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Results for “Vacuolar Proton-Translocating ATPases”

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At least 19 recordsLinked to original sources
PubMed1992

The vacuolar H(+)-ATPase--one of the most fundamental ion pumps in nature.

An electrochemical gradient of protons (PMF) is a universal high-energy intermediate in biological systems. Two related families of proton pumps, denoted F- and V-ATPases, are among the principal generators of a PMF from ATP and can form ATP at the expense of a PMF. The enzymes of these two families share a similar structure and subunit composition; some subunits in the two families evolved from common ancestors. Other subunits having no common ancestry were added independently to the various enzymes and defined the two separate families. The general mechanism for the proton pumping activity is similar in the two families. However, whereas F-ATPases can act in both proton pumping and ATP formation, the V-ATPases of eukaryotes function exclusively as ATP-dependent proton pumps. The catalytic and membrane sectors of F-ATPases and archaebacterial V-ATPases can separately catalyze their specific partial activities of ATPase and proton conduction. The catalytic and membrane sectors of the eukaryotic V-ATPases cannot act separately. This property is correlated with the presence of a large proteolipid that traverses the membrane four times. The gene duplication of the smaller proteolipid in the formation of the large proteolipid was one of the most important events in the evolution of the V-ATPases of eukaryotic cells.

Adenosine Triphosphatases↗
PubMed2026

A single small molecule-based human embryo model reveals V-ATPase requirement in mammalian blastocyst cavitation.

Human naïve pluripotent stem cells (nPSCs) can be induced by various combinations of signaling factors to generate blastocyst-like structures, termed blastoids. Despite rapid progress in human blastoid models, their potential to uncover fundamental mechanisms of early human development remains limited, leaving key morphogenetic processes poorly understood. Here, we describe a simple and robust system in which dimethyl sulfoxide (DMSO) alone induces blastoid formation from human nPSCs. This model recapitulates key pre- and post-implantation features and exhibits enhanced polar trophectoderm (TE) organization, more efficient attachment within an implantation-relevant window, improved epiblast lumenogenesis associated with amniotic cavity formation, and more robust, sustained expansion of embryonic lineages following attachment. Using this system, we reveal a previously unrecognized mechanism underlying TE cavitation and identify lysosome-associated genes - particularly subunits of the proton pump V-ATPase - as essential regulators of blastoid cavitation. DMSO treatment upregulates key V-ATPase subunits (ATP6V0A4 and ATP6V1B1), which are also enriched in the TE of human embryos. Genetic or pharmacological inhibition of V-ATPase activity disrupts lysosomal acidification, blocks intracellular vacuole formation, and impairs blastoid cavitation, whereas overexpression of V-ATPase subunits rescues this phenotype. Furthermore, genetic and pharmacological perturbations of V-ATPase function significantly compromise cavitation in both mouse and human blastocysts. Finally, DMSO treatment induces membrane biomechanical changes characteristic of early embryonic development, suggesting a mode of action distinct from conventional small-molecule, signaling pathway-based induction strategies. This simple DMSO-based blastoid model recapitulates key aspects of human blastocyst development and reveals a conserved requirement for V-ATPase-mediated lysosomal acidification during early mammalian embryogenesis.

Humans↗
PubMed2025

TCF25 serves as a nutrient sensor to orchestrate metabolic adaptation and cell death by enhancing lysosomal acidification under glucose starvation.

Cells adapt to nutrient limitation by activating catabolic and inhibiting anabolic pathways, yet prolonged stress may lead to cell death. How cells orchestrate metabolic adaptation and cell death to nutrient stress is poorly understood. We conduct a genome-wide CRISPR-Cas9 screen to identify regulators in glucose-starvation-induced cell death and find a group of genes in lysosomal pathway is enriched following glucose starvation. We focus on one candidate gene, Transcriptional Factor 25 (TCF25). We find TCF25 enhances lysosomal acidification by targeting V-ATPase, promoting autophagy and ATP generation under glucose starvation. However, prolonged glucose starvation constitutively activates ferritinophagy via TCF25, increasing lysosomal membrane permeability (LMP) and leading to lysosome-dependent cell death (LDCD). Knocking out TCF25 or V-ATPase components prevents cell death. Furthermore, TCF25 deficiency protects mice from hepatic ischemia-reperfusion injury. Our findings identify TCF25 as a crucial nutrient sensor that regulates lysosomal activity, offering potential therapeutic targets for metabolic and ischemic disorders.

Lysosomes↗
PubMed1992

The BPV-1 E5 protein, the 16 kDa membrane pore-forming protein and the PDGF receptor exist in a complex that is dependent on hydrophobic transmembrane interactions.

The E5 oncoprotein of bovine papillomavirus type 1 is a 44 amino acid, highly hydrophobic protein that induces the stable transformation of immortalized murine fibroblasts, presumably through its activation of growth factor receptors. Previous studies have shown that the E5 protein complexes with the 16 kDa (16k) pore-forming protein of vacuolar H(+)-ATPases. This integral membrane protein is essential for the acidification and function of subcellular compartments that process growth factor receptors. Using an SV40 expression system in COS cells, we analyzed whether the E5-16k complexes bind additional cellular proteins, including growth factor receptors. These studies demonstrate that E5 binds to both the 16k protein and the PDGF receptor and that this tri-component complex can be isolated with antibodies specific for each protein. Importantly, the 16k protein bound to the PDGF receptor in the absence of E5, suggesting that E5 binds to the PDGF receptor via its interaction with the 16k protein. An E5 mutant lacking the hydrophilic carboxyl-terminal 14 amino acids retained binding to both 16k and the PDGF receptor, indicating that E5 binds to these proteins through its hydrophobic, membrane-associating domain. These studies reveal that hydrophobic, intramembrane interactions govern the association of E5, 16k and the PDGF receptor, suggesting a ligand-independent mechanism for receptor activation and a potential link between receptor signal transduction pathways and membrane pore activity.

Adenosine Triphosphatases↗
PubMed2026

Whole exome sequencing identifies three novel variants and establishes the molecular diagnosis of ATP6V0A4-related distal renal tubular acidosis in a lebanese infant.

BACKGROUND: Distal renal tubular acidosis (dRTA) is a rare inherited disorder characterized by impaired urinary acidification, leading to metabolic acidosis, hypokalemia, nephrocalcinosis, and growth impairment. Pathogenic variants in ATP6V0A4 are among the most common genetic causes of autosomal recessive dRTA. METHODS AND RESULTS: We report a Lebanese infant presenting with failure to thrive, recurrent vomiting, severe hyperchloremic metabolic acidosis, hypokalemia, and bilateral nephrocalcinosis, in whom whole-exome sequencing (WES) was performed to establish the molecular diagnosis and perform a comprehensive genomic evaluation. WES identified three novel variants, including a novel homozygous likely pathogenic ATP6V0A4 variant, consistent with the patient's phenotype. Two additional novel variants in TTN and CEP290 were also detected. Family segregation analysis confirmed the inheritance pattern of all three variants and refined the interpretation of the additional genomic findings. The patient showed sustained clinical and biochemical improvement to alkali therapy, with normalization of biochemical abnormalities and improvement in growth during follow-up. CONCLUSIONS: This report expands the molecular spectrum of ATP6V0A4-related dRTA and illustrates the clinical utility of comprehensive WES combined with segregation analysis for accurate molecular diagnosis, variant interpretation, genetic counseling, and the evaluation of additional genomic findings in rare inherited disorders.

Humans↗
PubMed1992

Biochemistry of the renal V-ATPase.

In most eukaryotic cells, vacuolar H(+)-ATPases (V-ATPases) are present primarily or exclusively in intracellular membrane compartments, functioning in the acidification of the endocytic and secretory vacuolar apparatus necessary for constitutive cell function. V-ATPases also participate in renal hydrogen ion secretion in both the proximal and distal nephron, residing at high concentrations on the plasma membrane, where they are regulated physiologically to maintain the acid-base balance of the organism. Recent experiments have begun to reveal how the kidney controls transcellular proton transport while still maintaining acidification of intracellular compartments. Control may occur by recruitment of proton pumps to or away from the plasma membrane. The proton-transporting plasma membrane of intercalated cells is a specialized apparatus that translocates the enzyme between an intracellular membrane pool and the plasma membrane in response to physiological stimuli. Regulation may also occur by changes in the kinetics of the V-ATPase. V-ATPases are a family of structurally similar enzymes which differ in the composition of specific subunits. Cytosolic regulatory enzymes present in renal cells may preferentially affect V-ATPases in selective membrane compartments.

Adenosine Triphosphatases↗
PubMed1992

Chloride channels of intracellular organelles and their potential role in cystic fibrosis.

Chloride channels were previously purified from bovine kidney cortex membranes using a drug affinity column. Reconstitution of the purified proteins into artificial liposomes and planar bilayers yielded chloride channels. A 64 x 10(3) M(r) protein, p64, identified as a component of this chloride channel, was used to generate antibodies which depleted solubilized kidney membranes of all chloride channel activity. This antibody has now been used to identify a clone, H2B, from a kidney cDNA library. Antibodies, affinity-purified against the fusion protein of H2B, from a kidney cDNA library. Antibodies, affinity-purified against the fusion protein of H2B, also depleted solubilized kidney cortex from all chloride channel activity. The predicted amino acid sequence of p64 shows that it contains two and possibly four putative transmembrane domains and potential phosphorylation sites by protein kinases A and C. There was no significant homology to other protein (or DNA) sequences in the data base including other anion channels or the cystic fibrosis transmembrane conductance regulator. The protein is expressed in all cells tested and probably represents the chloride channel of intracellular organelles. Cystic fibrosis (CF) is associated with a defect in a cyclic-AMP-activated chloride channel in secretory epithelia which leads to decreased fluid secretion. In addition, many mucus glycoproteins show decreased sialylation but increased sulfation. We have recently shown that the pH of intracellular organelles is more alkaline in CF cells, an abnormality that is due to defective chloride conductance in the vesicle membranes. We postulate that the defect in the intracellular chloride channel, and hence the alkalization, could explain the glycosylation abnormalities since the pH optimum of Golgi sialyltransferase is acid while that of focusyl- and sulfotransferases is alkaline. Defects in sialyation of glycolipids might also generate receptors for Pseudomonas, which is known to colonize the respiratory tract of CF patients.

Adenosine Triphosphatases↗
PubMed1992

The osteoclast as a unicellular proton-transporting epithelium.

Osteoclasts dissolve bone mineral by the vectorial secretion of hydrogen ion at their osseous attachment site. To accomplish this, the osteoclast employs a vacuolar H+ ATPase that is polarized to the specialized proton-secreting plasma membrane domain, the ruffled border. Physiologically and biochemically, they resemble the specialized proton-secreting intercalated cells of the renal collecting duct, which also use a polarized vacuolar H+ ATPase to effect transepithelial hydrogen ion transport. Studies on the mechanism of hydrogen ion transport by the kidney may therefore provide insights into the control of acid secretion by the osteoclast.

Animals↗
PubMed1992

Inhibitory effects of HSP70 chaperones on nascent polypeptides.

Several of the major heat shock proteins (HSPs) function normally as molecular chaperones to prevent aggregation of immature polypeptides and thereby facilitate folding and oligomerization. To determine their effect on nascent polypeptides, we added purified preparations of different isoforms of HSP70 to in vitro translation reactions primed by the 26S mRNA of Sindbis virus, which encodes an autoprotease that functions cotranslationally, or by the mRNA encoding the yeast vacuolar H+ATPase, which is formed by a novel transpeptidase activity that removes the central region of the initial polypeptide. In the presence of HSP70s both the autoprotease and transpeptidase activities were inhibited, indicating that these chaperones can interact with nascent polypeptides and, in the cases studied here, perturb their normal structures.

Animals↗
PubMed1992

Plasma membrane Na(+)-H+ antiporter and H(+)-ATPase in the medullary thick ascending limb of rat kidney.

To characterize H+ transport mechanisms in a fresh suspension of rat medullary thick ascending limb (MTAL) tubules, we have monitored intracellular pH (pHi) with use of the fluorescent probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. First, a Na(+)-H+ antiporter was identified in bicarbonate-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered media at 25 degrees C. pHi recovery of Na-depleted acidified cells was dependent on extracellular sodium concentration, which was inhibited by amiloride in a manner consistent with simple competitive interaction with one external transport site (amiloride Ki = 1.5-2.1 x 10(-5) M); Na-induced pHi recovery of acidified cells was electroneutral since it was not affected by 5 or 100 mM extracellular potassium in the presence or absence of valinomycin. Second, at 37 degrees C, pHi recovery after acute intracellular acidification caused by 40 mM acetate addition to cell suspension was inhibited 36% by 200-400 nM bafilomycin A1, a macrolide antibiotic that specifically inhibits vacuolar-type H(+)-ATPase at submicromolar concentrations. In addition, amiloride-insensitive pHi recovery was inhibited by bafilomycin A1, 10(-3) M N-ethylmaleimide, and 10(-4) M preactivated omeprazole but not by 10(-5) M vanadate, 10(-4) M SCH 28080, or removal of extracellular potassium. Also, metabolic inhibition by absence of substrate, 10(-4) M KCN, or 5 x 10(-4) M iodoacetic acid inhibited amiloride-insensitive pHi recovery. The inhibitory effects of absence of metabolic substrate and iodoacetic acid were removed by reexposure to glucose and L-leucine and by exogenous ATP, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1992

Radiation-inactivation analysis of vacuolar H(+)-ATPase and H(+)-pyrophosphatase from Beta vulgaris L. Functional sizes for substrate hydrolysis and for H+ transport.

The functional sizes of the vacuolar H(+)-ATPase (V-ATPase; EC 3.6.1.34) and H(+)-pyrophosphatase (PPase; EC 3.6.1.1) from vacuolar membranes of red beet (Beta vulgaris L.) were estimated by radiation inactivation, both for substrate hydrolysis and for H+ transport. For the V-ATPase, the radiation-inactivation size for H+ transport was 446 (403-497) kDa and that for ATP hydrolysis was 394 (359-435) kDa. The low values of both of these estimates suggest that not all subunits which may co-purify with V-ATPases are required for either hydrolysis or transport. For the PPase, the radiation-inactivation size for hydrolysis was 91 (82-103) kDa, suggesting that the minimum functional unit for hydrolysis is the 81 kDa monomer. In contrast to the V-ATPase, the PPase gave a radiation-inactivation size for transport which was 3-4-fold larger than that for hydrolysis (two estimates for transport gave 307 and 350 kDa), indicating that a single catalytic subunit is insufficient for transport activity.

Adenosine Triphosphate↗
PubMed1992

H+ extrusion by an apical vacuolar-type H(+)-ATPase in rat renal proximal tubules.

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1992

Connexins and the vacuolar proteolipid-like 16-kDa protein are not directly associated with each other but may be components of similar or the same gap junctional complexes.

Gap junction preparations made from mouse liver plasma membranes by alkali extraction contain variable proportions of connexins (Cx32 and Cx26) and the 16-kDa protein which is closely related or may be identical to the 16-kDa proteolipid (subunit c) of the vacuolar H(+)-ATPase and the mediatophore complex. The absence of a stoichiometric relationship suggests that connexins and the 16-kDa protein are not subunits of the same channel complex, but analysis of alkali preparations by isopycnic centrifugation shows both types of protein are in membrane structures of the same buoyant density. Electron microscopic analysis of alkali preparations shows a homogeneous population of gap junctions of uniform morphology and width, suggesting the proteins are in the same or similar structures. The structures containing connexins and the 16-kDa protein can be separated by treatment of the plasma membranes with Triton X-100. After such treatment, the connexins remain associated with dense cellular or extracellular material and the gap junctional structures, after further extraction with N-lauroyl sarcosine and urea, contain only the 16-kDa protein. These detergent-extracted gap junctions are thinner (14.1 nm) than those in alkali preparations (18.4 nm).

Animals↗
PubMed1992

Analysis of the gene encoding a 16-kDa proteolipid subunit of the vacuolar H(+)-ATPase from Manduca sexta midgut and tubules.

Vacuolar ATPases (V-ATPases), originally characterised as components of endomembranes, have also been implicated in epithelial ion transport, both in vertebrates and in insects. The ATPase is particularly noteworthy in lepidopteran larvae, where it generates large transepithelial potential differences and short-circuit currents across the midgut epithelium. A cDNA library from Manduca sexta larval midguts and Malpighian tubules was screened with a Drosophila melanogaster cDNA encoding the 16-kDa proteolipid subunit of the V-ATPase, and a 1.4-kb cDNA sequenced in its entirety. The sequence contains a long open reading frame, encoding a putative peptide of 156 amino acids (aa) and with an M(r) of 15,967, in close agreement with values previously suggested by sodium dodecyl sulfate-polyacrylamide gels of M. sexta midgut proteins. Correspondence of the deduced aa sequence with those of other species, particularly D. melanogaster, was extremely close. Northern blots of M. sexta midgut mRNA at high stringency revealed two transcripts of 1.4 and 1.9 kb, whereas genomic Southern blots suggest that there is only a single copy of the gene in M. sexta. The possibility that members of the 16-kDa gene family might serve multiple roles in transport and membrane communication is discussed.

Amino Acid Sequence↗
PubMed1992

Vacuolar H(+)-translocating pyrophosphatases: a new category of ion translocase.

The membrane surrounding the central vacuole of plant cells contains an H(+)-translocating ATPase (H(+)-ATPase) and an H(+)-translocating inorganic pyrophosphatase (H(+)-PPase). Both enzymes are abundant and ubiquitous in plants but the H(+)-PPase is unusual in its exclusive use of inorganic pyrophosphate (PPi) as an energy source. The lack of sequence identity between the vacuolar H(+)-PPase and any other characterized ion pump implies a different evolutionary origin for this translocase. The existence of the vacuolar H(+)-PPase, in conjunction with increasing recognition of PPi as a key metabolite in plant systems, necessitates reconsideration of ATP as the primary energy source for membrane transport in plant cells.

Amino Acid Sequence↗
PubMed1992

Activation of vacuolar-type proton pumps by protein kinase C. Role in neutrophil pH regulation.

Activated neutrophils undergo a large burst of metabolic acid generation, yet maintain their cytosolic pH (pHi) within physiological limits. To analyze the underlying regulatory mechanisms, pHi was measured fluorimetrically in suspensions of human neutrophils. In acid loaded but otherwise unstimulated cells, pHi recovered rapidly via Na+/H+ exchange. Upon Na+ removal, recovery from an imposed acid load was negligible. Phorbol ester activation of acidified cells induced a rapid recovery of pHi partly due to a Zn(2+)-sensitive H(+)-conductive pathway. A third component of the regulatory response was apparent in Na(+)-free media containing Zn2+. Acid extrusion through this alternate pathway was voltage sensitive and capable of translocating H+ equivalents against their electrochemical gradient. This active H+ transport was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by nanomolar doses of bafilomycins A1 or B1, suggesting the involvement of vacuolar (V)-type H+ pumps. Cytosolic alkalinization was accompanied by extracellular acidification, indicative of translocation of H+ equivalents across the surface membrane and consistent with the sensitivity of the alkalinization to changes in plasma membrane potential. The activity of the V-type H+ pumps was virtually undetectable in resting cells, becoming apparent only after treatment with phorbol esters or other, chemically unrelated agonists of protein kinase C. These H+ pumps are likely to play a role in pHi homeostasis during the metabolic burst that accompanies neutrophil activation during infection and inflammation.

Adult↗