FIGURE 4.

Addition of 4% glucose to glucose-deprived pfk2Δ cells rescues V-ATPase reassembly and vacuolar acidification. A, V₁V_o reassembly is glucose-dose dependent. The cells were cultured in 2% glucose, converted to spheroplasts, and biosynthetically radiolabeled with Tran³⁵S. Chases were conducted in the presence of 2% glucose (+ G) for 20 min, after glucose depletion (− G) for 10 min, and after readdition of varied concentrations of glucose (0.1–4% glucose) (± G) for an additional 10 min. The V-ATPase complexes were immunoprecipitated with monoclonal antibodies to V₁ subunit B and V_o subunit a and separated by SDS-PAGE, and then the proportion of total V_o assembled in V₁V_o complexes was estimated. Data were analyzed in a Fuji scanner (FLA-5100) with Multi Gauge and GraphPad Prism 5 software. Results are presented as average from three independent experiments. Error bars are standard deviation. Statistically significant differences (*, p < 0.05; ***, p < 0.001) as compared with steady state (2% glucose) were determined by two-tailed unpaired t test. Error bars show mean ± S.D. B, readdition 4% glucose restores pfk2Δ vacuolar acidification after reassembly. Cells were stained with BCECF-AM and deprived of glucose for 10 min in 1 mm HEPE-MES (pH 5.0) buffer, and glucose was readded to a final concentration of 2% or 4% (arrow). The ratio of fluorescent emission (535 nm) excited at 490 nm and 450 nm was calculated, and the vacuolar pH was measured using calibration curves. Data represent three independent experiments. Error bars are standard deviation.