Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli

The twin arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. The active translocase is assembled on demand, with substrate-binding at a TatABC receptor complex triggering recruitment and assembly of multiple additional copies of TatA, however the molecular interactions mediating translocase assembly are poorly understood. A ‘polar cluster’ site on TatC transmembrane helix 5 was previously identified as binding to TatB. Here we use disulfide crosslinking and molecular modelling to identify a new binding site on TatC transmembrane helix 6, adjacent to the polar cluster site. We demonstrate that TatA and TatB each have the capacity to bind at both TatC sites, however in vivo this is regulated according to the activation state of the complex. In the resting state system, TatB binds the polar cluster site, with TatA occupying the transmembrane helix 6 site. However when the system is activated by overproduction of a substrate, TatA and TatB switch binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.