The TatBC complex of the Tat protein translocase in Escherichia coli and its transition to the substrate-bound TatABC complex.

The twin-arginine translocation (Tat) system serves to transport folded proteins across membranes of prokaryotes and plant plastids. In Escherichia coli, a complex consisting of multiple copies of TatB and TatC initiates the transport by binding the signal peptides of the Tat substrates. Using blue-native polyacrylamide gel electrophoresis, bands of TatBC-containing complexes can be detected at molecular masses of 440 and 580 kDa. We systematically analyzed the formation of Tat complexes with TatB or TatC variants that carried point mutations at selected positions. Several mutations resulted in specific disassembly patterns and alterations in the 440 kDa:580 kDa complex ratios. The 440 kDa complex contains only TatBC, whereas the 580 kDa complex consists of TatABC. Substrate binding results in a TatBC-Tat substrate complex at ~500 kDa and a TatABC-Tat substrate complex at ~600 kDa. Only the ~600 kDa complex was detected with nonrecombinant substrate levels and thus could be the physiologically most relevant species. The results suggest that some TatA is usually associated with TatBC, regardless of substrate binding.

The TatBC complex formation suppresses a modular TatB-multimerization in Escherichia coli.

Twin-arginine translocation (Tat) systems allow the translocation of folded proteins across biological membranes of most prokaryotes. In proteobacteria, a TatBC complex binds Tat substrates and initiates their translocation after recruitment of the component TatA. TatA and TatB belong to one protein family, but only TatB forms stable complexes with TatC. Here we show that TatB builds up TatA-like modular complexes in the absence of TatC. This TatB ladder ranges from about 100 to over 880 kDa with 105+/-10 kDa increments. TatC alone can form a 250 kDa complex which could be a scaffold that can recruit TatB to form defined TatBC complexes.

Topological studies on the twin-arginine translocase component TatC.

The twin-arginine translocation (Tat) system can translocate folded proteins across biological membranes. Among the known Tat-system components in Escherichia coli, TatC is the only protein with multiple trans-membrane domains. TatC is important for translocon interactions with Tat substrates. The knowledge of its membrane topology is therefore crucial for the understanding of substrate binding and translocon function. Recently, based on active PhoA reporter fusions to the second predicted cytoplasmic loop of TatC, a topology with four trans-membrane domains has been suggested, calling in silico predictions of six trans-membrane domains into question. Here we report studies with translational fusions of TatC to the topological marker enzymes PhoA and LacZ which provide strong evidence for a six-trans-membrane domain topology. The stop transfer capacity of the fourth trans-membrane domain was found to be strongly influenced by the succeeding cytoplasmic domain. The presence of linker sequences at PhoA-fusion sites of the cytoplasmic domain induced PhoA leakage. In the case of one tested fusion (S185-PhoA), the stop-transfer efficiency was already low due to the negative charge in the center of the fourth trans-membrane domain (E170). The results point to the importance of cytoplasmic loops for the stabilization of stop-transfer sequences and revoke evidence for only four trans-membrane domains of TatC.