V-ATPase Genes Research Articles

The proton pump inhibitor inhibits cell growth and induces apoptosis in human hepatoblastoma

In normal physiology, a vacuolar-type proton pump (V-ATPase) maintains an intracellular acid microenvironment in lysosome, endosome, and other endomembrane systems. Cancer cells overexpress V-ATPase compared with normal cells, and disturbances of the acid environment are thought to significantly impact the cancer cell infiltration and growth. Bafilomycin A1 (Baf-A1) is a specific inhibitor of the proton-pump inhibitor (PPI) V-ATPase. Neoplastic cells are reportedly more sensitive to Baf-A1 than normal cells, and the difference between the susceptibility to Baf-A1 in normal cells and that in cancer cells may become a target in the cancer therapy. With this in mind, we used cells of hepatoblastoma, the cancer type accounting for 80% of all childhood liver cancers, to investigate the effects of Baf-A1 as an inducer of cancer cell apoptosis and inhibitor of cancer cell reproduction Electron microscopy showed significant morphological change of the hepatoblastoma cells of the Baf-A1-treated group compared with hepatoblastoma cells of the Baf-A1-free group. The rate of the apoptotic cell increased, and cell reproduction was inhibited. Moreover, the analysis of hepatoblastoma cells using the gene Chip gene expression analysis arrays showed that three of the 27 V-ATPase-related transcripts (ATP6V0D2, ATP6V1B1, and ATP6V0A1) were more weakly expressed in the Baf-A1-treated cells than in the Baf-A1-free cells. In normal human hepatic cells, on the other hand, the inhibition of cell growth of the Baf-A1-treated cells was negligible compared to that of the cells without Baf-A1 treatment. The result of apoptotic cell detection by morphological observations and flow cytometry revealed that Baf-A1 inhibits hepatoblastoma cellular reproduction by inducing apoptosis. On the other hand, the Baf-A1-conferred inhibition of cell growth was negligible in normal human hepatocytes The V-ATPase inhibitor Baf-A1 has been proven to selectively inhibit the reproduction and induce the apoptosis of hepatoblastoma cells without adversely influencing normal hepatic cells. With these effects, V-ATPase inhibitors may hold promise as therapeutic agents for hepatoblastoma. Given that three V-ATPase-related genes (ATP6V0D2, ATP6V1B1, and ATP6V0A1) were more weakly expressed in the hepatoblastoma cells of the Baf-A1-treated group than in the Baf-A1-free cells, drug development targeting V-ATPase gene of hepatoblastomas is expected.

Identification of two partners from the bacterial Kef exchanger family for the apical plasma membrane V-ATPase of Metazoa.

The vital task of vectorial solute transport is often energised by a plasma membrane, proton-motive V-ATPase. However, its proposed partner, an apical alkali-metal/proton exchanger, has remained elusive. Here, both FlyAtlas microarray data and in situ analyses demonstrate that the bacterial kefB and kefC (members of the CPA2 family) homologues in Drosophila, CG10806 and CG31052, respectively, are both co-expressed with V-ATPase genes in transporting epithelia. Immunocytochemistry localises endogenous CG10806 and CG31052 to the apical plasma membrane of the Malpighian (renal) tubule. YFP-tagged CG10806 and CG31052 both localise to the plasma membrane of Drosophila S2 cells, and when driven in principal cells of the Malpighian tubule, they localise specifically to the apical plasma membrane. V-ATPase-energised fluid secretion is affected by overexpression of CG10806, but not CG31052; in the former case, overexpression causes higher basal rates, but lower stimulated rates, of fluid secretion compared with parental controls. Overexpression also impacts levels of secreted Na+ and K+. Both genes rescue exchanger-deficient (nha1 nhx1) yeast, but act differently; CG10806 is driven predominantly to the plasma membrane and confers protection against excess K+, whereas CG31052 is expressed predominantly on the vacuolar membrane and protects against excess Na+. Thus, both CG10806 and CG31052 are functionally members of the CPA2 gene family, colocalise to the same apical membrane as the plasma membrane V-ATPase and show distinct ion specificities, as expected for the Wieczorek exchanger.

Function and Subunit Interactions of the N-terminal Domain of Subunit a (Vph1p) of the Yeast V-ATPase

The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps that operate by a rotary mechanism in which ATP hydrolysis drives rotation of a ring of proteolipid subunits relative to subunit a within the integral V(0) domain. In vivo dissociation of the V-ATPase (an important regulatory mechanism) generates a V(0) domain that does not passively conduct protons. EM analysis indicates that the N-terminal domain of subunit a approaches the rotary subunits in free V(0), suggesting a possible mechanism of silencing passive proton transport. To test the hypothesis that the N-terminal domain inhibits passive proton flux by preventing rotation of the proteolipid ring in free V(0), factor Xa cleavage sites were introduced between the N- and C-terminal domains of subunit a (the Vph1p isoform in yeast) to allow its removal in vitro after isolation of vacuolar membranes. The mutant Vph1p gave rise to a partially uncoupled V-ATPase complex. Cleavage with factor Xa led to further loss of coupling of proton transport and ATP hydrolysis. Removal of the N-terminal domain by cleavage with factor Xa and treatment with KNO3 and MgATP did not, however, lead to an increase in passive proton conductance by free V(0), suggesting that removal of the N-terminal domain is not sufficient to facilitate passive proton conductance through V(0). Photoactivated cross-linking using the cysteine reagent maleimido benzophenone and single cysteine mutants of subunit a demonstrated the proximity of specific sites within the N-terminal domain and subunits E and G of the peripheral stalk. These results suggest that a localized region of the N-terminal domain (residues 347-369) is important in anchoring the peripheral stator in V1V0.

The V-ATPase in Paramecium: functional specialization by multiple gene isoforms

The vacuolar H(+)-ATPase (V-ATPase), a multisubunit, adenosine triphosphate (ATP)-driven proton pump, is essential for numerous cellular processes in all eukaryotes investigated so far. While structure and catalytic mechanism are similar to the evolutionarily related F-type ATPases, the V-ATPase's main function is to establish an electrochemical proton potential across membranes using ATP hydrolysis. The holoenzyme is formed by two subcomplexes, the transmembraneous V(0) and the cytoplasmic V(1) complexes. Sequencing of the whole genome of the ciliate Paramecium tetraurelia enabled the identification of virtually all the genes encoding V-ATPase subunits in this organism and the studying of the localization of the enzyme and roles in membrane trafficking and osmoregulation. Surprisingly, the number of V-ATPase genes in this free-living protozoan is strikingly higher than in any other species previously studied. Especially abundant are V(0)-a-subunits with as many as 17 encoding genes. This abundance creates the possibility of forming a large number of different V-ATPase holoenzymes by combination and has functional consequences by differential targeting to various organelles.

Vacuolar-type proton ATPase as regulator of membrane dynamics in multicellular organisms

Acidification inside membrane compartments is a common feature of all eukaryotic cells. The acidic milieu is involved in many physiological processes including secretion, protein processing, and others. However, its cellular relevance has not been well established beyond the results of in vitro studies involving cultured cell systems. In the last decade, human and mouse genetics have revealed that the acidification machinery is implicated in multiple pathophysiological disorders, and thus our understanding of physiological consequences of the defective acidification in multicellular organisms has improved. In invertebrates including Drosophila and nematodes, mutations of V-ATPase were found to lead the development of rather unexpected phenotypes. Studies have suggested that V-ATPase may be involved in membrane fusion and vesicle formation, important processes for membrane trafficking, and have further implied its involvement in cell-cell fusion. This rather novel idea arose from the phenotypes associated with genetic disorders involving V-ATPase genes in various genetic model systems. In this article, we focus and overview the non-classical, beyond proton-pumping function of the vacuolar-type ATPase in exo/endocytic systems.

Effect of hot water treatments on chilling injury and heat damage in ‘satsuma’ mandarins: Antioxidant enzymes and vacuolar ATPase, and pyrophosphatase

Susceptibility of ‘satsuma’ mandarin fruit (Citrus unshiu Marc) to chilling injury limits the marketability of stored fruit. In this study, the effect of hot water dip (HWD) treatments on chilling injury tolerance, fruit quality, activity of antioxidant enzymes, and gene expression of proton transporters in the vacuole were investigated in New Zealand. Some treatments suppressed chilling injury development, but none of them could prevent it completely. The major reduction in chilling injury incidence (proportion of unacceptable fruit) and severity (level of damage) was observed in HWD at 50 °C, for 2 min. Temperatures higher than 50 °C increased fruit peel damage. Chilling injury was paralleled by higher ethylene production and respiration rate in affected fruit, and an increase in the amount of ethanol and acetaldehyde emitted in juice headspace. Afterwards, values decreased gradually with increasing chilling damage. Treatments that reduced chilling injury suppressed anaerobic products, ethylene evolution and respiration. During storage at 2 °C for 8 weeks, a decline in catalase (CAT) activity was observed, while peroxidase (POX) activity increased. This rapid increase in POX activity was associated with increased peel damage due to both chilling injury and heat damage. Decreasing chilling injury in HWD was correlated with decreased POX activity and maintenance of CAT activity during storage. Amounts of vacuolar pyrophosphatase (V-PPase) and vacuolar ATPase (V-ATPase) gene transcript in fruit peel were reduced by heat treatment. Storage periods reduced V-PPase and induced V-ATPase gene transcript level in fruit treated at 50 °C, but this was not associated with changes in the acid content in the juice. A significant induction (5–6-fold) of ATPase and V-PPase occurred in juice sacs and this may be associated with compensation for a loss of tonoplast integrity in fruit treated at 55 °C compared to those at 50 °C. We also found that V-ATPase and V-PPase transcript levels in juice sacs responded rapidly to heat treatments but were largely recovered after 8 weeks cold storage for successful (non-damaging) heat treatments. The optimal HWD treatments were 47.5–50 °C for 2 min.

Transcript level regulation of the vacuolar H+-ATPase subunit isoforms VHA-a, VHA-E and VHA-G inArabidopsis thaliana

The presence of isogenes encoding V-ATPase subunits seems to be a characteristic for plants. Twenty-eight genes encode for the 13 different subunits in Arabidopsis thaliana, 23 genes each are known in tomato (Solanum lycopersicum) and can be identified in rice (Oryza sativa), respectively. In Arabidopsis the four subunits VHA-B, -E, -G and -a are encoded by three isogenes each. The transcript levels of these subunits were analysed by in silico evaluation of transcript pattern derived from the NASC-array database and exemplarily confirmed by semiquantitative RT-PCR. A tissue specifity was observed for the isoforms of VHA-E and VHA-G, whereas expression of VHA-a isoforms appeared independent of the tissue. Inflicting environmental stresses upon plants resulted in differentiated expression patterns of VHA-isoforms. Whereas salinity had minor effect on the expression of V-ATPase genes in A. thaliana, heat and drought stress led to alterations in transcript amount and preference of isoforms. Correlation analysis identified two clusters of isoforms, which were co-regulated on the transcript level.

Genome-wide survey of V-ATPase genes inDrosophilareveals a conserved renal phenotype for lethal alleles

V-ATPases are ubiquitous, vital proton pumps that play a multiplicity of roles in higher organisms. In many epithelia, they are the major energizer of cotransport processes and have been implicated in functions as diverse as fluid secretion and longevity. The first animal knockout of a V-ATPase was identified in Drosophila, and its recessive lethality demonstrated the essential nature of V-ATPases. This article surveys the entire V-ATPase gene family in Drosophila, both experimentally and in silico. Adult expression patterns of most of the genes are shown experimentally for the first time, using in situ hybridization or reporter gene expression, and these results are reconciled with published expression and microarray data. For each subunit, the single gene identified previously by microarray, as upregulated and abundant in tubules, is shown to be similarly abundant in other epithelia in which V-ATPases are known to be important; there thus appears to be a single dominant "plasma membrane" V-ATPase holoenzyme in Drosophila. This provides the most comprehensive view of V-ATPase expression yet in a multicellular organism. The transparent Malpighian tubule phenotype first identified in lethal alleles of vha55, the gene encoding the B-subunit, is shown to be general to those plasma membrane V-ATPase subunits for which lethal alleles are available, and to be caused by failure to accumulate uric acid crystals. These results coincide with the expression view of the gene family, in which 13 of the genes are specialized for epithelial roles, whereas others have spatially or temporally restricted patterns of expression.

Structural and functional characterization of two human V-ATPase subunit gene promoters

The vacuolar-ATPase (V-ATPase) is a multi-subunit enzyme that couples ATP hydrolysis to proton pumping across membranes. V-ATPase genes are considered to be housekeeping genes and are expressed in human neoplastic tissue and in cell lines. We have isolated and characterized several genomic clones containing the 5′-end of the human V-ATPase genes. DNA sequence analysis of the promoters of two V-ATPase subunit genes, encoding C ( ATP6C) and cʺ ( ATP6F), reveals GC-rich regions in the region of the first exon. Neither TATA- nor CCAAT-boxes were found in these promoters, but both GC-boxes and E-boxes were identified. Transient transfection analysis, using a series of 5′ nested deletions of promoter–luciferase constructs in human cancer cells, demonstrated that a positive cis-acting regulatory region was present in these TATA-less promoters. The regions between −79 and −40 of the ATP6C promoter and between −245 and −99 of the ATP6F promoter were identified as being likely to be extremely important for basal promoter activity. Electrophoretic mobility shift assays (EMSA) of these cis-regulatory regions revealed the basal promoter to be highly complex, with cooperative binding of several transcription factors, including Sp family members. These data identify the critical regulatory regions for both the ATP6C and ATP6F basal promoters and stress the functional importance of multiple protein complexes, involving the Sp family of transcription factors, in regulating gene expression.

Identification, conservation, and relative expression of V-ATPase cDNAs in tomato plants

Vacuolar H+-ATPases (V-ATPases) are multi-subunit enzymes that pump protons into intracellular compartments, thereby regulating pH levels. Because V-ATPase genes are highly conserved, using known sequences inArabidopsis to identify orthologous genes in other plants is possible. Using expressed sequence tags (ESTs) in the TIGR Tomato Gene Index, we have identified 23 distinct cDNAs representing all 13 known V-ATPase subunits. The subunits composing the 2-hexamer ring structures are the most highly conserved, whereas those forming peripheral stalks are the least conserved. Relative expression analyses using the same data set suggested several broad patterns of gene expression in tomato plants. V-ATPase ESTs were found in 26 of 27 libraries, normally within a 3-fold level of relative expression. EST frequencies were highest in roots, then in fruits, and essentially identical in all other tissues. In the libraries sampled, relative expression values for individual subunits varied widely and suggest that mRNAs encoding subunits a, e, and F are in low abundance in most tomato plant tissues. Finally, V-ATPase relative expression levels increased over the course of fruit ripening. The identification of cDNAs, description of conservation patterns, and preliminary expression analyses in this work should open the door for future V-ATPase studies in tomato plants.

Expression of Manduca sexta V-ATPase genes mvB, mvG and mvd is regulated by ecdysteroids.

V-ATPases are complex proteins consisting of a peripheral, ATP-hydrolysing V(1) complex and a membrane-bound H(+)-translocating V(o) complex. The plasma membrane V-ATPase from the tobacco hornworm (Manduca sexta) midgut is made up of eight different V(1) and four different V(o) subunits. During starvation and moulting, V-ATPase activity decreases as a result of the dissociation of the V(1) complex from the V(o) complex. To determine whether subunit biosynthesis is reduced during periods of enzyme inactivity, we measured the transcript levels and transcriptional activities of V-ATPase genes. Northern blots revealed the downregulation of almost all V-ATPase transcripts during starvation. During moulting, transcript levels of the three V-ATPase genes examined, mvB, mvG and mvd, also decreased, and this decrease was negatively correlated with the titre of 20-hydroxyecdysone (20-HE) and positively correlated with the titre of juvenile hormone (JH). To test the biological significance of these correlations, we injected both hormones into feeding larvae and measured transcript levels several hours later. A short-term increase and a long-term decrease in levels of mRNA were observed after 20-HE injection, whereas JH injection had no significant effect. Immunohistochemical studies of the midgut epithelium revealed that 20-HE injection led to changes in goblet cell morphology and in the subcellular distribution of the V(1) complex comparable with the situation during the moult and during starvation. Reporter gene assays in Sf21 cells using mvB, mvG and mvd promoters to initiate transcription of firefly luciferase led, after incubation of the cells with 20-HE, to results comparable with those obtained in the injection experiments. These findings suggest that putative ecdysone-responsive elements are present in all three promoters. Taken together, our results suggest that the expression of V-ATPase genes is controlled in a coordinated manner by ecdysteroids.

Linkage assignments of the porcineATP1A2,ATP1B1andV‐ATPase(CGI‐11) genes to chromosome 4

Linkage assignments of the porcine<i>ATP1A2</i>,<i>ATP1B1</i>and<i>V‐ATPase</i>(<i>CGI‐11</i>) genes to chromosome 4

Cloning and expression of cDNAs encoding plant V-ATPase subunits in the corresponding yeast null mutants

Complementation of yeast null mutants is widely used for cloning of homologous genes from heterologous sources. We have used this method to clone the relevant V-ATPase genes from lemon fruit and Arabidopsis thaliana cDNA libraries. The pH levels are very different in the vacuoles of the lemon fruit and the A. thaliana, yet both are the result of the activity of the same enzyme complex, namely the V-ATPase. In order to investigate the mechanism that enables the enzyme to maintain such differences in pH values, we have compared the subunit composition of the V-ATPase complex from both sources. Towards this end, we have constructed a cDNA library from lemon fruit and cloned it into a similar shuttle vector to the one of the A. thaliana cDNA library, which is commercially available. In this work, we report the cloning and expression of VMA10 from both sources, two isoforms of the lemon proteolipid ( VMA3) and the lemon homologue of yeast VPH1/STV1 subunit, LEMAC.

Disruption of vma-1, the Gene Encoding the Catalytic Subunit of the Vacuolar H+-ATPase, Causes Severe Morphological Changes in Neurospora crassa

By using the process of Repeat-induced Point mutation (Selker, E. U., and Garrett, P. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6870-6874), we inactivated vma-1, the gene encoding subunit A of the V-ATPase of Neurospora crassa. Two vma-1 mutant strains were characterized. One was mutated at multiple sites, did not make a protein product, and produced spores that only rarely germinated. The other had four point mutations, made a protein product, and produced viable spores. Neither strain had detectable V-ATPase activity. The vma-1 mutant strains did not grow in medium buffered to pH 7.0 or above or in medium supplemented with the cation Zn(2+). They were completely resistant to inhibition by concanamycin C, supporting our hypothesis that the V-ATPase is the in vivo target of this antibiotic. Inactivation of the vma-1 gene had a pronounced effect on morphology and development of the organism. In the mutants tip growth was inhibited, and multiple branching was induced. The vma-1 mutant strains could not differentiate conidia or perithecia. They could grow slowly as mycelia and could donate nuclei in a sexual cross. A mutation in the plasma membrane ATPase, which suppressed the sensitivity of wild type N. crassa to concanamycin, also proved effective in suppressing the sensitivity of a vma-1 null mutant to basic pH but did not correct the morphological defects.

The multifunctional Drosophila melanogaster V-ATPase is encoded by a multigene family.

In animals, V-ATPases are believed to play roles in the plasma membrane, as well as endomembrane. To understand these different functions, it is necessary to adopt a genetic approach in a physiologically tractable model organism. For this purpose, Drosophila melanogaster is ideal, because of the powerful genetics associated with the organism and because of the unusually informative epithelial phenotype provided by the Malpighian tubule. Recently, the first animal "knockouts" of a V-ATPase were described in Drosophila. The resulting phenotypes have general utility for our understanding of V-ATPase function and suggest a screen for novel subunits and associated proteins. Genome project resources have accelerated our knowledge of the V-ATPase gene family size and the new Drosophila genes vhaSFD, vha100-1, vha100-2, vha100-3, vha16-2, vha16-3, vha16-4, vhaPPA1, vhaPPA2, vhaM9.7.1, and vhaM9.7.2 are described. The Drosophila V-ATPase model is thus well-suited to both forward and reverse genetic analysis of this complex multifunctional enzyme.

Brain Ac39/physophilin: cloning, coexpression and colocalization with synaptophysin.

Physophilin is an oligomeric protein that binds the synaptic vesicle protein synaptophysin constituting a complex that has been hypothesized to form the exocytotic fusion pore. Microsequencing of several physophilin peptides putatively identified this protein as the Ac39 subunit of the V-ATPase. Ac39 has recently been shown to be present in a synaptosomal complex which, in addition to synaptophysin, includes the bulk of synaptobrevin II, and subunits c and Ac115 of the V0 sector of the V-ATPase. We have cloned physophilin from mouse brain and found a differential region of 12 amino acids when compared with the previously reported sequence of Ac39 from bovine adrenal medulla. RT-PCR cloning from the bovine adrenal medulla demonstrates that sequencing errors occurred in the previous cloning study, and shows that the amino acid sequences of physophilin and Ac39 are completely identical. In situ hybridization in rat brain reveals a largely neuronal distribution of Ac39/physophilin mRNA which spatio-temporally correlates with those of subunit c and synaptophysin. Immunohistochemical analysis shows that Ac39/physophilin is mostly concentrated in the neuropil with a pattern identical to subunit A and very similar to synaptophysin. Double-labelling immunofluorescence shows a complete colocalization of Ac39/physophilin with subunit A and a partial colocalization with synaptophysin in the neuropil. Our findings bring anatomical support for the in vivo occurrence of the synaptophysin-Ac39/physophilin interaction and further suggest a coordinated transcription of V-ATPase and synaptophysin genes. A putative role of Ac39/physophilin in the inactivation of the V-ATPase by disassembly of its V1 sector is also discussed.