for TatAd function with its partner TatCd subunit, and the same determinants are required for complementation of either tatA or tatB mutants in Escherichia coli. This is thus a critical domain in both TatA and TatB proteins. In contrast, substitution of a series of residues at the N-terminus specifically blocks the ability of TatAd to substitute for E. coli TatB. The results point to the presence of a universally conserved domain in the TatA/B-family, together with a separate N-terminal domain that is linked to the TatB-type function in Gram-negative bacteria.
Communicated by Wolfgang Buckel. J. P. Barnett J. Lawrence S. Mendel C. Robinson School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK C. Robinson (&) Department of Life Sciences, University of Warwick, Coventry CV4 7AL, UK e-mail: [email protected]
Keywords Tat Twin-arginine Protein transport Signal peptide
Introduction The Twin-Arginine Translocation (Tat) pathway is responsible for the export of a subset of periplasmic proteins across the bacterial plasma membrane. A notable attribute is its ability to transport proteins in a prefolded state, and this appears to be its primary role in bacteria (reviewed by Mu¨ller 2005; Robinson and Bolhuis 2004). Some Tat substrates are cofactor-containing redox enzymes involved in anaerobic respiratory pathways; these proteins require a co-factor to be inserted in the cytoplasm before translocation. Other cofactor-less proteins may fold either too rapidly or too tightly to be compatible with the alternative Sec-dependent protein export pathway (Mu¨ller 2005; Berks 1996; Santini et al. 1998; Weiner et al. 1998; Sargent et al. 1998). Most of our information on the Tat pathway has been obtained from studies on the Gram-negative bacterium Escherichia coli. E. coli contains three genes that are essential for Tat function: tatA, tatB and tatC. A homologue of the tatA gene, tatE, is expressed elsewhere in the genome and is though
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