Functional reconstitution of bacterial Tat translocation in vitro

The Tat (twin-arginine translocation) pathway is a Sec-independent mechanis m for translocating folded preproteins across or into the inner membrane of Escherichia coil, To study Tat translocation, we sought an in vitro transl ocation assay using purified inner membrane vesicles and in vitro synthesiz ed substrate protein. While membrane vesicles derived from wildtype cells t ranslocate the Sec-dependent substrate proOmpA, translocation of a Tat-depe ndent substrate, SufI, was not detected. We established that in vivo overex pression of SufI can saturate the Tat translocase, and that simultaneous ov erexpression of TatA, B and C relieves this SufI saturation. Using membrane vesicles derived from cells overexpressing TatABC, in vitro translocation of SufI was detected, Like translocation in vivo, translocation of SufI in vitro requires TatABC, an intact membrane potential and the twin-arginine t argeting motif within the signal peptide of SufI, In contrast to Sec transl ocase, we find that Tat translocase does not require ATP, The development o f an in vitro translocation assay is a prerequisite for further biochemical investigations of the mechanism of translocation, substrate recognition an d translocase structure.