The twin-arginine translocation (Tat) machinery mediates the transport of folded proteins across the cytoplasmic membranes of prokaryotes and thylakoid membranes of chloroplasts. In Gram-negative bacteria three integral membrane components, TatA, TatB and TatC, are essential for generating an active complex. Most Gram-positive bacteria however, have a minimalist Tat complex formed solely from TatA and TatC subunits. Bacillus subtilis encodes two TatAC systems; TatAdCd and TatAyCy. These complexes operate in parallel but have differing substrate specificities. To date, little is known about how the B. subtilis TatAC-complexes assemble, however, the Escherichia coli TatABC complex is well studied and a substrate-triggered positional exchange of TatA and TatB has been identified as the first step in the assembly of an active complex. In this study, we aim to identify site specific interactions taking place between the B. subtilis Tat components and how these interactions differ whilst the complex is engaged in transport of a substrate. Molecular modelling was used to identify likely interaction interfaces between TatA and TatC, and site-directed mutagenesis was used to generate single cysteine variants of both proteins. Subsequently in vivo disulfide crosslinking was undertaken in both E. coli and B.subtilis, in the presence and absence of overproduced Tat substrates, to determine changes that occur between resting state and activated complexes. Overall, this work will enable us to outline a possible mechanism of assembly in TatAC-complexes of Gram-positive bacteria and identify whether the two distinct translocases assemble in a similar fashion.

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