Studies on Na+ and H+ translocation through the Fo part of the Na(+)-translocating F1Fo ATPase from Propionigenium modestum: discovery of a membrane potential dependent step.
The purified ATPase of Propionigenium modestum (F1Fo) was incorporated into liposomes, and the F1 part was dissociated. The Fo-liposomes catalyzed proton uptake in response to a potassium diffusion potential (inside negative). Proton translocation was abolished by rebinding F1 to the Fo-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 Fo-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 Fo from P. modestum is therefore excluded. The Fo-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 delta pNa+ 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 Fo-liposomes catalyzed 22Na+ counterflow against a 28-fold concentration gradient. Uptake of 22Na+ into Fo-liposomes against delta pNa+ (counterflow) was completely prevented by imposing an inside-positive potassium diffusion potential of 90 mV. The catalysis of 22Na+ counterflow by Fo from P. modestum is a clear indication of a carrier (transporter)-type mechanism and excludes a channel mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Kluge
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C
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Dimroth
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P
D001421:Bacteria, Anaerobic / Q000201:enzymology / Q000378:metabolism*; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism*; D008081:Liposomes; D008564:Membrane Potentials; D011188:Potassium / Q000378:metabolism; D011510:Proteolipids; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*
D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; D006859:Hydrogen; D012964:Sodium; D006180:Proton-Translocating ATPases; D011188:Potassium