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J. Biol. Chem., Vol. 282, Issue 33, 23937-23945, August 17, 2007

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Cysteine Scanning Mutagenesis and Disulfide Mapping Studies of the TatA Component of the Bacterial Twin Arginine Translocase^*

Nicholas P. Greene, Ida Porcelli, Grant Buchanan, Matthew G. Hicks, Sonya M. Schermann, Tracy Palmer^¶, and Ben C. Berks¹

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

The Tat (twin arginine translocation) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The integral membrane proteins TatA, TatB, and TatC are essential components of the Tat pathway. TatA forms high order oligomers and is thought to constitute the protein-translocating unit of the Tat system. Cysteine scanning mutagenesis was used to systematically investigate the functional importance of residues in the essential N-terminal transmembrane and amphipathic helices of Escherichia coli TatA. Cysteine substitutions of most residues in the amphipathic helix, including all the residues on the hydrophobic face of the helix, severely compromise Tat function. Glutamine 8 was identified as the only residue in the transmembrane helix that is critical for TatA function. The cysteine variants in the transmembrane helix were used in disulfide mapping experiments to probe the oligomeric arrangement of TatA protomers within the larger TatA complex. Residues in the center of the transmembrane helix (including residues 10–16) show a distinct pattern of cross-linking indicating that this region of the protein forms well defined interactions with other protomers. At least two interacting faces were detected. The results of our TatA studies are compared with analogous data for the homologous, but functionally distinct, TatB protein. This comparison reveals that it is only in TatA that the amphipathic helix is sensitive to amino acid substitutions. The TatA amphipathic helix may play a role in forming and controlling the path of substrate movement across the membrane.

Received for publication, April 9, 2007 , and in revised form, June 8, 2007.

^* This work was supported by the Medical Research Council via a studentship (to N. G.) and a Senior Non-Clinical Fellowship award (to T. P.), by the Natural Sciences and Engineering Research Council of Canada, by a Fellowship (to S. S.), by the University of Oxford through a studentship (to I. P.), and by the Biotechnology and Biological Sciences and Research Council via a grant-in-aid to the John Innes Centre. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Tel.: 44-1865-275-250; Fax: 44-1865-275-259; E-mail: ben.berks{at}bioch.ox.ac.uk.

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