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 TatAd/TatCd proteins and involves a TatAd protein engaged in a cytosolic and a membrane-embedded localization. Because soluble TatAd 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 TatCd. By analyzing the distribution of TatAd and studying the mutual affinity with TatCd we have shown here that TatCd assists the membrane localization of TatAd. Besides detergent-solubilized TatCd, membrane-integrated TatCd showed affinity for soluble TatAd. By using a peptide library-specific binding of TatAd to cytosolic loops of membrane protein TatCd was demonstrated. Depletion of TatCd in B. subtilis resulted in a drastic reduction of TatAd, indicating a stabilizing effect of TatCd for TatAd. 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.