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

Claire Punginelli, Barbara Maldonado, Sabine Grahl, Rachael Jack, Meriem Alami, Juliane Schroeder, Ben C. Berks, Tracy Palmer

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    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.

    Original languageEnglish
    Pages (from-to)5482-5494
    Number of pages13
    JournalJournal of Bacteriology
    Issue number15
    Publication statusPublished - 2007

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