Precursor-specific requirements for SecA, SecB, and delta muH+ during protein export of Escherichia coli.

We compare translocation into inside-out plasma membrane vesicles (INV) of the in vitro synthesized outer membrane proteins LamB and OmpA and the periplasmic protein Skp of Escherichia coli and demonstrate a precursor-specific dependence on the export factors SecA, SecB, and the proton-motive force (delta mu H+). A partial reduction in soluble SecA caused a 50% decrease in translocation of preLamB. In contrast, removal of INV-bound SecA by urea extraction was required to see a decrease in translocation of preOmpA and preSkp, with 8% of preSkp still being translocated into urea-treated INV. Translocation of the three precursors into INV showed a corresponding differential sensitivity toward dissipation of delta mu H+ following removal of the F1-ATPase from the INV. While depletion of both F1 and SecA or simply lowering of the reaction temperature resulted in an inhibition of complete transmembrane translocation, it interfered less severely with signal sequence cleavage, indicating the formation of translocation intermediates under these conditions. The relative amounts of intermediate obtained were also different for the three preproteins correlating a low requirement for SecA and delta mu H+ with a facilitated initiation of translocation. Whereas preSkp was translocated independently of SecB, preLamB was not even targeted to the INV in its absence. Functional targeting of preOmpA required the presence of SecB during incubation of the precursor with INV and not during its synthesis. SecB, exogenously added during the period of synthesis, did not prevent the formation of translocation-incompetent preLamB. The latter results are consistent with an important targeting function of SecB, which so far has mostly been described as a molecular chaperone. The findings are discussed with respect to current models of bacterial protein export usually derived from the analysis of a single precursor.