Involvement of MoVMA11, a Putative Vacuolar ATPase c' Subunit, in Vacuolar Acidification and Infection-Related Morphogenesis of Magnaporthe oryzae.
ABSTRACT: 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 Magnaportheoryzae, 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 ?Movma11 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.
Project description:Subunit D of vacuolar H(+)-ATPase (V-ATPase) from bovine chromaffin granules was subjected to partial proteolysis and amino acid sequencing. A cDNA encoding this subunit was isolated and sequenced. The predicted open reading frame encodes a protein of 247 amino acids with a calculated molecular weight of 28,336. Northern blot analysis revealed an mRNA distribution with higher transcript amounts in tissues that are active in secretion. A homologous gene was identified as open reading frame 11 in chromosome V of Saccharomyces cerevisiae. The two proteins exhibit 55% identity with several conservative replacements. Interruption of the yeast gene, denoted as VMA8, resulted in the null mutant delta vma8::URA3 that, like all the other V-ATPase null mutants, did not grow on medium buffered at pH 7.5 and showed no accumulation of quinacrine into their vacuoles. Transformation of the null mutant with a plasmid containing the VMA8 gene restored the wild-type phenotype. This supports the conclusion that subunit D is an integral subunit of the catalytic sector of V-ATPase and its structural analysis suggests analogy to the gamma subunit of F-ATPases.
Project description:Hyperosmotic stress activates an array of cellular detoxification mechanisms, including the high-osmolarity glycerol (HOG) pathway. We report here that vacuolar H(+)-ATPase (V-ATPase) activity helps provide osmotic tolerance in Saccharomyces cerevisiae. V-ATPase subunit genes exhibit complex haploinsufficiency interactions with HOG pathway components. vma mutants lacking V-ATPase function are sensitive to high concentrations of salt and exhibit Hog1p activation even at low salt concentrations, as demonstrated by phosphorylation of Hog1p, a shift in Hog1-green fluorescent protein localization, transcriptional activation of a subset of HOG pathway effectors, and transcriptional inhibition of parallel mitogen-activated protein kinase pathway targets. vma2? hog1? and vma3? pbs2? double mutants have a synthetic growth phenotype, poor salt tolerance, and an aberrant, hyper-elongated morphology on solid media, accompanied by activation of a filamentous response element-LacZ construct, indicating cross talk into the filamentous growth pathway. Vacuoles isolated from wild-type cells briefly exposed to salt show higher levels of V-ATPase activity, and Na(+)/H(+) exchange in isolated vacuolar vesicles suggests a biochemical basis for the genetic interactions observed. V-ATPase activity is upregulated during salt stress by increasing assembly of the catalytic V(1) sector with the membrane-bound V(o) sector. Together, these data suggest that the V-ATPase acts in parallel with the HOG pathway in order to mediate salt detoxification.
Project description: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 vma2? 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 vma2? 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 tsa2? 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.
Project description:The regulator of ATPase of vacuoles and endosomes (RAVE) complex is implicated in vacuolar H(+)-translocating ATPase (V-ATPase) assembly and activity. In yeast, rav1 mutants exhibit a Vma(-) growth phenotype characteristic of loss of V-ATPase activity only at high temperature. Synthetic genetic analysis identified mutations that exhibit a full, temperature-independent Vma(-) growth defect when combined with the rav1 mutation. These include class E vps mutations, which compromise endosomal sorting. The synthetic Vma(-) growth defect could not be attributed to loss of vacuolar acidification in the double mutants, as there was no vacuolar acidification in the rav1 mutant. The yeast V-ATPase a subunit is present as two isoforms, Stv1p in Golgi and endosomes and Vph1p in vacuoles. Rav1p interacts directly with the N-terminal domain of Vph1p. STV1 overexpression suppressed the growth defects of both rav1 and rav1vph1, and allowed RAVE-independent assembly of active Stv1p-containing V-ATPases in vacuoles. Mutations causing synthetic genetic defects in combination with rav1 perturbed the normal localization of Stv1-green fluorescent protein. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vma(-) phenotypes arise from defects in Vph1p-containing complexes caused by rav1, combined with defects in Stv1p-containing V-ATPases caused by the second mutation. Thus RAVE is the first isoform-specific V-ATPase assembly factor.
Project description:During fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H(+)-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in ?vma2 and ?vma3 mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of vma mutants resulted from severe cytosolic acidification. Interestingly, the vma mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol.The yeast Saccharomyces cerevisiae has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains used in alcoholic fermentation. The results revealed that straight-chain alcohols induced cytosolic and vacuolar acidification through their membrane-permeabilizing effects. Contrary to expectations, a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress, but not in the maintenance of intracellular pH, seems to be important for protecting yeast cells against ethanol stress. These findings will expand our understanding of the mechanisms of ethanol tolerance and provide promising clues for the development of ethanol-tolerant yeast strains.
Project description:The vacuolar proton pyrophosphatase (H(+) -PPase) of Toxoplasma gondii (TgVP1), a membrane proton pump, localizes to acidocalcisomes and a novel lysosome-like compartment termed plant-like vacuole (PLV) or vacuolar compartment (VAC). We report the characterization of a T. gondii null mutant for the TgVP1 gene. Propagation of these mutants decreased significantly because of deficient attachment and invasion of host cells, which correlated with deficient microneme secretion. Processing of cathepsin L (CPL) in these mutants was deficient only when the parasites were incubated in the presence of low concentrations of the vacuolar H(+) -ATPase (V-H(+) -ATPase) inhibitor bafilomycin A1 , suggesting that either TgVP1 or the T. gondii?V-H(+) -ATPase (TgVATPase) are sufficient to support CPL processing. The lack of TgVP1 did not affect processing of micronemal proteins, indicating that it does not contribute to proMIC maturations. The TgVP1?null mutants were more sensitive to extracellular conditions and were less virulent in mice. We demonstrate that T. gondii tachyzoites possess regulatory volume decrease capability during hypo-osmotic stress and this ability is impaired in TgVP1?null mutants implicating TgVP1 in osmoregulation. We hypothesize that osmoregulation is needed for host cell invasion and that TgVP1 plays a role during the normal lytic cycle of T. gondii.
Project description: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 fab1? 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 fab1? 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 fab1? vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1? or vph1? fab1? 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.
Project description:Candida albicans vacuoles are central to many critical biological processes, including filamentation and in vivo virulence. The V-ATPase proton pump is a multisubunit complex responsible for organellar acidification and is essential for vacuolar biogenesis and function. To study the function of the V?B subunit of C. albicans V-ATPase, we constructed a tetracycline-regulatable VMA2 mutant, tetR-VMA2. Inhibition of VMA2 expression resulted in the inability to grow at alkaline pH and altered resistance to calcium, cold temperature, antifungal drugs, and growth on nonfermentable carbon sources. Furthermore, V-ATPase was unable to fully assemble at the vacuolar membrane and was impaired in proton transport and ATPase-specific activity. VMA2 repression led to vacuolar alkalinization in addition to abnormal vacuolar morphology and biogenesis. Key virulence-related traits, including filamentation and secretion of degradative enzymes, were markedly inhibited. These results are consistent with previous studies of C. albicans V-ATPase; however, differential contributions of the V-ATPase Vo and V? subunits to filamentation and secretion are observed. We also make the novel observation that inhibition of C. albicans V-ATPase results in increased susceptibility to osmotic stress. Notably, V-ATPase inhibition under conditions of nitrogen starvation results in defects in autophagy. Lastly, we show the first evidence that V-ATPase contributes to virulence in an acidic in vivo system by demonstrating that the tetR-VMA2 mutant is avirulent in a Caenorhabditis elegans infection model. This study illustrates the fundamental requirement of V-ATPase for numerous key virulence-related traits in C. albicans and demonstrates that the contribution of V-ATPase to virulence is independent of host pH.
Project description:In the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y transit from the Golgi to the lysosome-like vacuole via an endosome-like intermediate compartment. The vacuolar protein sorting (vps) mutant vps28, a member of the "class E" vps mutants, accumulates vacuolar, endocytic, and late Golgi markers in an aberrant endosome-like class E compartment. Sequence analysis of VPS28 revealed an open reading frame predicted to encode a hydrophilic protein of 242 amino acids. Consistent with this, polyclonal antiserum raised against Vps28p recognized a cytoplasmic protein of 28 kDa. Disruption of VPS28 resulted in moderate defects in both biosynthetic traffic and endocytic traffic destined for the vacuole. The transport of soluble vacuolar hydrolases to the vacuole was impaired in vps28 null mutant cells (approximately 40-50% carboxypeptidase Y missorted). Internalization of the endocytic marker FM 4-64, a vital lipophilic dye, resulted in intense staining of a small intracellular compartment adjacent to an enlarged vacuole in delta vps28 cells. Furthermore, the vacuolar H+-ATPase accumulated in the perivacuolar class E compartment in delta vps28 cells, as did a-factor receptor Ste3p that was internalized from the plasma membrane. Electron microscopic analysis revealed the presence of a novel compartment consisting of stacks of curved membrane cisternae. Immunolocalization studies demonstrated that the vacuolar H+-ATPase is associated with this cupped cisternal structure, indicating that it corresponds to the class E compartment observed by fluorescence microscopy. Our data indicate that kinetic defects in both anterograde and retrograde transport out of the prevacuolar compartment in vps28 mutants result in the accumulation of protein and membrane in an exaggerated multilamellar endosomal compartment. We propose that Vps28p, as well as other class E Vps proteins, may facilitate (possibly as coat proteins) the formation of transport intermediates required for efficient transport out of the prevacuolar endosome.
Project description:Vacuolar proton-translocating ATPase (V-ATPase) is located in fungal vacuolar membranes. It is involved in multiple cellular processes, including the maintenance of intracellular ion homeostasis by maintaining acidic pH within the cell. The importance of V-ATPase in virulence has been demonstrated in several pathogenic fungi, including Candida albicans. However, it remains to be determined in the clinically important fungal pathogen Candida glabrata. Increasing multidrug resistance of C. glabrata is becoming a critical issue in the clinical setting. In the current study, we demonstrated that the plecomacrolide V-ATPase inhibitor bafilomycin B1 exerts a synergistic effect with azole antifungal agents, including fluconazole and voriconazole, against a C. glabrata wild-type strain. Furthermore, the deletion of the VPH2 gene encoding an assembly factor of V-ATPase was sufficient to interfere with V-ATPase function in C. glabrata, resulting in impaired pH homeostasis in the vacuole and increased sensitivity to a variety of environmental stresses, such as alkaline conditions (pH 7.4), ion stress (Na+, Ca2+, Mn2+, and Zn2+ stress), exposure to the calcineurin inhibitor FK506 and antifungal agents (azoles and amphotericin B), and iron limitation. In addition, virulence of C. glabrata ?vph2 mutant in a mouse model of disseminated candidiasis was reduced in comparison with that of the wild-type and VPH2-reconstituted strains. These findings support the notion that V-ATPase is a potential attractive target for the development of effective antifungal strategies.