Affinity of TatCd for TatAd elucidates its receptor function in the Bacillus subtilis twin arginine translocation (Tat) translocase system.

Twin arginine translocation (Tat) systems catalyze the transport of folded proteins across the bacterial cytosolic membrane or the chloroplast thylakoid membrane. In the Tat systems of Escherichia coli and many other species TatA-, TatB-, and TatC-like proteins have been identified as essential translocase components. In contrast, the Bacillus subtilis phosphodiesterase PhoD-specific system consists only of a pair of TatA(d)/TatC(d) proteins and involves a TatA(d) protein engaged in a cytosolic and a membrane-embedded localization. Because soluble TatA(d) was able to bind the twin arginine signal peptide of prePhoD prior to membrane integration it could serve to recruit its substrate to the membrane via the interaction with TatC(d). By analyzing the distribution of TatA(d) and studying the mutual affinity with TatC(d) we have shown here that TatC(d) assists the membrane localization of TatA(d). Besides detergent-solubilized TatC(d), membrane-integrated TatC(d) showed affinity for soluble TatA(d). By using a peptide library-specific binding of TatA(d) to cytosolic loops of membrane protein TatC(d) was demonstrated. Depletion of TatC(d) in B. subtilis resulted in a drastic reduction of TatA(d), indicating a stabilizing effect of TatC(d) for TatA(d). In addition, the presence of the substrate prePhoD was the prerequisite for appropriate localization in the cytosolic membrane of B. subtilis as demonstrated by freeze-fracture experiments.