H+-pumping ATPase has little stimulatory effect on in vitro translocation of a model protein into Vibrio alginolyticus inside-out membrane vesicles.
We studied the effect of a H+ electrochemical potential generated by F1F0 ATPase on in vitro translocation of a model protein into Vibrio alginolyticus inside-out membrane vesicles. The F1F0 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 F1F0 ATPase was completely abolished by 30 microM N,N'-dicyclohexylcarbodiimide (DCCD) or 5 microM carbonylcyanide m-chlorophenylhydrazone (CCCP) at 30 degrees C. The treatment of membrane vesicles with 30 microM DCCD at 30 degrees C 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 F1F0 ATPase.
Kim
Y J
YJ
Department of Microbiology, Changwon National University, Korea.
Rhee
S K
SK
D000251:Adenosine Triphosphatases / Q000502:physiology; D000255:Adenosine Triphosphate / Q000502:physiology; D001425:Bacterial Outer Membrane Proteins / Q000502:physiology; D001426:Bacterial Proteins / Q000502:physiology; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D008074:Lipoproteins / Q000502:physiology; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism; D017494:Proton Pumps / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D014733:Vibrio / Q000201:enzymology* / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; C066293:OmpF-Lpp chimeric protein; D017494:Proton Pumps; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D016660:NAD(P)H Dehydrogenase (Quinone); D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D000081416:SecA Proteins