Fig. 4. Acridine Orange uptake into isolated vacuoles from stv1 and vph1 strains shows diminished activity. Yeast vacuoles were isolated by Ficoll gradient centrifugation and adjusted to a protein concentration of 2 mg ml^-1 (Uchida et al., 1985). ATP-dependent proton uptake was measured by following Acridine Orange absorption changes (A) at 491-540 nm. Vacuoles containing 10 µg of protein were assayed in each sample. Where indicated by an arrow, 10 µl of 0.1 mol l^-1 MgATP or 1 µl of 1 mmol l^-1 carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) was added.