Differential localization of vacuolar H+-ATPases containing a1, a2, a3, or a4 (ATP6V0A1-4) subunit isoforms along the nephron.

Vacuolar H(+)-ATPase are multi-subunit containing pumps important for several processes along the nephron such as receptor mediated endocytosis, acidification of intracellular organelles, bicarbonate reabsorption and secretion, and H(+)- extrusion. Mutations in the human a4 (ATP6V0A4) subunit cause distal renal tubular acidosis (dRTA). There are 4 known isoforms of the 'a' subunit (a1-a4). Here we investigated the expression and localization of all four isoforms in mouse kidney. Real-time PCR detected mRNAs encoding all four 'a' isoforms in mouse kidney with a relative abundance in the following order: a4>a2=a1>a3. Immunolocalization demonstrated expression of all 'a' subunits in the proximal tubule and in the intercalated cells of the collecting system. In intercalated cells a1 and a4 isoforms appeared on both the apical and basolateral side and were expressed in all subtypes of intercalated cells. In contrast, a2, and a3 were only found in the apical membrane. a1 and a4 were colocalized in the same cells with AE1 or pendrin, whereas a2 was only found in AE1 positive cells but absent from pendrin expressing intercalated cells. These results suggest that vacuolar H(+)-ATPases containing different 'a' isoforms may serve specific and distinct functions and may help explaining why loss of the a4 isoform causes only dRTA without an apparent defect in the proximal tubule.     

Molecular cloning and characterization of a novel form of the human vacuolar H+-ATPase e-subunit: an essential proton pump component

Several of the 13 subunits comprising mammalian H(+)-ATPases have multiple alternative forms, encoded by separate genes and with differing tissue expression patterns. These may play an important role in the intracellular localization and activity of H(+)-ATPases. Here we report the cloning of a previously uncharacterized human gene, ATP6V0E2, encoding a novel H(+)-ATPase e-subunit designated e2. We demonstrate that in contrast to the ubiquitously expressed gene encoding the e1 subunit (previously called e), this novel gene is expressed in a more restricted tissue distribution, particularly kidney and brain. We show by complementation studies in a yeast strain deficient for the ortholog of this subunit, that either form of the e-subunit is essential for proper proton pump function. The identification of this novel form of the e-subunit lends further support to the hypothesis that subunit differences may play a key role in the structure, site and function of H(+)-ATPases within the cell.     

The a-subunit of the V-type H+-ATPase interacts with phosphofructokinase-1 in humans

V-type or H+-ATPases are a family of ATP-dependent proton pumps that move protons across the plasma membrane at specialized sites such as kidney epithelial cells and osteoclasts as well as acidifying intracellular compartments. The 100-kDa polytopic a-subunit of this group of ATPases is suggested to play an important role in coupling the two functions of the pump, ATP hydrolysis and proton transport. In man, different a-subunit isoforms are encoded by four genes. ATP6V0A4 encodes a4, which is expressed apically in alpha-intercalated cells in both human and mouse kidney. We sought binding partners for the C terminus of a4 in order to address its potential role in the H+-ATPase complex. Random peptide phage display analysis revealed a consensus motif (WLELRP) with almost complete homology to part of the enzyme phosphofructokinase 1 (PFK-1). Activity of this enzyme is the rate-limiting step in glycolysis. Specificity of a4 binding to this peptide was confirmed by enzyme-linked immunosorbent assay. Protein-protein interaction was further demonstrated by co-immunoprecipitation of a4 with PFK-1 from solubilized human kidney membrane proteins. An in vitro bead-bound PFK-1 pull-down assay showed that this interaction was also true for the ubiquitously expressed a1 subunit. Finally, PFK-1 co-immunolocalized with a4 in alpha-intercalated cells in the collecting ducts of human kidney. These findings indicate a direct link between V-type H+-ATPases and glycolysis via the C-terminal region of the a-subunit of the pump and suggest a novel regulatory mechanism between H+-ATPase function and energy supply. This interaction between the a-subunit and PFK-1 also provides new evidence that the C terminus of this subunit lies cytoplasmically in vivo.     

Thyrotropin-releasing hormone-induced depletion of G(q)alpha/G(11)alpha proteins from detergent-insensitive membrane domains

Na+ transport through the F0 domain of Na(+)-F1F0-ATPases involves the combined action of subunits c and a but the residues involved in Na+ liganding in subunit a are unknown. As a first step towards the identification of these residues, we have cloned and sequenced the gene encoding subunit a of the Na(+)-F1F0-ATPase of Acetobacterium woodii. This is the second sequence available now for this subunit from Na(+)-F1F0-ATPases. A comparison of subunit a from Na(+)-F1F0-ATPases with those from H(+)-translocating enzymes unraveled structural similarity in a C-terminal segment including the ultimate and penultimate transmembrane helix. Seven residues are conserved in this region and, therefore, likely to be involved in Na+ liganding.     

Aldosterone stimulates vacuolar H(+)-ATPase activity in renal acid-secretory intercalated cells mainly via a protein kinase C-dependent pathway

Urinary acidification in the collecting duct is mediated by the activity of H(+)-ATPases and is stimulated by various factors including angiotensin II and aldosterone. Classically, aldosterone effects are mediated via the mineralocorticoid receptor. Recently, we demonstrated a nongenomic stimulatory effect of aldosterone on H(+)-ATPase activity in acid-secretory intercalated cells of isolated mouse outer medullary collecting ducts (OMCD). Here we investigated the intracellular signaling cascade mediating this stimulatory effect. Aldosterone stimulated H(+)-ATPase activity in isolated mouse and human OMCDs. This effect was blocked by suramin, a general G protein inhibitor, and GP-2A, a specific G(αq) inhibitor, whereas pertussis toxin was without effect. Inhibition of phospholipase C with U-73122, chelation of intracellular Ca(2+) with BAPTA, and blockade of protein kinase C prevented the stimulation of H(+)-ATPases. Stimulation of PKC by DOG mimicked the effect of aldosterone on H(+)-ATPase activity. Similarly, aldosterone and DOG induced a rapid translocation of H(+)-ATPases to the luminal side of OMCD cells in vivo. In addition, PD098059, an inhibitor of ERK1/2 activation, blocked the aldosterone and DOG effects. Inhibition of PKA with H89 or KT2750 prevented and incubation with 8-bromoadenosine-cAMP mildly increased H(+)-ATPase activity. Thus, the nongenomic modulation of H(+)-ATPase activity in OMCD-intercalated cells by aldosterone involves several intracellular pathways and may be mediated by a G(αq) protein-coupled receptor and PKC. PKA and cAMP appear to have a modulatory effect. The rapid nongenomic action of aldosterone may participate in the regulation of H(+)-ATPase activity and contribute to final urinary acidification.     

The amino-terminal domain of the E subunit of vacuolar H(+)-ATPase (V-ATPase) interacts with the H subunit and is required for V-ATPase function

Vacuolar H(+)-ATPases (V-ATPases) are highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although it is generally believed that V-ATPases transport protons by a rotary catalytic mechanism analogous to that used by F(1)F(0)-ATPases, the structure and subunit composition of the central or peripheral stalk of the multisubunit complex are not well understood. We searched for proteins that bind to the E subunit of V-ATPase using the yeast two-hybrid assay and identified the H subunit as an interacting partner. Physical association between the E and H subunits of V-ATPase was confirmed in vitro by precipitation assays. Deletion mapping analysis revealed that a 78-amino acid fragment at the amino terminus of the E subunit was sufficient for binding to the H subunit. Expression of the amino-terminal fragments of the E subunits from human and yeast as dominant-negative mutants resulted in dramatic decreases in bafilomycin A(1)-sensitive ATP hydrolysis and proton transport activities of V-ATPase. Our data demonstrate the physiological significance of the interaction between the E and H subunits of V-ATPase and extend previous studies on the arrangement of subunits on the peripheral stalk of V-ATPase.     

CDC50 proteins are critical components of the human class-1 P4-ATPase transport machinery

Members of the P(4) subfamily of P-type ATPases catalyze phospholipid transport and create membrane lipid asymmetry in late secretory and endocytic compartments. P-type ATPases usually pump small cations and the transport mechanism involved appears conserved throughout the family. How this mechanism is adapted to flip phospholipids remains to be established. P(4)-ATPases form heteromeric complexes with CDC50 proteins. Dissociation of the yeast P(4)-ATPase Drs2p from its binding partner Cdc50p disrupts catalytic activity (Lenoir, G., Williamson, P., Puts, C. F., and Holthuis, J. C. (2009) J. Biol. Chem. 284, 17956-17967), suggesting that CDC50 subunits play an intimate role in the mechanism of transport by P(4)-ATPases. The human genome encodes 14 P(4)-ATPases while only three human CDC50 homologues have been identified. This implies that each human CDC50 protein interacts with multiple P(4)-ATPases or, alternatively, that some human P(4)-ATPases function without a CDC50 binding partner. Here we show that human CDC50 proteins each bind multiple class-1 P(4)-ATPases, and that in all cases examined, association with a CDC50 subunit is required for P(4)-ATPase export from the ER. Moreover, we find that phosphorylation of the catalytically important Asp residue in human P(4)-ATPases ATP8B1 and ATP8B2 is critically dependent on their CDC50 subunit. These results indicate that CDC50 proteins are integral part of the P(4)-ATPase flippase machinery.     

The molecular chaperone calnexin associates with the vacuolar H(+)-ATPase from oat seedlings.

Acidification of endomembrane compartments by the vacuolar-type H(+)-ATPase (V-ATPase) is central to many cellular processes in eukaryotes, including osmoregulation and protein sorting. The V-ATPase complex consists of a peripheral sector (V1) and a membrane integral sector (V0); however, it is unclear how the multimeric enzyme is assembled. A 64-kD polypeptide that had copurified with oat V-ATPase subunits has been identified as calnexin, an integral protein on the endoplasmic reticulum. To determine whether calnexin interacted physically with the V-ATPase, microsomal membranes were Triton X-100 solubilized, and the protein-protein interaction was analyzed by coimmunoprecipitation. Monoclonal antibodies against calnexin precipitated both calnexin and V-ATPase subunits, including A and B and those of 44, 42, 36, 16, and 13 kD. A monoclonal antibody against subunit A precipitated the entire V-ATPase complex as well as calnexin and BiP, an endoplasmic reticulum lumen chaperone. The results support our hypothesis that both calnexin and BiP act as molecular chaperones in the folding and assembly of newly synthesized V1V0-ATPases at the endoplasmic reticulum.     

Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration

ClC-7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H(+)-ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC-7 or the H(+)-ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC-7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC-7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron-dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC-3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H(+)-ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H(+)-ATPase and ClC-7 can underlie human osteopetrosis.     

Defined sites of interaction between subunits E (Vma4p), C (Vma5p), and G (Vma10p) within the stator structure of the vacuolar H+-ATPase

Vacuolar H(+)-ATPases (V-ATPases) are multi-subunit membrane proteins that couple ATP hydrolysis to the extrusion of protons from the cytoplasm. Although they share a common macromolecular architecture and rotational mechanism with the F(1)F(0)-ATPases, the organization of many of the specialized V-ATPase subunits within this rotary molecular motor remains uncertain. In this study, we have identified sequence segments involved in linking putative stator subunits in the Saccharomyces V-ATPase. Precipitation assays revealed that subunits Vma5p (subunit C) and Vma10p (subunit G), expressed as glutathione-S-transferase fusion proteins in E. coli, are both able to interact strongly with Vma4p (subunit E) expressed in a cell-free system. GST-Vma10p also associated with Vma2p and Vma1p, the core subunits of the ATP-hydrolyzing domain, and was able to self-associate to form a dimer. Mutations within the first 19-residue region of Vma4p, which disrupted interaction with Vma5p in vitro, also prevented the Vma4p polypeptide from restoring V-ATPase function in a complementation assay in vivo. These mutations did not prevent assembly of Vma5p (subunit C) and Vma2p (subunit B) into an inactive complex at the vacuolar membrane, indicating that Vma5p must make multiple interactions involving other V-ATPase subunits. A second, highly conserved region of Vma4p between residues 19 and 38 is involved in binding Vma10p. This region is highly enriched in charged residues, suggesting a role for electrostatic effects in Vma4p-Vma10p interaction. These protein interaction studies show that the N-terminal region of Vma4p is a key factor not only in the stator structure of the V-ATPase rotary molecular motor, but also in mediating interactions with putative regulatory subunits.     

Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio)

In the skin of zebrafish embryo, the vacuolar H(+)-ATPase (V-ATPase, H(+) pump) distributed mainly in the apical membrane of H(+)-pump-rich cells, which pump internal acid out of the embryo and function similarly to acid-secreting intercalated cells in mammalian kidney. In addition to acid excretion, the electrogenic H(+) efflux via the H(+)-ATPases in the gill apical membrane of freshwater fish was proposed to act as a driving force for Na(+) entry through the apical Na(+) channels. However, convincing molecular physiological evidence in vivo for this model is still lacking. In this study, we used morpholino-modified antisense oligonucleotides to knockdown the gene product of H(+)-ATPase subunit A (atp6v1a) and examined the phenotype of the mutants. The H(+)-ATPase knockdown embryos revealed several abnormalities, including suppression of acid-secretion from skin, growth retardation, trunk deformation, and loss of internal Ca(2+) and Na(+). This finding reveals the critical role of H(+)-ATPase in embryonic acid -secretion and ion balance, as well.     

Immunochemical comparison of cardiac glycoside-sensitive (lamb) and -insensitive (rat) kidney (Na+ + K+)-ATPase

The immunological cross-reactivity of the ouabain-sensitive lamb kidney and the ouabain-insensitive rat kidney (Na+ + K+)-ATPase (EC 3.6.1.37) was examined using polyclonal and monoclonal antibodies. Studies using rabbit antisera prepared against both the lamb kidney and rat kidney holoenzymes showed the existence of substantial antigenic differences as well as similarities between the holoenzymes and the respective denatured alpha and beta subunits of these two enzymes. Quantitation of the extent of cross-reactivity using holoenzyme-directed antibodies showed a 40-60% cross-reactivity. In addition, rabbit antisera monospecific to the purified, denatured alpha and beta subunits of the lamb kidney enzyme showed about a 50% cross-reactivity towards the respective subunit of the rat enzyme. In contrast to the cross-reactivity observed using the polyclonal antibodies, six monoclonal antibodies specific for the alpha subunit of the lamb holoenzyme exhibited no cross-reactivity with the rat holoenzyme. Four of these monoclonal antibodies, however, showed substantial cross-reactivity with rat alpha subunit as resolved by SDS-polyacrylamide gel electrophoresis. A fifth antibody did not bind to the denatured alpha subunit of either the lamb or the rat enzyme. Another monoclonal antibody (M7-PB-E9), which is specific for an epitope previously implicated in the regulation of both ATP and ouabain binding to (Na+ + K+)-ATPase (Ball, W.J., Jr. (1984) Biochemistry 2275-2281) was found to bind to the denatured lamb alpha but not to the rat alpha. This antibody has identified a region of the lamb alpha that has an altered amino acid sequence in the ouabain-insensitive rat enzyme. These immunological studies indicate that there are substantial antigenic differences between the lamb and rat kidney (Na+ + K+)-ATPases. The majority of these antigenic differences appear to be due to variations in the tertiary structures rather than to variations in the primary structures of the alpha subunits.     

Monoclonal antibody HK4001 completely inhibits K(+)-dependent ATP hydrolysis and H+ transport of hog gastric H+,K(+)-ATPase

A monoclonal antibody (designated as HK4001) was prepared against hog gastric H+,K(+)-ATPase. It dose-dependently inhibited the H+,K(+)-ATPase activity, formation of the K(+)-sensitive phosphoenzyme, and proton uptake into gastric vesicles. The H+,K(+)-ATPase activity was completely inhibited by addition of the antibody at a molar ratio of 1:2 (antibody/catalytic subunit) at pH 7.8. The maximal inhibition decreased with decrease in pH of the medium (7.8 greater than 7.4 greater than 6.2). The Fab fragment obtained by digestion of the antibody with papain was also inhibitory. The antibody did not inhibit the K(+)-dependent p-nitrophenylphosphatase or the labeling of the enzyme with fluorescein isothiocyanate. It inhibited gastric H+,K(+)-ATPase from rabbits and rats, but did not cross-react with related cation-transport ATPases (Na+,K(+)-ATPase or Ca2(+)-ATPase) or H(+)-ATPase in the multivesicular body. From these and related findings, the antibody was suggested to recognize a highly specific site on the cytosolic surface of H+,K(+)-ATPase. The conformation of the epitope was conserved after treatment with Triton X-100, but not sodium dodecyl sulfate. In addition, judging from the stoichiometry of inactivation of H+,K(+)-ATPase by this antibody, the functional unit of H+,K(+)-ATPase was suggested to be a dimer or a tetramer (not a trimer) of the catalytic unit.     

The C-terminal 165 amino acids of the plasma membrane Ca(2+)-ATPase confer Ca2+/calmodulin sensitivity on the Na+,K(+)-ATPase alpha-subunit.

The C-terminal 165 amino acids of the rat brain plasma membrane (PM) Ca(2+)-ATPase II containing the calmodulin binding auto-inhibitory domain was connected to the C-terminus of the ouabain sensitive chicken Na+,K(+)-ATPase alpha 1 subunit. Expression of this chimeric molecule in ouabain resistant mouse L cells was assured by the high-affinity binding of [3H]ouabain. In the presence of Ca2+/calmodulin, this chimeric molecule exhibited ouabain inhibitable Na+,K(+)-ATPase activity; the putative chimeric ATPase activity was absent in the absence of Ca2+/calmodulin and activated by Ca2+/calmodulin in a dose-dependent manner. Furthermore, this chimeric molecule could bind monoclonal IgG 5 specific to the chicken Na+,K(+)-ATPase alpha 1 subunit only in the presence of Ca2+/calmodulin, suggesting that the epitope for IgG 5 in this chimera is masked in the absence of Ca2+/calmodulin and uncovered in their presence. These results propose a direct interaction between the calmodulin binding auto-inhibitory domain of the PM Ca(2+)-ATPase and the specific regions of the Na+,K(+)-ATPase alpha 1 subunit that are structurally homologous to the PM Ca(2+)-ATPase. A comparison of the deduced amino acid sequences revealed several possible regions within the Na+,K(+)-ATPase that might interact with the auto-inhibitory domain of the PM Ca(2+)-ATPase.     

Cloning and sequencing of the genes for A and B subunits of the V-type Na(+)-ATPase of a facultatively anaerobic alkaliphile

The structural genes for A and B subunits of the V-type Na(+)-ATPase from a facultatively anaerobic alkaliphile (Amphibacillus sp.), strain M-12, were cloned and sequenced. Transformation of Escherichia coli with the genes overexpressed two proteins, which crossreacted with an antiserum against A and B subunits of the V-type Na(+)-ATPase from Enterococcus hirae. The deduced amino acid sequence (594 amino acids; Mr, 66,144) of A subunit of the M-12 enzyme exhibited 73%, 51%, 49% and 53% identities with those of V-type ATPases from E. hirae, Thermus thermophilus, Neurospora crassa and Drosophila melanogaster, respectively. The amino acid sequence (458 amino acids; Mr, 51,308) of B subunit of the M-12 enzyme was 74%, 53%, 52% and 54% identical with those of the ATPases from E. hirae, T. thermophilus, N. crassa and D. melanogaster, respectively. The fact indicates that the amino acid sequences of A and B subunits of the M-12 enzyme exhibit significantly higher homologies with those of the E. hirae Na(+)-ATPase as compared with those of the H(+)-ATPases from T. thermophilus, N. crassa and D. melanogaster.     

Stoichiometry of the peripheral stalk subunits E and G of yeast V1-ATPase determined by mass spectrometry

The stoichiometry of yeast V(1)-ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V(1)-ATPase complex and each of the individual protein components, using native electrospray ionization-MS. The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 and 428,300 Da, respectively. Second, defined amounts of V(1)-ATPase purified from yeast grown on (14)N-containing medium were titrated with defined amounts of (15)N-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, (14)N- and (15)N-containing peptide pairs were used for quantification of subunit stoichiometry using matrix-assisted laser desorption/ionization-time of flight MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V(1)-ATPase is A(3)B(3)DE(3)FG(3)H.     

Targeting of two Arabidopsis H(+)-ATPase isoforms to the plasma membrane.

More than 11 different P-type H(+)-ATPases have been identified in Arabidopsis by DNA cloning. The subcellular localization for individual members of this proton pump family has not been previously determined. We show by membrane fractionation and immunocytology that a subfamily of immunologically related P-type H(+)-ATPases, including isoforms AHA2 and AHA3, are primarily localized to the plasma membrane. To verify that AHA2 and AHA3 are both targeted to the plasma membrane, we added epitope tags to their C-terminal ends and expressed them in transgenic plants. Both tagged isoforms localized to the plasma membrane, as indicated by aqueous two-phase partitioning and sucrose density gradients. In contrast, a truncated AHA2 (residues 1-193) did not, indicating that the first two transmembrane domains alone are not sufficient for plasma membrane localization. Two epitope tags were evaluated: c-myc, a short, 11-amino acid sequence, and beta-glucuronidase (GUS), a 68-kD protein. The c-myc tag is recommended for its sensitivity and specific immunodetection. GUS worked well as an epitope tag when transgenes were expressed at relatively high levels (e.g. with AHA2-GUS944); however, evidence suggests that GUS activity may be inhibited when a GUS domain is tethered to an H(+)-ATPase complex. Nevertheless, the apparent ability to localize a GUS protein to the plasma membrane indicates that a P-type H(+)-ATPase can be used as a delivery vehicle to target large, soluble proteins to the plasma membrane.     

Isolation and properties of bovine kidney brush border vacuolar H(+)-ATPase. A proton pump with enzymatic and structural differences from kidney microsomal H(+)-ATPase

Vacuolar H(+)-ATPase was isolated from highly purified bovine kidney brush border, using a previously described immunoaffinity method. The affinity purified enzyme had reconstitutively active ATP-induced acidification that was inhibited by N-ethylmaleimide. The brush border H(+)-ATPase had a single pH optimum of 7.3, and a single Km for ATP of 360 microM. The enzyme showed no lipid activation; it had a substrate preference of ATP greater than ITP greater than UTP greater than GTP much greater than CTP, with an ATP:GTP selectivity of 1.69. The brush border H(+)-ATPase required no monovalent anion or cation for activity and was inhibited by the oxyanions NO3(-1) much greater than SO4(-2); sulfite stimulated activity at low concentrations and inhibited at higher concentrations. The inhibition produced by nitrate could not be attributed to dissociation of subunits from the enzyme. The divalent or trivalent cation preference was Mn+2 much greater than Mg+2 much greater than Co+2 greater than Al+3 greater than Ca+2 much greater than Ba+2,Sr+2; 1 mM Zn+2 inhibited the enzyme completely, but Cu+2 inhibited only 49% of activity at concentrations up to 5 mM. Sodium dodecyl sulfate-polyacrylamide gels of the brush border H(+)-ATPase showed subunits at Mr 70,000, a doublet at 56,000, 45,000, 42,000, 38,000, 33,000, 31,000, 15,000, 14,000, and 12,000. On two-dimensional gels, the pl value for the Mr 70,000 subunit was 6.3, for the Mr 56,000 was 6.4, and for the Mr 31,000 was 7.5-8.5, and microheterogeneity was observed in the Mr 56,000 and 31,000 subunits. A comparison of kidney cortex brush border H(+)-ATPase with kidney cortex microsomal H(+)-ATPase revealed differences in pH optimum, Km for ATP, lipid dependence, substrate preference, divalent ion preference, copper sensitivity, and in microheterogeneity of the Mr 56,000 and 31,000 subunits, providing evidence that different functional and structural classes of vacuolar H(+)-ATPase are segregated to specific membrane compartments.     

Chromosomal localization of three vacuolar-H+ -ATPase 16 kDa subunit (ATP6V0C) genes in the murine genome

Vacuolar-H(+)-ATPase (V-H-ATPase) is a large multimeric protein composed of at least 12 distinct subunits. The 16-kDa hydrophobic proteolipid subunit (ATP6V0C; ATPase, H(+ )transporting, lysosomal 16 kDa, V0 subunit C) plays a central role in H(+) transport across cellular membranes. We have mapped three ATP6V0C genes (Atp6v0c, Atp6v0c-ps1 and Atp6voc-ps2) in the murine genome. Atp6v0c-ps1 and Atp6v0c-ps2 map to Chromosomes 7 and 6, respectively. Atp6v0c maps to Chromosome 17, closely linked to the Tsc2 locus and D17Mit55. This region of Chromosome 17 in mouse is homologous with chromosome 16 in human where the ATP6V0C gene is localized.