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Journal of Bacteriology, August 2007, p. 5482-5494, Vol. 189, No. 15
0021-9193/07/$08.00+0     doi:10.1128/JB.00647-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cysteine Scanning Mutagenesis and Topological Mapping of the Escherichia coli Twin-Arginine Translocase TatC Component

Claire Punginelli,¹ Bárbara Maldonado,^1,2 Sabine Grahl,¹ Rachael Jack,^1,2, Meriem Alami,³ Juliane Schröder,¹ Ben C. Berks,³ and Tracy Palmer^1,2*

Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom,¹ School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom,² Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom³

Received 25 April 2007/ Accepted 22 May 2007

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 the four suggested by a previous study (K. Gouffi, C.-L. Santini, and L.-F. Wu, FEBS Lett. 525:65-70, 2002). 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 temperatures. These results are consistent with the 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.

Published ahead of print on 1 June 2007.

Present address: Department of Molecular Microbiology, Karakorum International University, Gilgit, Northern Areas, Pakistan.