Vacuolar and Plasma Membrane Proton Pumps Collaborate to Achieve Cytosolic pH Homeostasis in Yeast

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 wildtype 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.The importance of V-ATPases 3 for acidification of the vacuole/lysosomes, Golgi apparatus, and endosomes of eukaryotic cells is well established (1, 2). Multiple cellular processes, including secondary transport of ions and metabolites, maturation of iron transporters, endocytic and biosynthetic protein sorting, and zymogen activation depend on compartment acidification and have been linked to V-ATPase activity (1, 3). In some cells such as macrophages, V-ATPases play specialized roles that clearly include regulation of cytosolic pH (4, 5). However, although V-ATPases pump protons from the cytosol into organelles in all cells, they are not generally believed to play a major role in cytosolic pH regulation.The yeast Saccharomyces cerevisiae has emerged as a major model system for eukaryotic V-ATPases. One reason for this is that yeast mutants lacking all V-ATPase activity (vma mutants) are viable, but loss of V-ATPase activity in eukaryotes other than fungi is lethal (6 -9). Yeast vma mutants do exhibit a set of distinctive phenotypes, however, that includes the inability to grow at pH values lower than 3 or higher than 7 and sensitivity to high extracellular calcium concentrations (2). This Vma Ϫ phenotype suggests a perturbation of pH homeostasis in these cells that is not fully understood. It has been suggested that vma mutants survive at low extracellular p...

Consequences of Loss of Vph1 Protein-containing Vacuolar ATPases (V-ATPases) for Overall Cellular pH Homeostasis

“…Using ratiometric fluorescent probes for vacuolar and cytosolic pH, we previously observed striking differences in pH homeostasis between wildtype cells and vma mutants (12). As expected, the mutants had elevated vacuolar pH, but they also exhibited lower cytosolic pH than wild-type cells, particularly upon resumption of glucose metabolism.…”

“…Measurement of Vacuolar, Cytosolic, and Golgi/Endosome pH-Vacuolar pH was measured using the ratiometric fluorescent dye BCECF-AM as described (12,16). Briefly, wild-type and mutant cells were grown to early to mid-log phase in YEPD buffered to pH 5 and then loaded with BCECF-AM in the same medium.…”

“…For fluorescence measurement, the cell suspension was diluted into 1 mM MES pH 5.0 buffer, and fluorescence intensity at emission wavelength 535 nm and at excitation wavelengths 450 and 495 nm was monitored continuously in a SPEX Fluorolog 3-21 fluorometer. The fluorescence ratio for each sample was calibrated for each strain in every experiment by clamping the pH to a range of values from 5.0 to 7.5 as described (12), and the resulting calibration curve was used to convert the experimental fluorescence ratios to vacuolar pH.…”

“…Yeast cells were transformed with this plasmid, and transformants were selected on SC medium lacking uracil (SC-uracil). Measurement of cytosolic pH responses was carried out as described (12,16,17). Cells were grown to mid-log phase in SC-uracil buffered to pH 5 with 50 mM MES, washed, and deprived of glucose for ϳ30 min.…”

“…As expected, the mutants had elevated vacuolar pH, but they also exhibited lower cytosolic pH than wild-type cells, particularly upon resumption of glucose metabolism. This may arise in part from internalization of the plasma membrane proton pump, Pma1, in the mutants (12)(13)(14). However, perturbation of organelle and cytosolic pH is observed without mislocalization of Pma1 when cells are treated with the V-ATPase inhibitor concanamycin A (12), indicating an unexpected level of coordination between plasma membrane and organellar proton pumps.…”