|
We studied the effect of a H^(+) electrochemical potential generated by F_(1)F_(0)ATPase on in vitro translocation of a model protein into Vibrio alginolyticus inside-out membrane vesicles. The F_(1)F_(0)ATPase of V. alginolyticus catalyzed the pumping of H^(+) coupled to ATP hydrolysis as measured in fluorescence quenching experiments. consequently, this enzyme leads to the generation of a H^(+) electrochemical potential. The H^(+) electrochemical potential generated by F_(1)F_(0) ATPase was completely abolished by 30μM N,N´-dicyclohexylcarbodiimide(DCCD) or 5μM carbonylcyanide m-chlorophenylhydrazone (CCCP) at 30℃. The treatment of membrane vesicles with 30μM DCCD at 30℃ had little influence on the translocation activity of uncleavable OmpF-Lpp, a model secretory protein, as compared to the intact membrane vesicles. On the other hand, the NADH:quinone oxidoreductase of V. alginolyticus is known to be a Na^(+) pump that leads to generation of a Na^(+) electrochemical potential. This Na^(+) electrochemical potential stimulates protein translocation into inside-out membrane vesicles prepared from V. alginolyticus in the presence of Escherichia coli SecA [Tokuda, H., Kim, Y. J., and Mizushima, S. (1990) FEBS Lett. 264, 10-12] From these results, it is evident that the stimulatory effect of the Na^(+) electrochemical potential on protein translocation in V. alginolyticus is not affected by the H^(+) electrochemical potential influence of F_(1)F_(0) ATPase.
|