A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli

The twin-arginine translocation (Tat) system transports folded proteins across membranes of prokaryotes, plant plastids, and some mitochondria. According to blue-native polyacrylamide gel electrophoresis after solubilization with digitonin, distinct interactions between the components TatA, TatB, and TatC result in two major TatBC-containing complexes in Escherichia coil that can bind protein substrates. We now report the first detection of a TatABC complex that likely represents the state at which transport occurs. This complex was initially found when the photo cross-linking amino acid p-benzoylL-phenylalanine (Bpa) was introduced at position 150 on the periplasmic side of the first trans-membrane domain of TatC. Cross-linking of TatC(150Bpa) resulted in TatC-TatC-crosslinks, indicating a close proximity to neighboring TatC in the complex. However, the new complex was not caused by cross-links but rather by non-covalent side chain interactions, as it was also detectable without UV-cross-linking or with an 150Y exchange. The new complex did not contain any detectable substrate. It was slightly upshifted relative to previously reported substrate-containing TatABC complexes. In the absence of TatA, an inactive TatBC(150Bpa), complex was formed of the size of wild-type substrate-containing TatABC complexes, suggesting that TatB occupies TatA-binding sites at TatC(150Bpa). When substrate binding was abolished by point mutations, this TatBC(150Bpa) complex shifted analogously to active TatABC(150Bpa) complexes, indicating that a defect substrate-binding site further enhances TatB association to TatA-binding sites. Only TatA could shift the complex with an intact substrate-binding site, which explains the TatA requirement for substrate transport by TatABC systems.