Co-assembly of SecYEG and SecA fully restores the properties of the native translocon.

In all cells a highly conserved channel transports proteins across membranes. In E.coli , that channel is SecYEG. Many investigations of this protein complex have used purified SecYEG reconstituted into proteoliposomes. How faithfully do activities of reconstituted systems reflect the properties of SecYEG in the native membrane environment? We investigated by comparing three in vitro systems: the native membrane environment of inner membrane vesicles and two methods of reconstitution. One method was the widely used reconstitution of SecYEG alone into lipid bilayers. The other was our method of co-assembly of SecYEG with SecA, the ATPase of the translocase. For nine different precursor species we assessed parameters that characterize translocation: maximal amplitude of competent precursor translocated, coupling of energy to transfer, and apparent rate constant. In addition we investigated translocation in the presence and absence of chaperone SecB. For all nine precursors, SecYEG co-assembled with SecA was as active as SecYEG in native membrane for each of the parameters studied. Effects of SecB on transport of precursors faithfully mimicked observations made in vivo From investigation of the nine different precursors, we conclude that the apparent rate constant, which reflects the step that limits the rate of translocation, is dependent on interactions with the translocon of portions of the precursors other than the leader. In addition, in some cases the rate-limiting step is altered by the presence of SecB. Candidates for the rate-limiting step that are consistent with our data are discussed.