V-ATPase Mutants Research Articles

The Role of Sch9 and the V-ATPase in the Adaptation Response to Acetic Acid and the Consequences for Growth and Chronological Lifespan.

Studies with Saccharomyces cerevisiae indicated that non-physiologically high levels of acetic acid promote cellular acidification, chronological aging, and programmed cell death. In the current study, we compared the cellular lipid composition, acetic acid uptake, intracellular pH, growth, and chronological lifespan of wild-type cells and mutants lacking the protein kinase Sch9 and/or a functional V-ATPase when grown in medium supplemented with different acetic acid concentrations. Our data show that strains lacking the V-ATPase are especially more susceptible to growth arrest in the presence of high acetic acid concentrations, which is due to a slower adaptation to the acid stress. These V-ATPase mutants also displayed changes in lipid homeostasis, including alterations in their membrane lipid composition that influences the acetic acid diffusion rate and changes in sphingolipid metabolism and the sphingolipid rheostat, which is known to regulate stress tolerance and longevity of yeast cells. However, we provide evidence that the supplementation of 20 mM acetic acid has a cytoprotective and presumable hormesis effect that extends the longevity of all strains tested, including the V-ATPase compromised mutants. We also demonstrate that the long-lived sch9Δ strain itself secretes significant amounts of acetic acid during stationary phase, which in addition to its enhanced accumulation of storage lipids may underlie its increased lifespan.

Loss of vacuolar-type H+-ATPase induces caspase-independent necrosis-like death of hair cells in zebrafish neuromasts.

ABSTRACTThe vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that regulates cellular pH. V-ATPase activity modulates several cellular processes, but cell-type-specific functions remain poorly understood. Patients with mutations in specific V-ATPase subunits can develop sensorineural deafness, but the underlying mechanisms are unclear. Here, we show that V-ATPase mutations disrupt the formation of zebrafish neuromasts, which serve as a model to investigate hearing loss. V-ATPase mutant neuromasts are small and contain pyknotic nuclei that denote dying cells. Molecular markers and live imaging show that loss of V-ATPase induces mechanosensory hair cells in neuromasts, but not neighboring support cells, to undergo caspase-independent necrosis-like cell death. This is the first demonstration that loss of V-ATPase can lead to necrosis-like cell death in a specific cell type in vivo. Mechanistically, loss of V-ATPase reduces mitochondrial membrane potential in hair cells. Modulating the mitochondrial permeability transition pore, which regulates mitochondrial membrane potential, improves hair cell survival. These results have implications for understanding the causes of sensorineural deafness, and more broadly, reveal functions for V-ATPase in promoting survival of a specific cell type in vivo.

Compensatory Internalization of Pma1 in V-ATPase Mutants in Saccharomyces cerevisiae Requires Calcium- and Glucose-Sensitive Phosphatases.

Loss of V-ATPase activity in organelles, whether through V-ATPase inhibition or V-ATPase (vma) mutations, triggers a compensatory downregulation of the essential plasma membrane proton pump Pma1 in Saccharomyces cerevisiae We have previously determined that the α-arrestin Rim8 and ubiquitin ligase Rsp5 are essential for Pma1 ubiquination and endocytosis in response to loss of V-ATPase activity. Here, we show that Pma1 endocytosis in V-ATPase mutants does not require Rim101 pathway components upstream and downstream of Rim8, indicating that Rim8 is acting independently in Pma1 internalization. We find that two phosphatases, the calcium-responsive phosphatase calcineurin and the glucose-sensitive phosphatase Glc7 (PP1), and one of the Glc7 regulatory subunits Reg1, exhibit negative synthetic genetic interactions with vma mutants, and demonstrate that both phosphatases are essential for ubiquitination and endocytic downregulation of Pma1 in these mutants. Although both acute and chronic loss of V-ATPase activity trigger the internalization of ∼50% of surface Pma1, a comparable reduction in Pma1 expression in a pma1-007 mutant neither compensates for loss of V-ATPase activity nor stops further Pma1 endocytosis. The results indicate that the cell surface level of Pma1 is not directly sensed and that internalized Pma1 may play a role in compensating for loss of V-ATPase-dependent acidification. Taken together, these results provide new insights into cross talk between two major proton pumps central to cellular pH control.

Effects of Disruption of PMC1 in the tfp1∆/∆ Mutant on Calcium Homeostasis, Oxidative and Osmotic Stress Resistance in Candida albicans.

The vacuolar-type H+-ATPase (V-ATPase) is essential for many cell processes. Our previous study has demonstrated that Tfp1 is a putative subunit of V-ATPase, loss of which causes disorders in calcium homeostasis and decreased resistance to oxidative stress. In this study, we found that further deletion of PMC1, a vacuolar calcium pump, in tfp1∆/∆ mutant led to more severe dysregulation of calcium homeostasis. Besides, the tfp1∆/∆pmc1∆/∆ mutant was more sensitive to H2O2 and had a higher ROS level. As is known, V-ATPase mutants are sensitive to NaCl, and PMC1 mutant is resistant against NaCl. However, the tfp1∆/∆pmc1∆/∆ mutant exhibited sensitivity to NaCl. Mechanism study demonstrated that their sensitivity was associated with reduced osmotic resistance caused by relatively low expression of GPD1. In addition, we first found that NaCl addition significantly declined ROS levels in tfp1∆/∆ and tfp1∆/∆pmc1∆/∆ mutants. In tfp1∆/∆ mutant, decreased ROS levels were relevant to enhanced antioxidant activities. However, in tfp1∆/∆pmc1∆/∆ mutant, reduced ROS resulted from decreased total calcium content, revealing that NaCl affected ROS levels in the two mutants through different mechanisms. Taken together, our data indicated that loss of both TFP1 and PMC1 further affected calcium homeostasis and other cellular processes in Candida albicans and provides a potential antifungal target.

Functional Analyses of V-Atpase Mutations in Follicular Lymphoma

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. Recently, frequent mutations (RRAGC, V-ATPase) in the amino acid sensing pathway connected to MTOR signaling have been described in FL. The RRAGC mutations activate MTOR. The V-ATPase is a multi-protein complex primarily involved in proton pumping and acidification of cellular compartments. Here, we report initial results from functional analyses of the common hotspot mutations p.Y371C and p.R400Q in the V-ATPase subunit ATP6V1B2.Methods: We introduced the ATP6V1B2 hotspot mutations by site-directed mutagenesis. The cDNAs were cloned into lentiviral or retroviral vectors and used to generate stable lymphoma cells lines and HEK293T cells. We also generated recombinant S. cerevisiae expressing a mutation homologous to human ATP6V1B2 p.R400Q at the endogenous VMA2locus (R381Q). We modeled the location of the mutations onto the existing 3D structures.Results: Some of the stable cell lines lost the expression of the ATP6V1B2 mutants over time, while others were more permissive. Given the critical role of V-ATPase in lysosomal acidification and autophagy we measured LC3-II levels in various recombinant cell lines and detected very elevated LC3-II levels in the V-ATPase mutant lines. Highly elevated LC3-II levels at steady state could indicate impairments in LC3-II metabolism as seen with chemical V-ATPase inhibition (bafilomycin A1 treatment) or elevated autophagic initiation and flux. We resorted to multiple experimental approaches to resolve this question: i) chemical MTOR inhibition strongly activates autophagy. Using rapamycin and Torin 2, we induced LC3-II to levels comparable to those seen with the V-ATPase mutants; ii) using inhibitors of lysosomal LC3-II degradation, we further increased already elevated LC3-II levels in V-ATPase mutant HEK293T cell lines; iii) in S. cerevisiae Vma2R381Qcells we found elevated autophagy activity based on analysis of the LC3 homolog Atg8, vacuolar/lysosomal processing of a GFP-Atg8 chimera, and enzymatic analysis of an autophagy marker, Pho8Δ60. Together, these findings currently allow for the working hypothesis that V-ATPase mutants strongly increase autophagic flux.We performed studies of the effects of V-ATPase mutants on MTOR activity as measured through RPS6KB/S6-kinase phosphorylation. In transient transfections in HEK293T cells and in stable HEK293T cells lines achieving expression slightly above endogenous V-ATPase levels, performed under conditions of leucine starvation or sufficiency, we detected MTOR activation.We mapped the mutated p.Y371C and p.R400Q residues onto the published electron microscopy-derived structures of yeast V-ATPase. We found that both residues are closely spaced and are located at the interface of the V1B2 and V1A subunits; the latter with potential effects on subunit interactions, the conformational state of the V1 complex (open, loose or tight) and possibly effects on ATPase activity.To gain insights into the physiological consequences of these findings, we measured growth and viability of a recombinant lymphoma cell line (SUDHL4) expressing wild type and mutant V-ATPase in the presence of lowered leucine concentration. We found improved growth and survival of cells expressing the ATP6V1B2 mutations. In a parallel experiment, we induced cellular stress by intentionally overgrowing these lines and measured survival over time. We found improved growth and survival of cells expressing the ATP6V1B2 mutations. Complementary measurements of the biochemical and functional consequences of V-ATPase mutations in isolated primary FL B cells are ongoing and will be updated at the meeting.Conclusion: Our initial efforts at functional characterization of the common FL-associated hotspot mutations p.Y371C and p.R400Q in the V-ATPase subunit ATP6V1B2 uncovered substantially elevated autophagic flux. To our knowledge, this is the first report of mutational activation of autophagy in follicular lymphoma. Our current working model is that this elevated flux promotes lysosomal amino acid generation that subsequently activates MTOR through previously proposed inside-out signaling pathways. Combined, these changes may allow for improved survival of FL cells under conditions of nutrient stress. These findings may allow for future therapeutic targeting of this pathway in FL. DisclosuresNo relevant conflicts of interest to declare.

V-ATPase activity in the TGN/EE is required for exocytosis and recycling in Arabidopsis.

In plants, vacuolar H(+)-ATPase (V-ATPase) activity acidifies both the trans-Golgi network/early endosome (TGN/EE) and the vacuole. This dual V-ATPase function has impeded our understanding of how the pH homeostasis within the plant TGN/EE controls exo- and endocytosis. Here, we show that the weak V-ATPase mutant deetiolated3 (det3) displayed a pH increase in the TGN/EE, but not in the vacuole, strongly impairing secretion and recycling of the brassinosteroid receptor and the cellulose synthase complexes to the plasma membrane, in contrast to mutants lacking tonoplast-localized V-ATPase activity only. The brassinosteroid insensitivity and the cellulose deficiency defects in det3 were tightly correlated with reduced Golgi and TGN/EE motility. Thus, our results provide strong evidence that acidification of the TGN/EE, but not of the vacuole, is indispensable for functional secretion and recycling in plants.

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes.

Involvement of MoVMA11, a Putative Vacuolar ATPase c' Subunit, in Vacuolar Acidification and Infection-Related Morphogenesis of Magnaporthe oryzae.

Many functions of vacuole depend on the activity of vacuolar ATPase which is essential to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. In filamentous fungus Magnaporthe oryzae , subcellular colocalization and quinacrine staining suggested that the V1V0 domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments. Targeted gene disruption of MoVMA11 gene, encoding the putative c’ subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not in the typical pH conditional lethality. Strikingly, aerial hyphae of the MoVMA11 null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated that MoVMA11 was involved in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of the ΔMovma11 mutant on onion epidermal cells. Finally, the MoVMA11 null mutant lost pathogenicity on both intact and wounded host leaves. Overall, our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions and highlighted that V-ATPase is essential for infection-related morphogenesis and pathogenesis in M . oryzae .

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.

Loss of Vacuolar H+-ATPase (V-ATPase) Activity in Yeast Generates an Iron Deprivation Signal That Is Moderated by Induction of the Peroxiredoxin TSA2

Vacuolar H(+)-ATPases (V-ATPases) acidify intracellular organelles and help to regulate overall cellular pH. Yeast vma mutants lack V-ATPase activity and allow exploration of connections between cellular pH, iron, and redox homeostasis common to all eukaryotes. A previous microarray study in a vma mutant demonstrated up-regulation of multiple iron uptake genes under control of Aft1p (the iron regulon) and only one antioxidant gene, the peroxiredoxin TSA2 (Milgrom, E., Diab, H., Middleton, F., and Kane, P. M. (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J. Biol. Chem. 282, 7125-7136). Fluorescent biosensors placing GFP under transcriptional control of either an Aft1-dependent promoter (P(FIT2)-GFP) or the TSA2 promoter (P(TSA2)-GFP) were constructed to monitor transcriptional signaling. Both biosensors were up-regulated in the vma2Δ mutant, and acute V-ATPase inhibition with concanamycin A induced coordinate up-regulation from both promoters. PTSA2-GFP induction was Yap1p-dependent, indicating an oxidative stress signal. Total cell iron measurements indicate that the vma2Δ mutant is iron-replete, despite up-regulation of the iron regulon. Acetic acid up-regulated P(FIT2)-GFP expression in wild-type cells, suggesting that loss of pH control contributes to an iron deficiency signal in the mutant. Iron supplementation significantly decreased P(FIT2)-GFP expression and, surprisingly, restored P(TSA2)-GFP to wild-type levels. A tsa2Δ mutation induced both nuclear localization of Aft1p and P(FIT2)-GFP expression. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis.

Inhibitors of V-ATPase Proton Transport Reveal Uncoupling Functions of Tether Linking Cytosolic and Membrane Domains of V0 Subunit a (Vph1p)

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 μM) and thonzonium bromide (EC(50) = 69 μM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 μM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.

Vacuolar H+-ATPase plays a key role in cell wall biosynthesis of Aspergillus niger

The identification of suitable targets is crucial for the discovery and development of new antifungals. Since the fungal cell wall is an essential organelle, the identification of genes involved in cell wall biosynthesis is expected to help discover new antifungal targets. From our previously obtained collection of cell wall mutants with a constitutively active cell wall stress response pathway, we selected a thermosensitive, osmotic-remediable mutant with decreased resistance to SDS for complementation analysis. The phenotypes of this mutant were complemented by a gene encoding a protein with high sequence similarity to subunit d of the eukaryotic Vacuolar-H+-ATPase (VmaD). Genetic analysis of this thermosensitive mutant revealed that the conditional mutant allele encodes a protein that lacks 12 amino acids at the C-terminus due to a point mutation that introduces a stop codon. Deletion of the entire gene resulted in very poor growth. The conditional mutant displayed several phenotypes that are typical for V-ATPase mutants, including increased sensitivity to zinc ions and reduced acidification of the vacuole as observed by quinacrine staining. Treatment of Aspergillus niger with the V-ATPase inhibitor bafilomycinB1 induced the expression of agsA and other cell wall related genes. Furthermore genes involved in cell wall reassembly like fksA, agsA and phiA were clearly up-regulated in the conditional mutant. Our results indicate that the ATP-driven transport of protons and acidification of the vacuole is crucial for the strength of the fungal cell wall and that reduced activity of the V-ATPase induces the cell wall stress response pathway.

The vacuolar ATPase is required for physiological as well as pathological activation of the Notch receptor.

Evidence indicates that endosomal entry promotes signaling by the Notch receptor, but the mechanisms involved are not clear. In a search for factors that regulate Notch activation in endosomes, we isolated mutants in Drosophila genes that encode subunits of the vacuolar ATPase (V-ATPase) proton pump. Cells lacking V-ATPase function display impaired acidification of the endosomal compartment and a correlated failure to degrade endocytic cargoes. V-ATPase mutant cells internalize Notch and accumulate it in the lysosome, but surprisingly also show a substantial loss of both physiological and ectopic Notch activation in endosomes. V-ATPase activity is required in signal-receiving cells for Notch signaling downstream of ligand activation but upstream of gamma-secretase-dependent S3 cleavage. These data indicate that V-ATPase, probably via acidification of early endosomes, promotes not only the degradation of Notch in the lysosome but also the activation of Notch signaling in endosomes. The results also suggest that the ionic properties of the endosomal lumen might regulate Notch cleavage, providing a rationale for physiological as well as pathological endocytic control of Notch activity.

A Simple and Specific Procedure to Permeabilize the Plasma Membrane ofSchizosaccharomyces pombe

Cu(2+)-treatment is a useful technique in selectively permeabilizing the fungal plasma membrane. We describe herein a practical application with Schizosaccharomyces pombe. Incubation of cells with 0.5 mM CuCl(2) at 30 degrees C for 20 min induced efficient leakage of cytosolic constituents. The kinetic characteristics of the calcium and amino acid flux from Cu(2+)-treated S. pombe cells suggested that the Cu(2+) treatment permeabilized the plasma membrane without loss of vacuolar function. As a further application of the method, the amino acid contents of Cu(2+)-treated and untreated cells were also determined. The amino acid pool of Cu(2+)-treated wild-type cells was enriched in basic amino acids but not in acidic amino acids, as is characteristic of the vacuolar amino acid pool of fungi, including Saccharomyces cerevisiae and Neurosporra crassa. The amino acid pool of the S. pombe V-ATPase mutant vma1Delta was also successfully determined. We conclude that the vacuolar amino acid pool of S. pombe can be measured using Cu(2+)-treated cells. The method is simple, inexpensive, and rapid relative to the isolation of vacuolar vesicles, making it useful in estimating vacuolar pools and transport across the vacuolar membrane.

The Vacuolar-ATPase Complex Regulates Retinoblast Proliferation and Survival, Photoreceptor Morphogenesis, and Pigmentation in the Zebrafish Eye

The vacuolar (v)-ATPase complex is a key regulator of the acidification of endosomes, lysosomes, and the luminal compartments of several cell types, tissues, and organs; however, little is know about the in vivo function of the v-ATPase complex or its roles during eye development. This study was conducted to characterize ocular defects in five zebrafish mutants in which core components of the v-ATPase complex were affected (atp6v1h, atp6v1f, atp6v1e1, atp6v0c, and atp6v0d1), as well as a sixth mutant in which a v-ATPase associated protein (atp6ap1) was affected. v-ATPase mutant zebrafish were characterized by histologic, molecular, and ultrastructural analyses. v-ATPase mutant zebrafish were oculocutaneous albinos and presented with defects in the formation and/or survival of melanosomes and with malformations in the retinal pigmented epithelium (RPE) that compromised melanosome distribution. They were microphthalmic, and BrdU incorporation assays indicated that retinoblast cell cycle exit and sustained proliferation in the ciliary marginal zone (CMZ) were compromised. v-ATPase mutants also possessed elevated levels of apoptotic neurons within their retinas and brains. Photoreceptor outer segment morphology was abnormal in the mutant eye with rosette structures forming adjacent to the affected regions of the RPE. Ultrastructural analyses indicate that RPE cells in v-ATPase mutants possess numerous membrane-bounded vacuoles containing undigested outer segment material. In situ hybridization analyses localized v-ATPase subunit transcripts within the RPE. These results demonstrate that the v-ATPase complex plays several critical roles during vertebrate eye development and maintenance, and they suggest that defects in v-ATPase complex function could possibly underlie human ocular disorders that affect the RPE.

The vacuolar V 1/V 0-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 yck3Δ phenotype, in which Vps41 accumulates in vacuolar dots, we observed that mutants in the V 0-part of the V 0/V 1-ATPase, in particular in vma16Δ, also accumulate Vps41. This accumulation is not due to a phosphorylation defect, but to reduced release of Vps41 from vma16Δ 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 V 0-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.

Genomic Screening in Vivo Reveals the Role Played by Vacuolar H+ ATPase and Cytosolic Acidification in Sensitivity to DNA-Damaging Agents Such as Cisplatin

Screening the Saccharomyces cerevisiae homozygous diploid deletion library against a sublethal concentration of cisplatin revealed 76 strains sensitive to the drug. As expected, the largest category of deletions, representing 40% of the sensitive strains, was composed of strains lacking genes involved in DNA replication and damage repair. Deletions lacking function of the highly conserved vacuolar H+ translocating ATPase (V-ATPase) composed the category representing the second largest number of sensitive strains. The effect on cell death exhibited by V-ATPase mutants was found to be a general response to various DNA damaging agents as opposed to being specific to cisplatin, as evidenced by sensitivity of the mutants to hydroxyurea (a DNA-alkylating agent) and UV irradiation. Loss of V-ATPase does not affect DNA repair, because double mutants defective for V-ATPase function and DNA repair pathways were more sensitive to cisplatin than the single mutants. V-ATPase mutants are more prone to DNA damage than wild-type cells, indicated by enhanced activation of the DNA damage checkpoint. Vacuole function per se is not cisplatin-sensitive, because vacuolar morphology and vacuolar acidification were unaffected by cisplatin in wild-type cells. V-ATPase also controls cytoplasmic pH, so the enhanced sensitivity to DNA damage may be associated with the drop in pHi associated with V-ATPase mutants. The increased loss in cell viability induced by cisplatin at lower pH in V-ATPase mutants supports this hypothesis. The loss in viability seen in wild-type cells under the same conditions was far less dramatic.

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.

Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to o...

Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis

Subunit a of V-ATPase in the yeast Saccharomyces cerevisiae, in contrast to its other subunits, is encoded by two genes VPH1 and STV1. While disruption of any other gene encoding the V-ATPase subunits results in growth arrest at pH 7.5, null mutants of Vph1p or Stv1p can grow at this pH. We used a polyclonal antibody to yeast Stv1p and a commercially available monoclonal antibody to Vph1p for analysis of yeast membranes by sucrose gradient fractionation, and two different vital dyes to characterize the phenotype of vph1 triangle up and stv1 triangle up mutants as compared to the double mutant and the wild-type cells. Immunological assays of sucrose gradient fractions revealed that the amount of Stv1p was elevated in the vph1 triangle up strain, and that vacuoles purified by this method with no detectable endosomal contamination contain an assembled V-ATPase complex, but with much lower activity than the wild type. These results suggest that Stv1p compensates for the loss of Vph1p in the vph1 triangle up strain. LysoSensor Green DND-189 was used as a pH sensor to demonstrate unexpected changes in vacuolar acidification in stv1 triangle up as the Vph1p-containing V-ATPase complex is commonly considered to acidify the vacuoles. In the vph1 triangle up strain, the dye revealed slight but definite acidification of the vacuole as well. The lipophilic dye FM4-64 was used as an endocytic marker. We show that the null V-ATPase mutants, as well as the vph1 triangle up one, markedly slow down endocytosis of the dye.