Fig. 3. The effect of the anti-HA antibody on ATP-dependent proton uptake activity in yeast expressing the HA-tagged subunits. Vacuoles were isolated from the various yeast strains and preincubated for 30 min at room temperature in 1 ml of the proton-uptake reaction mixture (see Materials and methods) in the presence or absence of 1, 2 or 5 µl of 1:1 diluted monoclonal anti-HA antibody (HA). The ATP-dependent proton uptake activity was measured by following the decrease in absorption difference at 490–540 nm of Acridine Orange. Where indicated, 1 µmol of MgATP or 1 nmol of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) was added to the reaction mixture. WT, wild type.