The yeast vacuolar proton-translocating ATPase contains a subunit homologous to the Manduca sexta and bovine e subunits that is essential for function

The yeast cwh36Delta mutant was identified in a screen for yeast mutants exhibiting a Vma(-) phenotype suggestive of loss of vacuolar proton-translocating ATPase (V-ATPase) activity. The mutation disrupts two genes, CWH36 and a recently identified open reading frame on the opposite strand, YCL005W-A. We demonstrate that disruption of YCL005W-A is entirely responsible for the Vma(-) growth phenotype of the cwh36Delta mutant. YCL005W-A encodes a homolog of proteins associated with the Manduca sexta and bovine chromaffin granule V-ATPase. The functional significance of these proteins for V-ATPase activity had not been tested, but we show that the protein encoded by YCL005W-A, which we call Vma9p, is essential for V-ATPase activity in yeast. Vma9p is localized to the vacuole but fails to reach the vacuole in a mutant lacking one of the integral membrane subunits of the V-ATPase. Vma9p is associated with the yeast V-ATPase complex in vacuolar membranes, as demonstrated by co-immunoprecipitation with known V-ATPase subunits and glycerol gradient fractionation of solubilized vacuolar membranes. Based on this evidence, we propose that Vma9p is a genuine subunit of the yeast V-ATPase and that e subunits may be a functionally essential part of all eukaryotic V-ATPases.     

Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium

Like numerous other eukaryotic organelles, the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. Organelle fission and fusion processes must be balanced to ensure organelle integrity. Coordination of vacuole fission and fusion depends on the interactions of vacuolar SNARE proteins and the dynamin-like GTPase Vps1p. Here, we identify a novel factor that impinges on the fusion-fission equilibrium: the vacuolar H(+)-ATPase (V-ATPase) performs two distinct roles in vacuole fission and fusion. Fusion requires the physical presence of the membrane sector of the vacuolar H(+)-ATPase sector, but not its pump activity. Vacuole fission, in contrast, depends on proton translocation by the V-ATPase. Eliminating proton pumping by the V-ATPase either pharmacologically or by conditional or constitutive V-ATPase mutations blocked salt-induced vacuole fragmentation in vivo. In living cells, fission defects are epistatic to fusion defects. Therefore, mutants lacking the V-ATPase display large single vacuoles instead of multiple smaller vacuoles, the phenotype that is generally seen in mutants having defects only in vacuolar fusion. Its dual involvement in vacuole fission and fusion suggests the V-ATPase as a potential regulator of vacuolar morphology and membrane dynamics.     

Vacuole membrane fusion: V0 functions after trans-SNARE pairing and is coupled to the Ca2+-releasing channel

Pore models of membrane fusion postulate that cylinders of integral membrane proteins can initiate a fusion pore after conformational rearrangement of pore subunits. In the fusion of yeast vacuoles, V-ATPase V0 sectors, which contain a central cylinder of membrane integral proteolipid subunits, associate to form a transcomplex that might resemble an intermediate postulated in some pore models. We tested the role of V0 sectors in vacuole fusion. V0 functions in fusion and proton translocation could be experimentally separated via the differential effects of mutations and inhibitory antibodies. Inactivation of the V0 subunit Vph1p blocked fusion in the terminal reaction stage that is independent of a proton gradient. Deltavph1 mutants were capable of docking and trans-SNARE pairing and of subsequent release of lumenal Ca2+, but they did not fuse. The Ca2+-releasing channel appears to be tightly coupled to V0 because inactivation of Vph1p by antibodies blocked Ca2+ release. Vph1 deletion on only one fusion partner sufficed to severely reduce fusion activity. The functional requirement for Vph1p correlates to V0 transcomplex formation in that both occur after docking and Ca2+ release. These observations establish V0 as a crucial factor in vacuole fusion acting downstream of trans-SNARE pairing.     

Association of L-type calcium channels with a vacuolar H(+)-ATPase G2 subunit

The class C L-type calcium (Ca(2+)) channels have been implicated in many important physiological processes. Here, we have identified a mouse vacuolar H(+)-ATPase (V-ATPase) G2 subunit protein that bound to the C-terminal domain of the pore-forming alpha(1C) subunit using a yeast two-hybrid screen. Protein-protein interaction between the V-ATPase G subunit and the alpha(1C) subunit was confirmed using in vitro GST pull-down assays and coimmunoprecipitation from intact cells. Moreover, treatment of cells expressing L-type Ca(2+) channels with a specific inhibitor of the V-ATPase blocked proper targeting of the channels to the plasma membrane.     

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.     

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.     

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.     

Microtubules are involved in glucose-dependent dissociation of the yeast vacuolar [H+]-ATPase in vivo

The vacuolar [H(+)]-ATPases (V-ATPases) are composed of a peripheral V(1) domain and a membrane-embedded V(0) domain. Reversible dissociation of the V(1) and V(0) domains has been observed in both yeast and insects and has been suggested to represent a general regulatory mechanism for controlling V-ATPase activity in vivo. In yeast, dissociation of the V-ATPase is triggered by glucose depletion, but the signaling pathways that connect V-ATPase dissociation and glucose metabolism have not been identified. We have found that nocodazole, an agent that disrupts microtubules, partially blocked dissociation of the V-ATPase in response to glucose depletion in yeast. By contrast, latrunculin, an agent that disrupts actin filaments, had no effect on glucose-dependent dissociation of the V-ATPase complex. Neither nocodazole nor latrunculin blocked reassembly of the V-ATPase upon re-addition of glucose to the medium. The effect of nocodazole appears to be specifically through disruption of microtubules since glucose-dependent dissociation of the V-ATPase was not blocked by nocodazole in yeast strains bearing a mutation in tubulin that renders it resistant to nocodazole. Because nocodazole has been shown to arrest cells in the G(2) phase of the cell cycle, it was of interest to determine whether nocodazole exerted its effect on dissociation of the V-ATPase through cell cycle arrest. Glucose-dependent dissociation of the V-ATPase was examined in four yeast strains bearing temperature-sensitive mutations that arrest cells in different stages of the cell cycle. Because dissociation of the V-ATPase occurred normally at both the permissive and restrictive temperatures in these mutants, the results suggest that in vivo dissociation is not dependent upon cell cycle phase.     

The Ras/cAMP/protein kinase A pathway regulates glucose-dependent assembly of the vacuolar (H+)-ATPase in yeast

Vacuolar (H+)-ATPases (V-ATPases) are ubiquitous, ATP-driven proton pumps that acidify organelles or the extracellular space. A rapid and effective mechanism for regulating V-ATPase activity involves reversible dissociation of the two functional domains of the pump, V1 and V0. This process is best characterized in yeast, where V-ATPases are reversibly disassembled in response to glucose depletion. To identify regulators that control this process in vivo, a genetic screen was performed in yeast to search for mutants that cannot disassemble their V-ATPases when grown in the absence of glucose. This screen identified IRA1 (inhibitory regulator of the Ras/cAMP pathway 1) and IRA2 as essential genes for regulating V-ATPase dissociation in vivo. IRA1 and IRA2 encode GTPase-activating proteins that negatively regulate Ras in nutrient-poor conditions. Down-regulation of Ras lowers cAMP levels by reducing adenylate cyclase activity. Decreased cAMP levels in turn lead to reduced activity of protein kinase A (PKA). Our results show that targeted deletion of IRA2 results in defective disassembly of the V-ATPase in response to glucose depletion, and reexpression of the gene rescues this phenotype. Glucose-dependent dissociation is also blocked in strains expressing the dominant active RAS2val19 allele or in strains deficient for the regulatory subunit of PKA, both of which lead to constitutively active PKA. These results reveal a role for PKA in controlling glucose-dependent V-ATPase assembly in yeast.     

Hph1 and Hph2 are novel components of the Sec63/Sec62 posttranslational translocation complex that aid in vacuolar proton ATPase biogenesis

Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1Δ hph2Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1Δ hph2Δ and hph1Δ hph2Δ sec71Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1Δ hph2Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.     

A LAPF/phafin1-like protein regulates TORC1 and lysosomal membrane permeabilization in response to endoplasmic reticulum membrane stress

Lysosomal membrane permeabilization (LMP) is a poorly understood regulator of programmed cell death that involves leakage of luminal lysosomal or vacuolar hydrolases into the cytoplasm. In Saccharomyces cerevisiae, LMP can be induced by antifungals and endoplasmic reticulum stressors when calcineurin also has been inactivated. A genome-wide screen revealed Pib2, a relative of LAPF/phafin1 that regulates LMP in mammals, as a pro-LMP protein in yeast. Pib2 associated with vacuolar and endosomal limiting membranes in unstressed cells in a manner that depended on its FYVE domain and on phosphatidylinositol 3-phosphate (PI(3)P) biosynthesis. Genetic experiments suggest that Pib2 stimulates the activity of TORC1, a vacuole-associated protein kinase that is sensitive to rapamycin, in a pathway parallel to the Ragulator/EGO complex containing the GTPases Gtr1 and Gtr2. A hyperactivating mutation in the catalytic subunit of TORC1 restored LMP to the gtr1∆ and pib2∆ mutants and also prevented the synthetic lethality of the double mutants. These findings show novel roles of PI(3)P and Pib2 in the regulation of TORC1, which in turn promoted LMP and nonapoptotic death of stressed cells. Rapamycin prevented the death of the pathogenic yeast Candida albicans during exposure to fluconazole plus a calcineurin inhibitor, suggesting that TORC1 broadly promotes sensitivity to fungistats in yeasts.     

Novel calcineurin interacting protein-2: the functional characterization of CNP-2 in Caenorhabditis elegans

Calcineurin (Cn) is a serine/threonine phosphatase implicated in a wide variety of biological responses. To identify proteins that mediate Cn signaling pathway effects, we used yeast two-hybrid assays to screen for Cn interacting proteins, discovering a protein encoded by the gene, cnp-2 (Y46G5A.10). Utilizing serially deleted forms of Cn as baits, we demonstrated that the catalytic domain of Cn (TAX-6) binds with CNP-2, and this physical interaction was able to be reconstituted in vitro, supporting our yeast two-hybrid results. cnp-2 is a nematode-specific novel gene found in C. elegans as well as its closest relative, C. briggsae. CNP-2 was strongly expressed in the intestine of C. elegans. To study the function of cnp-2, we performed cnp-2 RNAi knock-down and characterized phenotypes associated with Cn mutants. However, no gross defects were revealed in these RNAi experiments. CNP-2 was proven to be a Cn binding protein; however, its role remains to be elucidated.     

Formation and dissociation of proteasome storage granules are regulated by cytosolic pH

The 26S proteasome is the major protein degradation machinery of the cell and is regulated at many levels. One mode of regulation involves accumulation of proteasomes in proteasome storage granules (PSGs) upon glucose depletion. Using a systematic robotic screening approach in yeast, we identify trans-acting proteins that regulate the accumulation of proteasomes in PSGs. Our dataset was enriched for subunits of the vacuolar adenosine triphosphatase (V-ATPase) complex, a proton pump required for vacuole acidification. We show that the impaired ability of V-ATPase mutants to properly govern intracellular pH affects the kinetics of PSG formation. We further show that formation of other protein aggregates upon carbon depletion also is triggered in mutants with impaired activity of the plasma membrane proton pump and the V-ATPase complex. We thus identify cytosolic pH as a specific cellular signal involved both in the glucose sensing that mediates PSG formation and in a more general mechanism for signaling carbon source exhaustion.     

Cellular environment is important in controlling V-ATPase dissociation and its dependence on activity

One mechanism of regulating V-ATPase activity in vivo involves reversible dissociation into its component V(1) and V(0) domains, which in yeast occurs in response to glucose depletion. V-ATPase complexes containing the Vph1p isoform of subunit a (VCC) are targeted to the vacuole, and Stv1p-containing complexes (SCC) are targeted to the Golgi. Overexpression of Stv1p results in mistargeting of SCC to the vacuole. We have investigated the role of the a subunit isoform and cellular environment in controlling dissociation using vacuolar protein sorting (vps) mutants that accumulate proteins in either the prevacuolar compartment (PVC) (vps27Delta) or a post-Golgi compartment (PGC) (vps21Delta). Dissociation of both VCC and SCC depends upon cellular environment, with dissociation most complete in the vacuole and least complete in the PVC. The dependence of dissociation on V-ATPase activity was also investigated using both concanamycin and inactivating mutations. Concanamycin partly blocks dissociation of both VCC and SCC in all three compartments, with inhibition generally greater for SCC than VCC. The R735Q mutant of Vph1p results in loss of both ATPase and proton transport, whereas the R735K mutant lacks proton transport but has 10% of wild type ATPase activity. For VCC in the vacuole, dissociation is completely blocked for the R735Q but not the R735K mutant. Significant dissociation of VCC is observed for both mutants in the PVC and PGC, indicating that V-ATPase activity is not absolutely required for dissociation. Similar results were obtained for SCC, although dissociation of SCC is again generally more sensitive to activity than VCC. These results suggest that the cellular environment is important both in controlling in vivo dissociation of the V-ATPase and the dependence of this process on catalytic activity. Moreover, catalytic activity is not absolutely required for V-ATPase dissociation.     

A Cytotoxic Type III Secretion Effector of Vibrio parahaemolyticus Targets Vacuolar H+-ATPase Subunit c and Ruptures Host Cell Lysosomes

Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H⁺-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death.     

vac2: a yeast mutant which distinguishes vacuole segregation from Golgi-to-vacuole protein targeting.

We have isolated four yeast mutants that are unable to partition maternal vacuoles into growing buds. Three of these vacuole segregation (vac) mutants also mislocalize the vacuolar protease carboxypeptidase Y (CPY) to the cell surface, a phenotype previously reported for vac strains. A fourth mutant, vac2-1, exhibits a temperature-sensitive defect in vacuole segregation but does not show a defect in protein targeting from the Golgi apparatus to the vacuole. Haploid vac2-1 cells grown at the non-permissive temperature do not secrete CPY or a second vacuolar protease, proteinase A (PrA). Furthermore, newly synthesized precursors of CPY are converted to mature forms with similar kinetics in both vac2-1 and wild-type cells. In addition, invertase is secreted normally from vac2-1 cells, indicating that post-Golgi steps in the secretory pathway are not blocked in this mutant. These results suggest that VAC2 function is necessary for vacuole division and segregation in yeast but is not involved in vacuole protein sorting events at the Golgi apparatus.     

Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae

The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property.     

Formation of Hydrogen Sulfide from Cysteine in Saccharomyces cerevisiae BY4742: Genome Wide Screen Reveals a Central Role of the Vacuole

Discoveries on the toxic effects of cysteine accumulation and, particularly, recent findings on the many physiological roles of one of the products of cysteine catabolism, hydrogen sulfide (H₂S), are highlighting the importance of this amino acid and sulfur metabolism in a range of cellular activities. It is also highlighting how little we know about this critical part of cellular metabolism. In the work described here, a genome-wide screen using a deletion collection of Saccharomyces cerevisiae revealed a surprising set of genes associated with this process. In addition, the yeast vacuole, not previously associated with cysteine catabolism, emerged as an important compartment for cysteine degradation. Most prominent among the vacuole-related mutants were those involved in vacuole acidification; we identified each of the eight subunits of a vacuole acidification sub-complex (V₁ of the yeast V-ATPase) as essential for cysteine degradation. Other functions identified included translation, RNA processing, folate-derived one-carbon metabolism, and mitochondrial iron-sulfur homeostasis. This work identified for the first time cellular factors affecting the fundamental process of cysteine catabolism. Results obtained significantly contribute to the understanding of this process and may provide insight into the underlying cause of cysteine accumulation and H₂S generation in eukaryotes.