Translocation of ProOmpA possessing an intramolecular disulfide bridge into membrane vesicles of Escherichia coli. Effect of membrane energization.

In the absence of delta mu H+, the in vitro translocation of proOmpA resulted in the stable accumulation of a possible translocation intermediate in addition to a transiently accumulating one. The stable intermediate was detected on a polyacrylamide gel as two proteinase K-resistant bands corresponding to a molecular weight of about 28,000. The appearance of the bands was appreciably enhanced when proOmpA was oxidized with ferricyanide. No mature OmpA appeared. When proOmpA reduced with dithiothreitol was used, on the other hand, the bands did not appear at all. Upon the replacement of Cys302 of OmpA with Gly, the intermediate accumulation was abolished. The proOmpA treated with dithiothreitol was labeled with N-[3H]-ethylmaleimide, whereas that treated with ferricyanide was not. The ferricyanide-treated proOmpA was translocated into membrane vesicles in the presence of delta mu H+. The mature OmpA thus translocated and processed was not labeled with N-[3H]ethylmaleimide. It is concluded that proOmpA possessing the Cys290-Cys302 disulfide bridge can be translocated without cleavage of the bridge, when delta mu H+ is imposed. The accumulation of the disulfide bridge-containing intermediate was ATP-dependent, whereas its conversion to the translocated mature form was not blocked in the presence of adenosine 5'-(beta, gamma-imino)triphosphate. It is concluded that the early and late stages of the translocation reaction require ATP and delta mu H+ differently.