Cysteine-scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC component

Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom; School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom; Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom

^* To whom correspondence should be addressed. Email: tracy.palmer{at}bbsrc.ac.uk.

	Abstract

The TatC protein is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. It is a polytopic membrane protein that forms a complex with TatB, together acting as the receptor for Tat substrates. In this study we have constructed 57 individual cysteine substitutions throughout the protein. Each of the substitutions resulted in a TatC protein that was competent to support Tat-dependent protein translocation. Accessibility studies with membrane-permeant and -impermeant thiol-reactive reagents demonstrated that TatC has six transmembrane helices rather than four suggested by a previous study (Gouffi, K., Santini, C.-L., and L.-F. Wu. 2002. FEBS Lett. 525:65-70). Disulfide cross-linking experiments with TatC proteins containing single cysteine residues showed that each transmembrane domain of TatC was able to interact with the same domain from a neighboring TatC protein. Surprisingly, only three of these cysteine variants retained the ability to cross-link at low temperature. These results are consistent with likelihood that most of the disulfide cross-links are between TatC proteins in separate TatBC complexes, suggesting that TatC is located on the periphery of the complex.