Electrogenic proton translocation coupled to ATP hydrolysis by the plasma membrane Mg2+-dependent ATPase of yeast in reconstituted proteoliposomes.
The purified plasma membrane Mg2+-dependent ATPase of the yeast Schizosaccharomyces pombe was incorporated in liposomes using a cholate-dialysis method. The ATPase activity of the incorporated enzyme was stimulated by the H+-conducting agent carbonyl cyanide m-chlorophenylhydrazone and to a much lower extent of the K+-ionophore valinomycin in the presence of potassium. The K+/H+ exchanger nigericin (plus K+) did not stimulate ATPase activity, whereas the combined addition of both nigericin plus valinomycin was strongly stimulatory. The incorporated ATPase activity was controlled by the generated electrochemical H+ gradient since only conditions which collapse both the membrane potential and the pH gradient stimulated fully the ATPase activity of the incorporated enzyme. Direct measurement of proton movement with a pH glass electrode showed a fast and transient proton entry into the proteoliposomes upon addition of MgATP in the presence of the charge-compensating cation K+ (plus valinomycin). Moreover, during the steady state ATP hydrolysis, a H+ entry was again observed when the membrane potential was collapsed upon addition of valinomycin in the presence of K+. These data demonstrate that the plasma membrane ATPase of yeast cells is involved in electrogenic H+ translocation coupled to ATP hydrolysis since the purified enzyme incorporated in the liposomes is virtually free of mitochondrial F1F0-ATPase contaminant.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001203:Ascomycota / Q000201:enzymology*; D017301:Ca(2+) Mg(2+)-ATPase; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D004579:Electron Transport; D007700:Kinetics; D008854:Microscopy, Electron; D009550:Nigericin / Q000494:pharmacology; D009840:Oligomycins / Q000494:pharmacology; D010086:Oxidative Phosphorylation Coupling Factors / Q000378:metabolism; D011510:Proteolipids; D006180:Proton-Translocating ATPases; D012568:Schizosaccharomyces / Q000201:enzymology*; D013367:Submitochondrial Particles / Q000201:enzymology; D014634:Valinomycin / Q000494:pharmacology
D009840:Oligomycins; D010086:Oxidative Phosphorylation Coupling Factors; D011510:Proteolipids; C020485:proteoliposomes; D014634:Valinomycin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; D009550:Nigericin