Diploids heterozygous for a vma13Delta mutation in Saccharomyces cerevisiae highlight the importance of V-ATPase subunit balance in supporting vacuolar acidification and silencing cytosolic V1-ATPase activity

The V-ATPase H subunit (encoded by the VMA13 gene) activates ATP-driven proton pumping in intact V-ATPase complexes and inhibits MgATPase activity in cytosolic V1 sectors (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767). Yeast diploids heterozygous for a vma13Delta mutation show the pH- and calcium-dependent conditional lethality characteristic of mutants lacking V-ATPase activity, although they still contain one wild-type copy of VMA13. Vacuolar vesicles from this strain have approximately 50% of the ATPase activity of those from a wild-type diploid but do not support formation of a proton gradient. Compound heterozygotes with a second heterozygous deletion in another V1 subunit gene exhibit improved growth, vacuolar acidification, and ATP-driven proton transport in vacuolar vesicles. In contrast, compound heterozygotes with a second deletion in a Vo subunit grow even more poorly than the vma13Delta heterozygote, have very little vacuolar acidification, and have very low levels of V-ATPase subunits in isolated vacuoles. In addition, cytosolic V1 sectors from this strain and from the strain containing only the heterozygous vma13Delta mutation have elevated MgATPase activity. The results suggest that balancing levels of subunit H with those of other V-ATPase subunits is critical, both for allowing organelle acidification and for preventing unproductive hydrolysis of cytosolic ATP.     

The vacuolar V1/V0-ATPase is involved in the release of the HOPS subunit Vps41 from vacuoles, vacuole fragmentation and fusion

At yeast vacuoles, phosphorylation of the HOPS subunit Vps41 depends on the Yck3 kinase. In a screen for mutants that mimic the yck3Delta phenotype, in which Vps41 accumulates in vacuolar dots, we observed that mutants in the V0-part of the V0/V1-ATPase, in particular in vma16Delta, also accumulate Vps41. This accumulation is not due to a phosphorylation defect, but to reduced release of Vps41 from vma16Delta vacuoles. One reason could be a connection to vacuole fission, which is blocked in V-ATPase mutants. Vacuole fusion is not impaired between vacuoles lacking the V0-subunits Vma16 or Vma6 and wild-type vacuoles, whereas fusion between mutant vacuoles is reduced. Our data suggest a connection between vacuole biogenesis and membrane fusion.     

Cytosolic Ca2+ homeostasis is a constitutive function of the V-ATPase in Saccharomyces cerevisiae

The vacuole is the major site of intracellular Ca(2+) storage in yeast and functions to maintain cytosolic Ca(2+) levels within a narrow physiological range via a Ca(2+) pump (Pmc1p) and a H(+)/Ca(2+) antiporter (Vcx1p) driven by the vacuolar H(+)-ATPase (V-ATPase). We examined the function of the V-ATPase in cytosolic Ca(2+) homeostasis by comparing responses to a brief Ca(2+) challenge of a V-ATPase mutant (vma2Delta) and wild-type cells treated with the V-ATPase inhibitor concanamycin A. The kinetics of the Ca(2+) response were determined using transgenic aequorin as an in vivo cytosolic Ca(2+) reporter system. In wild-type cells, the V-ATPase-driven Vcx1p was chiefly responsible for restoring cytosolic Ca(2+) concentrations after a brief pulse. In cells lacking V-ATPase activity, brief exposure to elevated Ca(2+) compromised viability, even when there was little change in the final cytosolic Ca(2+) concentration. vma2Delta cells were more efficient at restoring cytosolic [Ca(2+)] after a pulse than concanamycin-treated wild-type cells, suggesting long term loss of V-ATPase triggers compensatory mechanisms. This compensation was dependent on calcineurin, and was mediated primarily by Pmc1p.     

Vacuolar H+-ATPase is involved in preventing heavy metal-induced oxidative stress in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, vacuolar H⁺-ATPase (V-ATPase) involved in the regulation of intracellular pH homeostasis has been shown to be important for tolerances to cadmium, cobalt and nickel. However, the molecular mechanism underlying the protective role of V-ATPase against these metals remains unclear. In this study, we show that cadmium, cobalt and nickel disturbed intracellular pH balance by triggering cytosolic acidification and vacuolar alkalinization, likely via their membrane permeabilizing effects. Since V-ATPase plays a crucial role in pumping excessive cytosolic protons into the vacuole, the metal-sensitive phenotypes of the Δvma2 and Δvma3 mutants lacking V-ATPase activity were supposed to result from highly acidified cytosol. However, we found that the metal-sensitive phenotypes of these mutants were caused by increased production of reactive oxygen species, likely as a result of decreased expression and activities of manganese superoxide dismutase and catalase. In addition, the loss of V-ATPase function led to aberrant vacuolar morphology and defective endocytic trafficking. Furthermore, the sensitivities of the Δvma mutants to other chemical compounds (i.e. acetic acid, H₂O₂, menadione, tunicamycin and cycloheximide) were a consequence of increased endogenous oxidative stress. These findings, therefore, suggest the important role of V-ATPase in preventing endogenous oxidative stress induced by metals and other chemical compounds.     

Structural and functional separation of the N- and C-terminal domains of the yeast V-ATPase subunit H

The H subunit of the yeast V-ATPase is an extended structure with two relatively independent domains, an N-terminal domain consisting of amino acids 1-348 and a C-terminal domain consisting of amino acids 352-478. We have expressed these two domains independently and together in a yeast strain lacking the H subunit (vma13Delta mutant). The N-terminal domain partially complements the growth defects of the mutant and supports approximately 25% of the wild-type Mg(2+)-dependent ATPase activity in isolated vacuolar vesicles, but surprisingly, this activity is both largely concanamycin-insensitive and uncoupled from proton transport. The C-terminal domain does not complement the growth defects, and supports no ATP hydrolysis or proton transport, even though it is recruited to the vacuolar membrane. Expression of both domains in a vma13Delta strain gives better complementation than either fragment alone and results in higher concanamycin-sensitive ATPase activity and ATP-driven proton pumping than the N-terminal domain alone. Thus, the two domains make complementary contributions to structural and functional coupling of the peripheral V(1) and membrane V(o) sectors of the V-ATPase, but this coupling does not require that they be joined covalently. The N-terminal domain alone is sufficient for activation of ATP hydrolysis in V(1), but the C-terminal domain is essential for proper communication between the V(1) and V(o) sectors.     

Consequences of loss of Vph1 protein-containing vacuolar ATPases (V-ATPases) for overall cellular pH homeostasis

In yeast cells, subunit a of the vacuolar proton pump (V-ATPase) 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 vph1Δ 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 vph1Δ and stv1Δ 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 and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast

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.     

The H subunit (Vma13p) of the yeast V-ATPase inhibits the ATPase activity of cytosolic V1 complexes

V-ATPases are composed of a peripheral complex containing the ATP-binding sites, the V(1) sector, attached to a membrane complex containing the proton pore, the V(o) sector. In vivo, free, inactive V(1) and V(o) sectors exist in dynamic equilibrium with fully assembled, active V(1) V(o) complexes, and this equilibrium can be perturbed by changes in carbon source. Free V(1) complexes were isolated from the cytosol of wild-type yeast cells and mutant strains lacking V(o) subunit c (Vma3p) or V(1) subunit H (Vma13p). V(1) complexes from wild-type or vma3Delta mutant cells were very similar, and contained all previously identified yeast V(1) subunits except subunit C (Vma5p). These V(1) complexes hydrolyzed CaATP but not MgATP, and CaATP hydrolysis rapidly decelerated with time. V(1) complexes from vma13Delta cells contained all V(1) subunits except C and H, and had markedly different catalytic properties. The initial rate of CaATP hydrolysis was maintained for much longer. The complexes also hydrolyzed MgATP, but showed a rapid deceleration in hydrolysis. These results indicate that the H subunit plays an important role in silencing unproductive ATP hydrolysis by cytosolic V(1) complexes, but suggest that other mechanisms, such as product inhibition, may also play a role in silencing in vivo.     

The Histoplasma capsulatum vacuolar ATPase is required for iron homeostasis, intracellular replication in macrophages and virulence in a murine model of histoplasmosis

Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (Mphi). To identify specific genes required for intracellular survival, we utilized Agrobacterium tumefaciens-mediated mutagenesis, and screened for H. capsulatum insertional mutants that were unable to survive in human Mphi. One colony was identified that had an insertion within VMA1, the catalytic subunit A of the vacuolar ATPase (V-ATPase). The vma1 mutant (vma1::HPH) grew normally on iron-replete medium, but not on iron-deficient media. On iron-deficient medium, the growth of the vma1 mutant was restored in the presence of wild-type (WT) H. capsulatum yeasts, or the hydroxamate siderophore, rhodotorulic acid. However, the inability to replicate within Mphi was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28 degrees C. Complementation of the mutant (vma/VMA1) restored its ability to replicate in Mphi, grow on iron-poor medium and grow as a mold at 28 degrees C. The vma1::HPH mutant was avirulent in a mouse model of histoplasmosis, whereas the vma1/VMA1 strain was as pathogenic as WT yeasts. These studies demonstrate the importance of V-ATPase function in the pathogenicity of H. capsulatum, in iron homeostasis and in fungal dimorphism.     

PKR1 encodes an assembly factor for the yeast V-type ATPase

Deletion of the yeast gene PKR1 (YMR123W) results in an inability to grow on iron-limited medium. Pkr1p is localized to the membrane of the endoplasmic reticulum. Cells lacking Pkr1p show reduced levels of the V-ATPase subunit Vph1p due to increased turnover of the protein in mutant cells. Reduced levels of the V-ATPase lead to defective copper loading of Fet3p, a component of the high affinity iron transport system. Levels of Vph1p in cells lacking Pkr1p are similar to cells unable to assemble a functional V-ATPase due to lack of a V0 subunit or an endoplasmic reticulum (ER) assembly factor. However, unlike yeast mutants lacking a V0 subunit or a V-ATPase assembly factor, low levels of Vph1p present in cells lacking Pkr1p are assembled into a V-ATPase complex, which exits the ER and is present on the vacuolar membrane. The V-ATPase assembled in the absence of Pkr1p is fully functional because the mutant cells are able to weakly acidify their vacuoles. Finally, overexpression of the V-ATPase assembly factor Vma21p suppresses the growth and acidification defects of pkr1Delta cells. Our data indicate that Pkr1p functions together with the other V-ATPase assembly factors in the ER to efficiently assemble the V-ATPase membrane sector.     

Vma21p is a yeast membrane protein retained in the endoplasmic reticulum by a di-lysine motif and is required for the assembly of the vacuolar H(+)-ATPase complex.

The yeast vacuolar proton-translocating ATPase (V-ATPase) is a multisubunit complex comprised of peripheral membrane subunits involved in ATP hydrolysis and integral membrane subunits involved in proton pumping. The yeast vma21 mutant was isolated from a screen to identify mutants defective in V-ATPase function. vma21 mutants fail to assemble the V-ATPase complex onto the vacuolar membrane: peripheral subunits accumulate in the cytosol and the 100-kDa integral membrane subunit is rapidly degraded. The product of the VMA21 gene (Vma21p) is an 8.5-kDa integral membrane protein that is not a subunit of the purified V-ATPase complex but instead resides in the endoplasmic reticulum. Vma21p contains a dilysine motif at the carboxy terminus, and mutation of these lysine residues abolishes retention in the endoplasmic reticulum and results in delivery of Vma21p to the vacuole, the default compartment for yeast membrane proteins. Our findings suggest that Vma21p is required for assembly of the integral membrane sector of the V-ATPase in the endoplasmic reticulum and that the unassembled 100-kDa integral membrane subunit present in delta vma21 cells is rapidly degraded by nonvacuolar proteases.     

Mutations in the Yeast KEX2 Gene Cause a Vma−-Like Phenotype: a Possible Role for the Kex2 Endoprotease in Vacuolar Acidification

Mutants of Saccharomyces cerevisiae that lack vacuolar proton-translocating ATPase (V-ATPase) activity show a well-defined set of Vma⁻ (stands for vacuolar membrane ATPase activity) phenotypes that include pH-conditional growth, increased calcium sensitivity, and the inability to grow on nonfermentable carbon sources. By screening based on these phenotypes and the inability of vma mutants to accumulate the lysosomotropic dye quinacrine in their vacuoles, five new vma complementation groups (vma41 to vma45) were identified. The VMA45 gene was cloned by complementation of the pH-conditional growth of the vma45-1 mutant strain and shown to be allelic to the previously characterized KEX2 gene, which encodes a serine endoprotease localized to the late Golgi compartment. Both vma45-1 mutants and kex2 null mutants exhibit the full range of Vma⁻ growth phenotypes and show no vacuolar accumulation of quinacrine, indicating loss of vacuolar acidification in vivo. However, immunoprecipitation of the V-ATPase from both strains under nondenaturing conditions revealed no defect in assembly of the enzyme, vacuolar vesicles isolated from a kex2 null mutant showed levels of V-ATPase activity and proton pumping comparable to those of wild-type cells, and the V-ATPase complex purified from kex2 null mutants was structurally indistinguishable from that of wild-type cells. The results suggest that kex2 mutations exert an inhibitory effect on the V-ATPase in the intact cell but that the ATPase is present in the mutant strains in a fully assembled state, potentially capable of full enzymatic activity. This is the first time a mutation of this type has been identified.     

Degradation of the gluconeogenic enzyme fructose-1, 6-bisphosphatase is dependent on the vacuolar ATPase

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced during glucose starvation. After the addition of glucose, inactivated FBPase is selectively targeted to Vid (vacuolar import and degradation) vesicles and then to the vacuole for degradation. To identify proteins involved in this pathway, we screened various libraries for mutants that failed to degrade FBPase. Via these approaches, subunits of the vacuolar- H+ -ATPase (V-ATPase) have been identified repeatedly. The V-ATPase has established roles in endocytosis, sorting of carboxypeptidase Y and homotypic vacuole fusion. Here, we show that mutants lacking Stv1p, Vph1p, and other subunits of the V-ATPase are defective for FBPase degradation. FBPase was detected in Vid vesicles. However, most FBPase was resistant to proteinase K digestion in the Deltavph1 or vma mutants, whereas the majority of FBPase was sensitive to proteinase K digestion in the Deltastv1 mutant. Therefore, STV1 and VPH1 have distinct functions in FBPase degradation. In cells lacking V0 genes, Vma2p and Vma5p were still detected on Vid vesicles and vacuoles, suggesting that the distribution of V1 proteins is independent of V0 genes. The V0 and V1 domains are assembled following a glucose shift and the assembly is not regulated by protein kinase A and RAV genes. Assembly of the V0 complex is necessary for FBPase trafficking, since mutants that block the assembly and transport of V0 out of the ER were defective in FBPase degradation.     

Cysteine scanning mutagenesis of the noncatalytic nucleotide binding site of the yeast V-ATPase

To investigate residues involved in the formation of the noncatalytic nucleotide binding sites of the vacuolar proton-translocating adenosine triphosphatase (V-ATPase), cysteine scanning mutagenesis of the VMA2 gene that encodes the B subunit in yeast was performed. Replacement of the single endogenous cysteine residue at position 188 gave rise to a Cys-less form of the B subunit (Vma2p) which had near wild-type levels of activity and which was used in the construction of 16 single cysteine-containing mutants. The ability of adenine nucleotides to prevent reaction of the introduced cysteine residues with the sulfhydryl reagent 3-(N-maleimidopropionyl)biocytin (biotin-maleimide) was evaluated by Western blot. Biotin-maleimide labeling of the purified V-ATPase from the wild-type and the mutants S152C, L178C, N181C, A184C, and T279C was reduced after reaction with the nucleotide analog 3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (BzATP). These results suggest the proximity of these residues to the nucleotide binding site on the B subunit. In addition, we have examined the level of endogenous nucleotide bound to the wild-type V-ATPase and to a mutant (the A subunit mutant R483Q) which is postulated to be altered at the noncatalytic site and which displays a marked nonlinearity in ATP hydrolysis (MacLeod, K. J., Vasilyeva, E., Baleja, J. D., and Forgac, M. (1998) J. Biol. Chem. 273, 150-156). The R483Q mutant contained 2.6 mol of ATP/mol of V-ATPase compared with the wild-type enzyme, which contained 0.8 mol of ATP/mol of V-ATPase. These results suggest that binding of additional ATP to the noncatalytic sites may modulate the catalytic activity of the enzyme.     

Functional complementation of yeast vma1 delta cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H(+)-ATPase activity

The ability of a vacuolar H(+)-ATPase (V-ATPase) subunit homolog (subunit A) from plants to rescue the vma mutant phenotype of yeast was investigated as a first step towards investigating the structure and function of plant subunits in molecular detail. Heterologous expression of cotton cDNAs encoding near-identical isoforms of subunit A in mutant vma1 delta yeast cells successfully rescued the mutant vma phenotype, indicating that subunit A of plants and yeast have retained elements essential to V-ATPases during the course of evolution. Although vacuoles become acidified, the plant-yeast hybrid holoenzyme only partially restored V-ATPase activity (approximately 60%) in mutant yeast cells. Domain substitution of divergent N- or C-termini only slightly enhanced V-ATPase activity, whereas swapping both domains acted synergistically, increasing coupled ATP hydrolysis and proton translocation by approximately 22% relative to the native plant subunit. Immunoblot analysis indicated that similar amounts of yeast, plant or plant-yeast chimeric subunits are membrane-bound. These results suggest that subunit A terminal domains contain structural information that impact V-ATPase structure and function.     

Regulation of yeast ectoapyrase ynd1p activity by activator subunit Vma13p of vacuolar H+-ATPase

CD39-like ectoapyrases are involved in protein and lipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. By using a two-hybrid screen, we found that an activator subunit (Vma13p) of yeast vacuolar H(+)-ATPase (V-ATPase) binds to the cytoplasmic domain of Ynd1p, a yeast ectoapyrase. Interaction of Ynd1p with Vma13p was demonstrated by direct binding and co-immunoprecipitation. Surprisingly, the membrane-bound ADPase activity of Ynd1p in a vma13Delta mutant was drastically increased compared with that of Ynd1p in VMA13 cells. A similar increase in the apyrase activity of Ynd1p was found in a vma1Delta mutant, in which the catalytic subunit A of V-ATPase is missing, and the membrane peripheral subunits including Vma13p are dissociated from the membranes. However, the E286Q mutant of VMA1, which assembles inactive V-ATPase complex including Vma13p in the membrane, retained wild type levels of Ynd1p activity, demonstrating that the presence of Vma13p rather than the function of V-ATPase in the membrane represses Ynd1p activity. These results suggest that association of Vma13p with the cytoplasmic domain of Ynd1p regulates its apyrase activity in the Golgi lumen.     

Photoaffinity labeling of wild-type and mutant forms of the yeast V-ATPase A subunit by 2-azido-[(32)P]ADP.

Molecular modeling studies have previously suggested the possible presence of four aromatic residues (Phe(452), Tyr(532), Tyr(535), and Phe(538)) near the adenine binding pocket of the catalytic site on the yeast V-ATPase A subunit (MacLeod, K. J., Vasilyeva, E., Baleja, J. D., and Forgac, M. (1998) J. Biol. Chem. 273, 150-156). To test the proximity of these aromatic residues to the adenine ring, the yeast V-ATPase containing wild-type and mutant forms of the A subunit was reacted with 2-azido-[(32)P]ADP, a photoaffinity analog that stably modifies tyrosine but not phenylalanine residues. Mutant forms of the A subunit were constructed in which the two endogenous tyrosine residues were replaced with phenylalanine and in which a single tyrosine was introduced at each of the four positions. Strong ATP-protectable labeling of the A subunit was observed for the wild-type and the mutant containing tyrosine at 532, significant ATP-protectable labeling was observed for the mutants containing tyrosine at positions 452 and 538, and only very weak labeling was observed for the mutants containing tyrosine at 535 or in which all four residues were phenylalanine. These results suggest that Tyr(532) and possibly Phe(452) and Tyr(538) are in close proximity to the adenine ring of ATP bound to the A subunit. In addition, the effects of mutations at Phe(452), Tyr(532), Tyr(535), and Glu(286) on dissociation of the peripheral V(1) and integral V(0) domains both in vivo and in vitro were examined. The results suggest that in vivo dissociation requires catalytic activity while in vitro dissociation requires nucleotide binding to the catalytic site.     

Saccharomyces cerevisiae Mutants Affected in Vacuole Assembly or Vacuolar H+-ATPase are Hypersensitive to Lead (Pb) Toxicity

Lead is an important environmental pollutant. The role of vacuole, in Pb detoxification, was studied using a vacuolar protein sorting mutant strain (vps16Δ), belonging to class C mutants. Cells disrupted in VPS16 gene, did not display a detectable vacuolar-like structure. Based on the loss of cell proliferation capacity, it was found that cells from vps16Δ mutant exhibited a hypersensitivity to Pb-induced toxicity, compared to wild type (WT) strain. The function of vacuolar H⁺-ATPase (V-ATPase), in Pb detoxification, was evaluated using mutants with structurally normal vacuoles but defective in subunits of catalytic (vma1Δ or vma2Δ) or membrane domain (vph1Δ or vma3Δ) of V-ATPase. All mutants tested, lacking a functional V-ATPase, displayed an increased susceptibility to Pb, comparatively to cells from WT strain. Modification of vacuolar morphology, in Pb-exposed cells, was visualized using a Vma2p-GFP strain. The treatment of yeast cells with Pb originated the fusion of the medium size vacuolar lobes into one enlarged vacuole. In conclusion, it was found that vacuole plays an important role in the detoxification of Pb in Saccharomyces cerevisiae; in addition, a functional V-ATPase was required for Pb compartmentalization.     

A genomic screen for yeast vacuolar membrane ATPase mutants

V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification and the basis of these growth phenotypes, approximately 4700 yeast deletion mutants were screened for growth defects at pH 7.5 in 60 mm CaCl(2). In addition to 13 of 16 mutants lacking known V-ATPase subunits or assembly factors, 50 additional mutants were identified. Sixteen of these also grew poorly in nonfermentable carbon sources, like the known V-ATPase mutants, and were analyzed further. The cwh36Delta mutant exhibited the strongest phenotype; this mutation proved to disrupt a previously uncharacterized V-ATPase subunit. A small subset of the mutations implicated in vacuolar protein sorting, vps34Delta, vps15Delta, vps45Delta, and vps16Delta, caused both Vma- growth phenotypes and lower V-ATPase activity in isolated vacuoles, as did the shp1Delta mutation, implicated in both protein sorting and regulation of the Glc7p protein phosphatase. These proteins may regulate V-ATPase targeting and/or activity. Eight mutants showed a Vma- growth phenotype but no apparent defect in vacuolar acidification. Like V-ATPase-deficient mutants, most of these mutants rely on calcineurin for growth, particularly at high pH. A requirement for constitutive calcineurin activation may be the predominant physiological basis of the Vma- growth phenotype.