STUDIES ON NA+ AND H+ TRANSLOCATION THROUGH THE F0 PART OF THE NA+-TRANSLOCATING F1F0 ATPASE FROM PROPIONIGENIUM-MODESTUM - DISCOVERY OF A MEMBRANE-POTENTIAL DEPENDENT STEP

The purified ATPase of Propionigenium modestum (F1F(o)) was incorporated into liposomes, and the F1 part was dissociated. The F(o)-liposomes catalyzed proton uptake in response to a potassium diffusion potential (inside negative). Proton translocation was abolished by rebinding F1 to the F(o)-liposomes or after incubation with the c-subunit-specific inhibitor dicyclohexylcarbodiimide (DCCD). Proton uptake was also sensitive to the presence of external Na+ or Li+ ions and was completely abolished at 2 mM NaCl or 150 mM LiCl, respectively. However, the same concentrations of these salts in the internal volume of the F(o)-liposomes were without effect, suggesting that the cation binding site is not accessible from both sides of the membrane simultaneously. An open channel-type of transport through F(o) from P. modestum is therefore excluded. The F(o)-liposomes also catalyzed Na+ influx or efflux in response to a K+ diffusion potential that was negative on the inside or outside, respectively. These Na+ fluxes could not be created, however, by DELTApNa+ of about 60-180 mV. The initial rate of Na+ uptake depended strongly on the size of the membrane potential with no significant conductivity below -40 mV, followed by a proportional increase up to about -115 mV. In the absence of a membrane potential, the F(o)-liposomes catalyzed Na-22+ counterflow against a 28-fold concentration gradient. Uptake of Na-22+ into F(o)-liposomes against DELTApNa+ (counterflow) was completely prevented by imposing an inside-positive potassium diffusion potential of 90 mV. The catalysis of Na-22+ counterflow by F(o) from P. modestum is a clear indication of a carrier (transporter)-type mechanism and excludes a channel mechanism. On the basis of our data, we propose a model of Na+ translocation by F(o) that involves a minimum of four steps: (1) binding of Na+ from a defined surface of the membrane; (2) translocation of the binary complex to the outer surface; (3) release of Na+; (4) return of the unloaded carrier to the original surface. The first three steps that are sufficient for Na+ counterflow are assumed to be independent of the membrane potential, while the fourth step that is specifically required for Na+ influx or Na+ efflux seems to represent the membrane potential dependent reaction step.