Abstract
Vacuolar proton-translocating ATPases (V-ATPases) are responsible for organelle acidification in all eukaryotic cells. The yeast V-ATPase, known to be regulated by reversible disassembly in response to glucose deprivation, was recently reported to be regulated by extracellular pH as well (Padilla-López, S., and Pearce, D. A. (2006) J. Biol. Chem. 281, 10273-10280). Consistent with those results, we find 57% higher V-ATPase activity in vacuoles isolated after cell growth at extracellular pH of 7 than after growth at pH 5 in minimal medium. Remarkably, under these conditions, the V-ATPase also becomes largely insensitive to reversible disassembly, maintaining a low vacuolar pH and high levels of V(1) subunit assembly, ATPase activity, and proton pumping during glucose deprivation. Cytosolic pH is constant under these conditions, indicating that the lack of reversible disassembly is not a response to altered cytosolic pH. We propose that when alternative mechanisms of vacuolar acidification are not available, maintaining V-ATPase activity becomes a priority, and the pump is not down-regulated in response to energy limitation. These results also suggest that integrated pH and metabolic inputs determine the final assembly state and activity of the V-ATPase.
Highlights
Under these conditions, disassembly of the V-ATPase in response to glucose deprivation is largely suppressed. These results suggest that activity of VATPases on intracellular organelles can respond to extracellular pH conditions and that retention of V-ATPase activity may become a cellular priority at high pHext, even under conditions of energy limitation
Cells were converted to spheroplasts, recovered for 20 –30 min in glucose-containing medium buffered to pH 5 or 7, and lysed, and vacuolar vesicles were isolated without further pH adjustment
The specific V-ATPase activity in vacuolar vesicles isolated from cells grown in the pH 7 medium was significantly higher than the activity in vacuoles from cells grown in pH 5 medium (Fig. 1A)
Summary
Strains and Media—Wild type yeast strain SF838-5A (MAT␣ leu112 ura ade his4-519) was used for all experiments except the cytosolic and vacuolar pH measurements in Figs. 5 and 6, which generally use BY4741 (MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0) background. (Cells were deprived of glucose for a total of 20 –30 min before measurement.) 20 l of cell suspension was added to 2 ml of 1 mM MES/TEA, pH 5 or 7, and the mixture was stirred in the cuvette at 30 °C. Cytosolic pH measurements were performed on wild type strain BY4741, as described previously (5, 30), but used a different plasmid, which expresses the pH-sensitive, ratiometric green fluorescent protein, yeast pHluorin, under control of the phosphoglycerate kinase promoter (a generous gift from Dr Rajini Rao, The Johns Hopkins University). The rate of proton pumping is represented by the initial rate (first 15 s following the addition of MgATP) of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching in the presence or absence of concanamycin A and is normalized to the amount of vacuolar protein (determined by a Lowry assay) added. Immunofluorescence imaging was done using a Zeiss Imager.Z1 fluorescent microscope, under Nomarski optics and fluorescein fluorescence optics with equal exposure time for all fluorescence samples
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