PRECURSOR-SPECIFIC REQUIREMENTS FOR SECA, SECB, AND DELTA-MU(H-COLI()DURING PROTEIN EXPORT OF ESCHERICHIA)

We compare translocation into inside-out plasma membrane vesicles (INV ) of the in vitro synthesized outer membrane proteins LamB and OmpA an d the periplasmic protein Skp of Escherichia coli and demonstrate a pr ecursor-specific dependence on the export factors SecA, SecB, and the proton-motive force (Delta mu(H+)). A partial reduction in soluble Sec A caused a 50% decrease in translocation of preLamB. In contrast, remo val of INV-bound SecA by urea extraction was required to see a decreas e in translocation of preOmpA and preSkp, with 8% of preSkp still bein g translocated into ureatreated INV. Translocation of the three precur sors into INV showed a corresponding differential sensitivity toward d issipation of Delta mu(H+) following removal of the F-1- ATPase from t he INV. While depletion of both F-1 and SecA or simply lowering of the reaction temperature resulted in an inhibition of complete transmembr ane translocation, it interfered less severely with signal sequence cl eavage, indicating the formation of translocation intermediates under these conditions. The relative amounts of intermediate obtained were a lso different for the three preproteins correlating a low requirement for SecA and Delta mu(H+) with a facilitated initiation of translocati on. 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 precurs or with INV and not during its synthesis. SecB, exogenously added duri ng the period of synthesis, did not prevent the formation of transloca tion-incompetent preLamB. The latter results are consistent with an im portant targeting function of SecB, which so far has mostly been descr ibed as a molecular chaperone. The findings are discussed with respect to current models of bacterial protein export usually derived from th e analysis of a single precursor.