Wild-type and mutant vacuolar membranes support pH-dependent reassembly of the yeast vacuolar H+-ATPase in vitro.
Treatment of the yeast vacuolar proton-translocating ATPase (H+-ATPase) with 300 mM KI in the presence of 5 mM MgATP results in a 90% inhibition of ATPase activity accompanied by removal of at least five of the peripheral subunits of the enzyme from the membrane. Functional reassembly of the enzyme, as indicated by reattachment of the peripheral subunits and a partial (30-70%) recovery of ATPase activity, could be achieved by dialysis of the stripped wild-type membranes to remove the KI and MgATP, but proved to be strongly pH-dependent, with optimal reassembly and recovery of activity occurring after dialysis at pH 5.5. Vacuolar membranes isolated from vma2Delta mutants, which lack one of the peripheral subunits of the enzyme, do not contain any of the peripheral subunits but are shown to contain assembled membrane (Vo) complexes. The vma2Delta mutant vacuoles are demonstrated to be competent for attachment of KI-stripped peripheral subunits and reactivation of ATPase activity. The results indicate that previously assembled Vo complexes are capable of inducing assembly of the peripheral subunits, both with each other and with the membrane subunits, and of activating the ATPase activity that resides in the peripheral subunits in a pH-dependent manner.
Parra
K J
KJ
Department of Biochemistry and Molecular Biology, State University of New York Health Science Center at Syracuse, Syracuse, New York 13210, USA.
Kane
P M
PM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004793:Enzyme Reactivators / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D009154:Mutation; D011193:Potassium Iodide / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014617:Vacuoles / Q000201:enzymology*
D004791:Enzyme Inhibitors; D004793:Enzyme Reactivators; D011193:Potassium Iodide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases