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...

In yeast cells, subunit a of the vacuolar proton pump (VATPase) is encoded by two organelle-specific isoforms, VPH1 and STV1. V-ATPases containing Vph1 and Stv1 localize predominantly to the vacuole and the Golgi apparatus/endosomes, respectively. Ratiometric measurements of vacuolar pH confirm that loss of STV1 has little effect on vacuolar pH. Loss of VPH1 results in vacuolar alkalinization that is even more rapid and pronounced than in vma mutants, which lack all V-ATPase activity. Cytosolic pH responses to glucose addition in the vph1⌬ mutant are similar to those in vma mutants. The extended cytosolic acidification in these mutants arises from reduced activity of the plasma membrane proton pump, Pma1p. Pma1p is mislocalized in vma mutants but remains at the plasma membrane in both vph1⌬ and stv1⌬ mutants, suggesting multiple mechanisms for limiting Pma1 activity when organelle acidification is compromised. pH measurements in early prevacuolar compartments via a pHluorin fusion to the Golgi protein Gef1 demonstrate that pH responses of these compartments parallel cytosolic pH changes. Surprisingly, these compartments remain acidic even in the absence of V-ATPase function, possibly as a result of cytosolic acidification. These results emphasize that loss of a single subunit isoform may have effects far beyond the organelle where it resides.Vacuolar proton-translocating ATPases (V-ATPases) 3 acidify multiple organelles, including mammalian lysosomes, plant and fungal vacuoles, the Golgi apparatus, endosomes, and regulated secretory granules. Through their effects on organelle acidification, V-ATPases impact numerous cellular processes including protein sorting, macromolecular degradation, cytosolic pH and ion homeostasis, and nutrient storage and mobilization (1, 2). Consistent with these diverse roles, complete loss of V-ATPase function is lethal in most organisms. Fungi, however, can tolerate a complete loss of V-ATPase function, and Saccharomyces cerevisiae has emerged as a major model system for mechanistic studies of V-ATPases (3). Yeast mutants lacking V-ATPase activity (vma mutants) show a well defined set of Vma Ϫ growth phenotypes, including sensitivity to high extracellular pH, high Ca 2ϩ concentrations, and heavy metals (4).V-ATPases are highly conserved both at the level of individual subunit sequences and at an overall structural level. A complex of peripheral membrane subunits containing the sites of ATP hydrolysis, V 1 , is attached to an integral membrane complex, V o , containing the proton pore. In higher eukaryotes, many of the subunits are present as multiple isoforms, encoded as multiple genes and/or splice variants (5). These subunit isoforms exhibit tissue-specific expression and/or organelle-specific localization, and in some cases, impart different biochemical characteristics on V-ATPases, possibly tuning their activity to the requirements of different locales (2). Subunit a of the V o sector is present as multiple isoforms in many organisms. Humans have four different subunit a genes (...

No abstract

V-ATPases2 acidify multiple organelles in all eukaryotic cells and, through their role in organelle acidification, are linked to cellular functions, including protein sorting and degradation, ion homeostasis, and viral entry (1). In addition, certain polarized cells have high levels of apical plasma membrane V-ATPases that pump protons out of the cell; defects in specific plasma membrane V-ATPases have been linked to the human diseases distal renal tubule acidosis and osteopetrosis (1, 2). All eukaryotic V-ATPases consist of 13 or 14 different subunits assembled into a peripheral membrane sector containing the sites of ATP hydrolysis, V 1 , and a membrane sector containing the proton pore, V o (1, 3). The individual subunit sequences and the overall subunit composition of the enzyme are very similar in fungi, plants, and animals and also bear considerable resemblance to the V-or A-type ATPases found in certain eubacteria and archaebacteria (4, 5).The yeast V-ATPase has emerged as the predominant model for eukaryotic V-ATPases, in part because complete loss of V-ATPase activity is conditionally lethal in fungi but is lethal in higher eukaryotes. Loss of V-ATPase activity in Saccharomyces cerevisiae results in the Vma Ϫ phenotype, characterized by sensitivity to elevated pH and calcium concentrations, inability to grow on nonfermentable carbon sources, and sensitivity to many heavy metals, along with an array of other phenotypes (3). Although they exhibit some growth defects under all conditions, yeast vma mutant strains grow optimally at pH 5 and fail to grow at pH 7.5 (6). This pH-dependent growth phenotype has permitted biochemical characterization of multiple loss of function mutations, ranging from full deletions to point mutations in individual subunit genes. Virtually all of these studies in yeast have been carried out in haploid cells in which the sole copy of V-ATPase subunit genes was deleted or mutated.In addition to loss of function mutations, altering subunit ratios has been shown to result in a Vma Ϫ phenotype in certain cases. For example, overexpression of wild-type V 1 subunit G in yeast destabilizes V 1 subunit, E, and results in a Vma Ϫ phenotype (7). Low level overexpression of subunits C and H also results in a Vma Ϫ phenotype, whereas high level overexpression of these subunits appeared to be lethal (8). These results suggest that balanced stoichiometry of certain V-ATPase subunits is critical to the cell, and perturbing this balance can be more detrimental than a simple loss of V-ATPase function would explain.Disrupting subunit balance in the F-ATP synthase, the mitochondrial ATP synthase that is evolutionarily related to VATPases (9), can also have dramatic consequences. S. cerevisiae can tolerate loss of F-ATPase activity by relying solely on fermentative growth, so mutants lacking F-ATPase activity can grow on fermentable, but not on nonfermentable, carbon * This work was supported by National Institutes of Health Grant R01 GM50322 (to P. M. K.). The costs of publication of this article w...

Permeable spheroplasts were prepared from two strains of Saccharomyces cerevisiae by incubating with zymolyase without a permeabilizing agent. The loss of the plasma membrane barrier was confirmed by the nucleotide release, the activity of glucose 6-phosphate dehydrogenase with external substrates and by the effects on respiration of mitochondrial substrates and ADP. Mitochondrial integrity was maintained, as shown by respiration with lactate, pyruvate, glucose and ethanol, and its acceleration by ADP showed a coupled respiration. Potassium uptake into the vacuole was measured with a selective electrode and found to be taken up effectively by spheroplasts only in the presence of Mg-ATP; it was reverted by CCCP and PCP and inhibited by bafilomycin A 1 , but not by sodium vanadate or sodium azide. Potassium ions did not alter of the vacuole, followed with oxonol V, but caused vacuolar alkalinization, as followed with pyranine. The increase of vacuolar pH was non-selective and observed at 50-200 mM of several monovalent cations. Isolated vacuoles with pyranine inside showed similar changes of the internal pH in the presence of KCl. Results indicate that some strains do not require a permeabilizing agent to directly access the vacuole in spheroplasts prepared with zymolyase. The hypothesis about the existence of a K + /H + antiporter in the vacuolar membrane of S. cerevisiae is discussed.