Karlett J. Parra

Reversible Association between the V1 and V0 Domains of Yeast Vacuolar H+-ATPase Is an Unconventional Glucose-Induced Effect

ABSTRACT The yeast vacuolar H+-ATPase (V-ATPase) is a multisubunit complex responsible for organelle acidification. The enzyme is structurally organized into two major domains: a peripheral domain (V1), containing the ATP binding sites, and an integral membrane domain (V0), forming the proton pore. Dissociation of the V1 and V0 domains inhibits ATP-driven proton pumping, and extracellular glucose concentrations regulate V-ATPase activity in vivo by regulating the extent of association between the V1 and V0 domains. To examine the mechanism of this response, we quantitated the extent of V-ATPase assembly in a variety of mutants with known effects on other glucose-responsive processes. Glucose effects on V-ATPase assembly did not involve the Ras-cyclic AMP pathway, Snf1p, protein kinase C, or the general stress response protein Rts1p. Accumulation of glucose 6-phosphate was insufficient to maintain or induce assembly of the V-ATPase, suggesting that further glucose metabolism is required. A transient decrease in ATP concentration with glucose deprivation occurs quickly enough to help trigger disassembly of the V-ATPase, but increases in cellular ATP concentrations with glucose readdition cannot account for reassembly. Disassembly was inhibited in two mutant enzymes lacking ATPase and proton pumping activities or in the presence of the specific V-ATPase inhibitor, concanamycin A. We propose that glucose effects on V-ATPase assembly occur by a novel mechanism that requires glucose metabolism beyond formation of glucose 6-phosphate and generates a signal that can be sensed efficiently only by a catalytically competent V-ATPase.

Characterization of a Temperature-sensitive Yeast Vacuolar ATPase Mutant with Defects in Actin Distribution and Bud Morphology

The 27-kDa E subunit, encoded by theVMA4 gene, is a peripheral membrane subunit of the yeast vacuolar H+-ATPase. We have randomly mutagenized theVMA4 gene in order to examine the structure and function of the 27-kDa subunit. Cells lacking a functional VMA4 gene are unable to grow at pH > 7 or in elevated concentrations of CaCl2. Plasmid-borne, mutagenized vma4 genes were screened for failure to complement these phenotypes. Mutants producing Vma4 proteins detectable by immunoblot were selected; one (vma4–1 ts ) is temperature conditional, exhibiting the Vma− phenotype only at elevated temperature (37 °C). Sequencing revealed that a single point mutation, D145G, was responsible for the phenotypes of thevma4-1 ts allele. The unassembled 27-kDa subunit made in the vma4-1 ts cells is rapidly degraded, particularly at 37 °C, but can be protected from degradation by prior assembly into the V-ATPase complex. In purified vacuolar vesicles from the mutant cells, the peripheral subunits are localized to the vacuolar membrane at decreased levels and a comparably decreased level of ATPase activity (14% of the activity in wild-type vesicles) is observed. When vma4-1 ts mutant cells are shifted to pH 7.5 medium at 37 °C, the cells become enlarged and exhibit multiple large buds, elongated buds, and other abnormal morphologies, together with delocalization of actin and chitin, within 4 h. These phenotypes suggest connections between the vacuolar ATPase, bud morphology, and cytokinesis that had not been recognized previously.

Inhibitors of vacuolar ATPase proton pumps inhibit human prostate cancer cell invasion and prostate-specific antigen expression and secretion.

Vacuolar ATPases (V‐ATPases) comprise specialized and ubiquitously distributed pumps that acidify intracellular compartments and energize membranes. To gain new insights into the roles of V‐ATPases in prostate cancer (PCa), we studied the effects of inhibiting V‐ATPase pumps in androgen‐dependent (LNCaP) and androgen‐independent (C4‐2B) cells of a human PCa progression model. Treatment with nanomolar concentrations of the V‐ATPase inhibitors bafilomycin A or concanamycin A reduced the in vitro invasion in both cell types by 80%, regardless that V‐ATPase was prominent at the plasma membrane of C4‐2B cells and only traces were detected in the low‐metastatic LNCaP parental cells. In both cell types, intracellular V‐ATPase was excessive and co‐localized with prostate‐specific antigen (PSA) in the Golgi compartment. V‐ATPase inhibitors reversibly excluded PSA from the Golgi and led to the accumulation of largely dispersed PSA‐loaded vesicles of lysosomal composition. Inhibition of acridine orange staining and transferrin receptor recycling suggested defective endosomal and lysosomal acidification. The inhibitors, additionally, interfered with the AR‐PSA axis under conditions that reduced invasion. Bafilomycin A significantly reduced steady‐state and R1881‐induced PSA mRNA expression and secretion in the LNCaP cells which are androgen‐dependent, but not in the C4‐2B cells which are androgen ablation‐resistant. In the C4‐2B cells, an increased susceptibility to V‐ATPase inhibitors was detected after longer treatments, as proliferation was reduced and reversibility of bafilomycin‐induced responses impaired. These findings make V‐ATPases attractive targets against early and advanced PCa tumors.

Wild-type and Mutant Vacuolar Membranes Support pH-dependent Reassembly of the Yeast Vacuolar H+-ATPase in Vitro

Treatment of the yeast vacuolar proton-translocating ATPase (H+-ATPase) with 300 mM KI in the presence of 5 mM MgATP results in a 90% inhibition of ATPase activity accompanied by removal of at least five of the peripheral subunits of the enzyme from the membrane. Functional reassembly of the enzyme, as indicated by reattachment of the peripheral subunits and a partial (30-70%) recovery of ATPase activity, could be achieved by dialysis of the stripped wild-type membranes to remove the KI and MgATP, but proved to be strongly pH-dependent, with optimal reassembly and recovery of activity occurring after dialysis at pH 5.5. Vacuolar membranes isolated from vma2Δ mutants, which lack one of the peripheral subunits of the enzyme, do not contain any of the peripheral subunits but are shown to contain assembled membrane (Vo) complexes. The vma2Δ mutant vacuoles are demonstrated to be competent for attachment of KI-stripped peripheral subunits and reactivation of ATPase activity. The results indicate that previously assembled Vo complexes are capable of inducing assembly of the peripheral subunits, both with each other and with the membrane subunits, and of activating the ATPase activity that resides in the peripheral subunits in a pH-dependent manner.

Identification of a Domain in the Vo Subunit d That Is Critical for Coupling of the Yeast Vacuolar Proton-translocating ATPase

Vacuolar proton-translocating ATPase pumps consist of two domains, V1 and Vo. Subunit d is a component of Vo located in a central stalk that rotates during catalysis. By generating mutations, we showed that subunit d couples ATP hydrolysis and proton transport. The mutation F94A strongly uncoupled the enzyme, preventing proton transport but not ATPase activity. C-terminal mutations changed coupling as well; ATPase activity was decreased by 59-72%, whereas proton transport was not measurable (E328A) or was moderately reduced (E317A and C329A). Except for W325A, which had low levels of V1Vo, mutations allowed wild-type assembly regardless of the fact that subunits E and d were reduced at the membrane. N- and C-terminal deletions of various lengths were inhibitory and gradually destabilized subunit d, limiting V1Vo formation. Both N and C terminus were required for Vo assembly. The N-terminal truncation 2-19Δ prevented V1Vo formation, although subunit d was available. The C terminus was required for retention of subunits E and d at the membrane. In addition, the C terminus of its bacterial homolog (subunit C from T. thermophilus) stabilized the yeast subunit d mutant 310-345Δ and allowed assembly of the rotor structure with subunits A and B. Structural features conserved between bacterial and eukaryotic subunit d and the significance of domain 3 for vacuolar proton-translocating ATPase function are discussed.

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

Background: Vacuolar ATPase (V-ATPase) proton pumps maintain pH homeostasis. Results: We discovered new V-ATPase inhibitors that uncouple the proton transport and ATPase activity of the pump. Conclusion: Residues at the tether connecting V0 subunit a to the membrane give uncoupling potential to V-ATPases. Significance: The tether may offer new mechanisms to regulate V-ATPase and cellular pH in vivo by uncoupling the pump. 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 (EC50 = 39 μm) and thonzonium bromide (EC50 = 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 V0 were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V1V0 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.

Deletion of Vacuolar Proton-translocating ATPase Voa Isoforms Clarifies the Role of Vacuolar pH as a Determinant of Virulence-associated Traits in Candida albicans

Background: V-ATPase regulates pH, and Candida albicans virulence is pH-dependent. Results: Deletion of V-ATPase Voa subunit Vph1p, but not Stv1p, alkalinizes vacuoles; several virulence-related traits remain unaffected. Conclusion: Vacuolar acidification is not essential for in vitro filamentation, biofilm formation, and macrophage killing in C. albicans. Significance: Stv1p in non-vacuolar organelles may play important roles in C. albicans infectivity, particularly if Vph1p is not functional. Vacuolar proton-translocating ATPase (V-ATPase) is a central regulator of cellular pH homeostasis, and inactivation of all V-ATPase function has been shown to prevent infectivity in Candida albicans. V-ATPase subunit a of the Vo domain (Voa) is present as two fungal isoforms: Stv1p (Golgi) and Vph1p (vacuole). To delineate the individual contribution of Stv1p and Vph1p to C. albicans physiology, we created stv1Δ/Δ and vph1Δ/Δ mutants and compared them to the corresponding reintegrant strains (stv1Δ/ΔR and vph1Δ/ΔR). V-ATPase activity, vacuolar physiology, and in vitro virulence-related phenotypes were unaffected in the stv1Δ/Δ mutant. The vph1Δ/Δ mutant exhibited defective V1Vo assembly and a 90% reduction in concanamycin A-sensitive ATPase activity and proton transport in purified vacuolar membranes, suggesting that the Vph1p isoform is essential for vacuolar V-ATPase activity in C. albicans. The vph1Δ/Δ cells also had abnormal endocytosis and vacuolar morphology and an alkalinized vacuolar lumen (pHvph1Δ/Δ = 6.8 versus pHvph1Δ/ΔR = 5.8) in both yeast cells and hyphae. Secreted protease and lipase activities were significantly reduced, and M199-induced filamentation was impaired in the vph1Δ/Δ mutant. However, the vph1Δ/Δ cells remained competent for filamentation induced by Spider media and YPD, 10% FCS, and biofilm formation and macrophage killing were unaffected in vitro. These studies suggest that different virulence mechanisms differentially rely on acidified vacuoles and that the loss of both vacuolar (Vph1p) and non-vacuolar (Stv1p) V-ATPase activity is necessary to affect in vitro virulence-related phenotypes. As a determinant of C. albicans pathogenesis, vacuolar pH alone may prove less critical than originally assumed.

Candida albicans VMA3 is necessary for V-ATPase assembly and function and contributes to secretion and filamentation.

ABSTRACT The vacuolar membrane ATPase (V-ATPase) is a protein complex that utilizes ATP hydrolysis to drive protons from the cytosol into the vacuolar lumen, acidifying the vacuole and modulating several key cellular response systems in Saccharomyces cerevisiae. To study the contribution of V-ATPase to the biology and virulence attributes of the opportunistic fungal pathogen Candida albicans, we created a conditional mutant in which VMA3 was placed under the control of a tetracycline-regulated promoter (tetR-VMA3 strain). Repression of VMA3 in the tetR-VMA3 strain prevents V-ATPase assembly at the vacuolar membrane and reduces concanamycin A-sensitive ATPase-specific activity and proton transport by more than 90%. Loss of C. albicans V-ATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. The tetR-VMA3 strain also displays abnormal vacuolar morphology, indicative of defective vacuolar membrane fission. The tetR-VMA3 strain has impaired aspartyl protease and lipase secretion, as well as attenuated virulence in an in vitro macrophage killing model. Repression of VMA3 suppresses filamentation, and V-ATPase-dependent filamentation defects are not rescued by overexpression of RIM8, MDS3, EFG1, CST20, or UME6, which encode positive regulators of filamentation. Specific chemical inhibition of Vma3p function also results in defective filamentation. These findings suggest either that V-ATPase functions downstream of these transcriptional regulators or that V-ATPase function during filamentation involves independent mechanisms and alternative signaling pathways. Taken together, these data indicate that V-ATPase activity is a fundamental requirement for several key virulence-associated traits in C. albicans.

Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry.

Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC(50)=26 microM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved.

Saccharomyces cerevisiae Vacuolar H+-ATPase Regulation by Disassembly and Reassembly: One Structure and Multiple Signals

ABSTRACT Vacuolar H+-ATPases (V-ATPases) are highly conserved ATP-driven proton pumps responsible for acidification of intracellular compartments. V-ATPase proton transport energizes secondary transport systems and is essential for lysosomal/vacuolar and endosomal functions. These dynamic molecular motors are composed of multiple subunits regulated in part by reversible disassembly, which reversibly inactivates them. Reversible disassembly is intertwined with glycolysis, the RAS/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, and phosphoinositides, but the mechanisms involved are elusive. The atomic- and pseudo-atomic-resolution structures of the V-ATPases are shedding light on the molecular dynamics that regulate V-ATPase assembly. Although all eukaryotic V-ATPases may be built with an inherent capacity to reversibly disassemble, not all do so. V-ATPase subunit isoforms and their interactions with membrane lipids and a V-ATPase-exclusive chaperone influence V-ATPase assembly. This minireview reports on the mechanisms governing reversible disassembly in the yeast Saccharomyces cerevisiae, keeping in perspective our present understanding of the V-ATPase architecture and its alignment with the cellular processes and signals involved.