Biochemical support for the V-ATPase rotary mechanism: antibody against HA-tagged Vma7p or Vma16p but not Vma10p inhibits activity.
V-ATPase null mutants in yeast have a distinct, conditionally lethal phenotype that can be obtained through disruption of any one of its subunits. This enables supplementation of this mutant with the relevant subunit tagged with an epitope against which an antibody is available. In this system, the effect of antibody on the activity of the enzyme can be analyzed. Towards this end we used HA to tag subunits Vma7p, Vma10p and Vma16p, which are assumed to represent, respectively, the shaft, stator and turbine of the enzyme, and used them to supplement the corresponding yeast V-ATPase null mutants. The anti-HA epitope antibody inhibited both the ATP-dependent proton uptake and the ATPase activities of the Vma16p-HA and Vma7p-HA containing complexes, in intact vacuoles and in the detergent-solubilized enzyme. Neither of these activities was inhibited by the antibody in Vma10p-HA containing enzyme. These results support the function of Vma10p as part of the stator, while the other tagged subunits are part of the rotor apparatus. The HA-tag was attached to the N terminus of Vma16p; thus the antibody inhibition points to its accessibility outside the vacuolar membrane. This assumption is supported by the supplementation of the yeast mutant by the homologues of Vma16p isolated from Arabidopsis thaliana and lemon fruit c-DNA. Contrary to yeast, which has five predicted helices, the plant subunit Vma16p has only four. Our results confirm a recent report that only four of the yeast Vma16p complexes are actually transmembrane helices.
Aviezer-Hagai
Keren
K
Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Padler-Karavani
Vered
V
Nelson
Nathan
N
D000911:Antibodies, Monoclonal / Q000378:metabolism* / Q000494:pharmacology; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D015151:Immunoblotting; D008954:Models, Biological; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018076:DNA, Complementary; D029701:Saccharomyces cerevisiae Proteins; C089600:VMA7 protein, S cerevisiae; C485339:VMA10 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases