Partial assembly of the yeast vacuolar H(+)-ATPase in mutants lacking one subunit of the enzyme.
Partial assembly of the peripheral and integral membrane sectors of the yeast vacuolar H(+)-ATPase has been detected in mutants lacking one subunit of the enzyme. Assembled complexes of the vacuolar H(+)-ATPase could be immunoprecipitated from biosynthetically labeled wild-type cells using monoclonal antibodies specific for the 69- and 60-kDa subunits of the enzyme, and assembled membrane (V0) sectors could be immunoprecipitated using a monoclonal antibody against the 100-kDa subunits. Parallel immunoprecipitations from mutant cells lacking one subunit of the vacuolar H(+)-ATPase revealed different degrees of assembly depending on the subunit that was missing. Partially assembled complexes of the peripheral subunits could also be detected in a soluble, cytoplasmic fraction from wild-type and mutant cells following glycerol gradient fractionation. The results indicate that the peripheral (V1) sector and integral membrane (V0) sectors of the yeast vacuolar H(+)-ATPase can assemble independently. The 69-, 60-, and 27-kDa subunits all appear to be necessary for any assembly of the V1 sector to occur, but these subunits and the 32-kDa subunit can assemble into a complex in the absence of the 42-kDa peripheral subunit. The implications of the results for the structure and assembly of the yeast vacuolar H(+)-ATPase are discussed.
Doherty
R D
RD
Department of Chemistry, College of William and Mary, Williamsburg, Virginia 23185.
Kane
P M
PM
D002498:Centrifugation; D009154:Mutation; D011233:Precipitin Tests; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D012995:Solubility; D014617:Vacuoles / Q000201:enzymology*; D015003:Yeasts / Q000201:enzymology* / Q000235:genetics
D006180:Proton-Translocating ATPases