Abstract
Vacuolar proton-translocating ATPases (V-ATPases) play a central role in organelle acidification in all eukaryotic cells. To address the role of the yeast V-ATPase in vacuolar and cytosolic pH homeostasis, ratiometric pH-sensitive fluorophores specific for the vacuole or cytosol were introduced into wild-type cells and vma mutants, which lack V-ATPase subunits. Transiently glucose-deprived wild-type cells respond to glucose addition with vacuolar acidification and cytosolic alkalinization, and subsequent addition of K(+) ion increases the pH of both the vacuole and cytosol. In contrast, glucose addition results in an increase in vacuolar pH in both vma mutants and wild-type cells treated with the V-ATPase inhibitor concanamycin A. Cytosolic pH homeostasis is also significantly perturbed in the vma mutants. Even at extracellular pH 5, conditions optimal for their growth, cytosolic pH was much lower, and response to glucose was smaller in the mutants. In plasma membrane fractions from the vma mutants, activity of the plasma membrane proton pump, Pma1p, was 65-75% lower than in fractions from wild-type cells. Immunofluorescence microscopy confirmed decreased levels of plasma membrane Pma1p and increased Pma1p at the vacuole and other compartments in the mutants. Pma1p was not mislocalized in concanamycin-treated cells, but a significant reduction in cytosolic pH under all conditions was still observed. We propose that short-term, V-ATPase activity is essential for both vacuolar acidification in response to glucose metabolism and for efficient cytosolic pH homeostasis, and long-term, V-ATPases are important for stable localization of Pma1p at the plasma membrane.
Highlights
Ification and have been linked to V-ATPase activity [1, 3]
Vma Mutants Poorly Regulate Both Vacuolar and Cytosolic pH—One of the defining phenotypes of vma mutants is their sensitivity to external pH, but relatively little is known about how cytosolic and vacuolar pH are regulated in response to changes in extracellular pH, even in wild-type cells
Changes to the vacuolar pH were partially reciprocal; the vacuolar pH dropped in response to glucose (Fig. 1)
Summary
Materials—2Ј,7Ј-Bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) was purchased from Invitrogen, and the yeast pHluorin plasmid was a generous gift from Dr Rajini Rao (Johns Hopkins University). Cells were grown to log phase in YEPD, pH 5 medium, washed, and resuspended as described above. One liter of cells in log phase (absorbance 0.6 – 0.8) were harvested by centrifugation (5 min, 5000 ϫ g), resuspended in 80 ml of 0.4 M sucrose in buffer A (25 mM imidazole-HCl, pH 7, containing 1 g/ml pepstatin A, 2 g/ml chymostatin, 1 mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, and 1 g/ml leupeptin). For assessment of protein levels of Pma1p, Pep12p, and alkaline phosphatase, plasma membrane fractions were solubilized, separated by SDS-PAGE, and transferred to nitrocellulose as described [42], except that a portion of the samples to be blotted for Pma1p were solubilized in 100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% -mercaptoethanol rather than cracking buffer. Stained cells were visualized under fluorescein fluorescence optics on a Zeiss Axioplan 2 fluorescence microscope
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