Substrate-triggered position-switching of TatA and TatB is an essential step in the Escherichia coli Tat protein export pathway.

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. Here we use disulfide crosslinking and molecular modelling to show there are two binding sites for TatA/B proteins on TatC. TatA and TatB are each able to occupy both sites if they are the only TatA/B protein present. However, under resting conditions the sites are differentially occupied with TatB occupying the polar cluster site while TatA binds adjacently at the TatC transmembrane helix 6 binding site. When the Tat system is activated by the overproduction of a substrate, TatA and TatB switch their binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.