FIGURE 2.
Disassembly of the V-ATPase in response to glucose deprivation is suppressed at high extracellular pH. A, vacuolar pH was measured using the ratiometric fluorescent dye BCECF-AM loaded into the vacuoles of log phase cells grown in SC buffered to pH 5 or 7 with 50 mm MES and maintained at the same pH during the measurement. Cells were deprived of glucose for 20–30 min before the initial pH measurement (−), and then glucose was added to a final concentration of 50 mm, and the vacuolar pH was measured again 5 min after the glucose addition (+), as described under “Experimental Procedures.” Fluorescence measurements were performed at the same extracellular pH as the growth medium. Vacuolar pH is plotted as mean ± S.E. (error bars) for n = 4 independent measurements. B and C, cells were grown as described in A, converted to spheroplasts, and then incubated for 30 min in SC containing 1.2 m sorbitol and buffered to pH 5 or 7, either in the presence of 50 mm glucose (+) or in the absence of added glucose (−). After this incubation, spheroplasts were lysed, and vacuolar vesicles were isolated as described under “Experimental Procedures.” V-ATPase activity (B) and proton pumping (C) were assayed as described in the legend to Fig. 1 and under “Experimental Procedures.” RFU, relative fluorescence units. D, immunoblot of vacuolar vesicles prepared as described in B and C. Vesicles were solubilized, separated by SDS-PAGE, and transferred to nitrocellulose as described under “Experimental Procedures.” For detection of the V1 A and B subunits, 1 μg of vacuolar vesicle protein was loaded on the gel, whereas for detection of V0 a and V1 C, 4 μg was loaded from each preparation. The blots were probed with subunit-specific mouse monoclonal antibodies (see “Experimental Procedures”), followed by alkaline phosphatase-conjugated goat anti-mouse antibody and colorimetric development.