Regulation of the Lemon-Fruit V-ATPase by Variable Stoichiometry and Organic Acids

The lemon-fruit V-ATPase can exist in two forms: nitrate-sensitive and nitrate-insensitive. Here we report the results of measurements of H+ /ATP stoichiometries using two kinetic methods: one based on steady-state DpH and one based on initial rates of H+-pumping. Our findings indicate that the nitrate-insensitive fruit V-ATPase has an H+ /ATP stoichiometry of ~1, while both the nitrate-sensitive fruit V-ATPase and the epicotyl V-ATPase have stoichiometries of 2, under zero-load conditions. As DpH increases, the stoichiometry of the nitrate-sensitive fruit V-ATPase decreases to 1. Under similar conditions, the stoichiometry of the epicotyl enzyme remains 2. Thus, the pH-dependent variable stoichiometry of the lemon-fruit V-ATPase may represent a key factor in juice sac vacuolar hyperacidification. On the other hand, the H+ /ATP stoichiometry of the epicotyl V-ATPase can decrease from 2 to 1 in the presence of a membrane potential. The low pH of the fruit vacuole is not due solely to the lower H+/ATP stoichiometry of its pump. We show that lumenal citrate and malate improve the coupling of both the epicotyl and fruit V-ATPases and enhance their ability to generate a pH gradient. Since citrate accumulation is restricted to fruit vacuoles, it may be another important determinant of vacuolar pH.     

Characterization of the functional coupling of bovine brain vacuolar-type H(+)-translocating ATPase. Effect of divalent cations,phospholipids, and subunit H (SFD)

Vacuolar-type H+-translocating ATPases (V-ATPases or V-pumps) are complex proteins containing multiple subunits and are organized into two functional domains: a peripheral catalytic sector V1 and a membranous proton channel V0. The functional coupling of ATP hydrolysis activity to proton transport in V-pumps requires a regulatory component known as subunit H (SFD) as has been shown both in vivo and in vitro (Ho, M. N., Hirata, R., Umemoto, N., Ohya, Y., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1993) J. Biol. Chem. 268, 18286-18292; Xie, X. S., Crider, B. P., Ma, Y. M., and Stone, D. K. (1994) J. Biol. Chem. 269, 25809-25815). Ca2+ is thought to uncouple V-pumps because it is found to support ATP hydrolysis but not proton transport, while Mg2+ supports both activities. The direct effect of phospholipids on the coupling of V-ATPases has not been reported, likely due to the fact that phospholipids are constituents of biological membranes. We now report that Ca2+-induced uncoupling of the bovine brain V-ATPase can be reversed by imposition of a favorable membrane potential. Furthermore we report a simple "membrane-free" assay system using the V0 proton channel-specific inhibitor bafilomycin as a probe to detect the coupling of V-ATPase under certain conditions. With this system, we have characterized the functional effect of subunit H, divalent cations, and phospholipids on bovine brain V-ATPase and have found that each of these three factors plays a critical role in the functional coupling of the V-pump.     

Close-Up and Genomic Views of the Yeast Vacuolar H<Superscript>+</Superscript>-ATPase

The yeast V-ATPase has emerged as an excellent model for other eukaryotic V-ATPases. In this review, recent biochemical and genomic studies of the yeast V-ATPase are described, with a focus on: 1) the role of V₁ subunit H in coupling ATP hydrolysis and proton pumping and 2) identification of the full set of yeast haploid deletion mutants that exhibit the pH and calcium-sensitive growth characteristic of loss of V-ATPase activity. The combination of “close-up” biochemical views of V-ATPase structure and mechanism and “geomic” views of its functional reach promises to provide new insights into the physiological of V-ATPases.     

Structural bases of physiological functions and roles of the vacuolar H(+)-ATPase

Vacuolar-type H⁺-ATPases (V-ATPases) is a large multi-protein complex containing at least 14 different subunits, in which subunits A, B, C, D, E, F, G, and H compose the peripheral 500-kDa V₁ responsible for ATP hydrolysis, and subunits a, c, c′, c″, and d assembly the 250-kDa membrane-integral V₀ harboring the rotary mechanism to transport protons across the membrane. The assembly of V-ATPases requires the presence of all V₁ and V₀ subunits, in which the V₁ must be completely assembled prior to association with the V₀, accordingly the V₀ failing to assemble cannot provide a membrane anchor for the V₁, thereby prohibiting membrane association of the V-ATPase subunits. The V-ATPase mediates acidification of intracellular compartments and regulates diverse critical physiological processes of cell for functions of its numerous functional subunits. The core catalytic mechanism of the V-ATPase is a rotational catalytic mechanism. The V-ATPase holoenzyme activity is regulated by the reversible assembly/disassembly of the V₁ and V₀, the targeting and recycling of V-ATPase-containing vesicles to and from the plasma membrane, the coupling ratio between ATP hydrolysis and proton pumping, ATP, Ca²⁺, and its inhibitors and activators.     

Inhibition of the H+-ATPase in bovine adrenal chromaffin granule ghosts by diethylstilbestrol. Evidence for a tight coupling between ATP hydrolysis and proton translocation

Diethylstilbestrol (DES) was found to inhibit reversibly the hydrolysis of MgATP (80% at 100 microM) and proton pump activity (I50 approximately equal to 15 microM, complete at 100 microM) in chromaffin granule ghosts. The parallel inhibition suggests a tight kinetic coupling between the two activities. The Mg2+-ATPase activity, but not proton pumping, was partially restored by N,N'-dicyclohexylcarbodiimide, indicating that the two inhibitors in combination cause a partial uncoupling. The non-competitive type of inhibition shows that the action of DES is distal to the site of ATP binding and hydrolysis. Although unspecific, the interaction of DES with the chromaffin granule membrane seems primarily to affect the H+-ATPase.     

The C-H Peripheral Stalk Base: A Novel Component in V1-ATPase Assembly

Vacuolar ATPases (V-ATPases) are molecular machines responsible for creating electrochemical gradients and preserving pH-dependent cellular compartments by way of proton translocation across the membrane. V-ATPases employ a dynamic rotary mechanism that is driven by ATP hydrolysis and the central rotor stalk. Regulation of this rotational catalysis is the result of a reversible V₁V_o-domain dissociation that is required to preserve ATP during instances of cellular starvation. Recently the method by which the free V₁-ATPase abrogates the hydrolytic breakdown of ATP upon dissociating from the membrane has become increasingly clear. In this instance the central stalk subunit F adopts an extended conformation to engage in a bridging interaction tethering the rotor and stator components together. However, the architecture by which this mechanism is stabilized has remained ambiguous despite previous work. In an effort to elucidate the method by which the rotational catalysis is maintained, the architecture of the peripheral stalks and their respective binding interactions was investigated using cryo-electron microscopy. In addition to confirming the bridging interaction exuded by subunit F for the first time in a eukaryotic V-ATPase, subunits C and H are seen interacting with one another in a tight interaction that provides a base for the three EG peripheral stalks. The formation of a CE₃G₃H sub-assembly appears to be unique to the dissociated V-ATPase and highlights the stator architecture in addition to revealing a possible intermediate in the assembly mechanism of the free V₁-ATPase.     

ATP binding to the ϵ subunit of thermophilic ATP synthase is crucial for efficient coupling of ATPase and H+ pump activities

ATP binding to the ? subunit of F1-ATPase, a soluble subcomplex of TFoF1 (FoF1-ATPase synthase from the thermophilic Bacillus strain PS3), affects the regulation of F1-ATPase activity by stabilizing the compact, ATPase-active, form of the ? subunit [Kato, S., Yoshida, M. and Kato-Yamada, Y. (2007) J. Biol. Chem. 282, 37618-37623]. In the present study, we report how ATP binding to the ? subunit affects ATPase and H+ pumping activities in the holoenzyme TFoF1. Wild-type TFoF1 showed significant H+ pumping activity when ATP was used as the substrate. However, GTP, which bound poorly to the ? subunit, did not support efficient H+ pumping. Addition of small amounts of ATP to the GTP substrate restored coupling between GTPase and H+ pumping activities. Similar uncoupling was observed when TFoF1 contained an ATP-binding-deficient ? subunit, even with ATP as a substrate. Further analysis suggested that the compact conformation of the ? subunit induced by ATP binding was required to couple ATPase and H+ pumping activities in TFoF1 unless the ? subunit was in its extended-state conformation. The present study reveals a novel role of the ? subunit as an ATP-sensitive regulator of the coupling of ATPase and H+ pumping activities of TFoF1.     

The glycolytic enzyme aldolase mediates assembly, expression, and activity of vacuolar H+-ATPase

Vacuolar H(+)-ATPases (V-ATPases) are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. How ATP is supplied for V-ATPase-mediated hydrolysis and for coupling of proton transport is poorly understood. We have reported that the glycolytic enzyme aldolase physically associates with V-ATPase. Here we show that aldolase interacts with three different subunits of V-ATPase (subunits a, B, and E). The binding sites for the V-ATPase subunits on aldolase appear to be on distinct interfaces of the glycolytic enzyme. Aldolase deletion mutant cells were able to grow in medium buffered at pH 5.5 but not at pH 7.5, displaying a growth phenotype similar to that observed in V-ATPase subunit deletion mutants. Abnormalities in V-ATPase assembly and protein expression observed in aldolase deletion mutant cells could be fully rescued by aldolase complementation. The interaction between aldolase and V-ATPase increased dramatically in the presence of glucose, suggesting that aldolase may act as a glucose sensor for V-ATPase regulation. Taken together, these findings provide functional evidence that the ATP-generating glycolytic pathway is directly coupled to the ATP-hydrolyzing proton pump through physical interaction between aldolase and V-ATPase.     

Function, structure and regulation of the vacuolar (H+)-ATPases

The vacuolar ATPases (or V-ATPases) are ATP-driven proton pumps that function to both acidify intracellular compartments and to transport protons across the plasma membrane. Intracellular V-ATPases function in such normal cellular processes as receptor-mediated endocytosis, intracellular membrane traffic, prohormone processing, protein degradation and neurotransmitter uptake, as well as in disease processes, including infection by influenza and other viruses and killing of cells by anthrax and diphtheria toxin. Plasma membrane V-ATPases are important in such physiological processes as urinary acidification, bone resorption and sperm maturation as well as in human diseases, including osteopetrosis, renal tubular acidosis and tumor metastasis. V-ATPases are large multi-subunit complexes composed of a peripheral domain (V₁) responsible for hydrolysis of ATP and an integral domain (V₀) that carries out proton transport. Proton transport is coupled to ATP hydrolysis by a rotary mechanism. V-ATPase activity is regulated in vivo using a number of mechanisms, including reversible dissociation of the V₁ and V₀ domains, changes in coupling efficiency of proton transport and ATP hydrolysis and changes in pump density through reversible fusion of V-ATPase containing vesicles. V-ATPases are emerging as potential drug targets in treating a number of human diseases including osteoporosis and cancer.     

Subunit D (Vma8p) of the yeast vacuolar H+-ATPase plays a role in coupling of proton transport and ATP hydrolysis

To investigate the function of subunit D in the vacuolar H(+)-ATPase (V-ATPase) complex, random and site-directed mutagenesis was performed on the VMA8 gene encoding subunit D in yeast. Mutants were selected for the inability to grow at pH 7.5 but the ability to grow at pH 5.5. Mutations leading to reduced levels of subunit D in whole cell lysates were excluded from the analysis. Seven mutants were isolated that resulted in pH-dependent growth but that contained nearly wild-type levels of subunit D and nearly normal assembly of the V-ATPase as assayed by subunit A levels associated with isolated vacuoles. Each of these mutants contained 2-3 amino acid substitutions and resulted in loss of 60-100% of proton transport and 58-93% of concanamycin-sensitive ATPase activity. To identify the mutations responsible for the observed effects on activity, 14 single amino acid substitutions and 3 double amino acid substitutions were constructed by site-directed mutagenesis and analyzed as described above. Six of the single mutations and all three of the double mutations led to significant (>30%) loss of activity, with the mutations having the greatest effects on activity clustering in the regions Val(71)-Gly(80) and Lys(209)-Met(221). In addition, both M221V and the double mutant V71D/E220V led to significant uncoupling of proton transport and ATPase activity, whereas the double mutant G80D/K209E actually showed increased coupling efficiency. Both a mutant showing reduced coupling and a mutant with only 6% of wild-type proton transport activity showed normal dissociation of the V-ATPase complex in vivo in response to glucose deprivation. These results suggest that subunit D plays an important role in coupling of proton transport and ATP hydrolysis and that only low rates of turnover of the enzyme are required to support in vivo dissociation.     

Temperature dependence and mercury inhibition of tonoplast-type H+-ATPase

The effects of changing temperature on ATP hydrolysis and proton pumping associated with the H+-ATPase of tonoplast membrane vesicles isolated from the maize root microsomal fraction were determined. In the range 5 to 45 degrees C, the maximal initial rate of ATP hydrolysis obeyed a simple Arrhenius model and the activation energy determined was approximately 14 kcal/mol. On the other hand, the initial proton pumping rate showed a bell-shaped temperature dependence, with maximum activity around 25 degrees C. Lineweaver-Burke analysis of the activities showed that the Km of ATP hydrolysis, unlike that of proton pumping, was relatively insensitive to temperature changes. Detailed kinetic analysis of the proton pumping process showed that the increase in membrane leakage to protons during the pumping stage constituted a major reason for the decreased transport. Nitrate-sensitive ATPase activities of the tonoplast vesicles were found to be inhibited by the presence of micromolar concentrations of Hg2+. The proton pumping process was more sensitive to the presence of Hg2+. Double-reciprocal analysis of kinetic data indicated that Hg2+ was a noncompetitive inhibitor of proton pumping but was an uncompetitive inhibitor of ATP hydrolysis. Further kinetic analysis of Hg2+ effects revealed that the lower proton transport did not result from enhanced membrane leakage but rather from reduced coupling between H+ pumping and ATP hydrolysis.     

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.     

Luminal and cytosolic pH feedback on proton pump activity and ATP affinity of V-type ATPase from Arabidopsis

Proton pumping of the vacuolar-type H(+)-ATPase into the lumen of the central plant organelle generates a proton gradient of often 1-2 pH units or more. Although structural aspects of the V-type ATPase have been studied in great detail, the question of whether and how the proton pump action is controlled by the proton concentration on both sides of the membrane is not understood. Applying the patch clamp technique to isolated vacuoles from Arabidopsis mesophyll cells in the whole-vacuole mode, we studied the response of the V-ATPase to protons, voltage, and ATP. Current-voltage relationships at different luminal pH values indicated decreasing coupling ratios with acidification. A detailed study of ATP-dependent H(+)-pump currents at a variety of different pH conditions showed a complex regulation of V-ATPase activity by both cytosolic and vacuolar pH. At cytosolic pH 7.5, vacuolar pH changes had relative little effects. Yet, at cytosolic pH 5.5, a 100-fold increase in vacuolar proton concentration resulted in a 70-fold increase of the affinity for ATP binding on the cytosolic side. Changes in pH on either side of the membrane seem to be transferred by the V-ATPase to the other side. A mathematical model was developed that indicates a feedback of proton concentration on peak H(+) current amplitude (v(max)) and ATP consumption (K(m)) of the V-ATPase. It proposes that for efficient V-ATPase function dissociation of transported protons from the pump protein might become higher with increasing pH. This feature results in an optimization of H(+) pumping by the V-ATPase according to existing H(+) concentrations.     

Physical interaction between aldolase and vacuolar H+-ATPase is essential for the assembly and activity of the proton pump

Vacuolar proton-translocating ATPases (V-ATPases) are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although V-ATPases are involved in a number of cellular processes, how the proton pumps are regulated under physiological conditions is not well understood. We have reported that the glycolytic enzyme aldolase mediates V-ATPase assembly and activity by physical association with the proton pump (Lu, M., Holliday, L. S., Zhang, L., Dunn, W. A., and Gluck, S. L. (2001) J. Biol. Chem. 276, 30407-30413 and Lu, M., Sautin, Y., Holliday, L. S., and Gluck, S. L. (2004) J. Biol. Chem. 279, 8732-8739). In this study, we generate aldolase mutants that lack binding to the B subunit of V-ATPase but retain normal catalytic activities. Functional analysis of the aldolase mutants shows that disruption of binding between aldolase and the B subunit of V-ATPase results in disassembly and malfunction of V-ATPase. In contrast, aldolase enzymatic activity is not required for V-ATPase assembly. Taken together, these findings strongly suggest an important role for physical association between aldolase and V-ATPase in the regulation of the proton pump.     

Structure,subunit function and regulation of the coated vesicle and yeast vacuolar (H(+))-ATPases

The vacuolar (H(+))-ATPases (or V-ATPases) are ATP-dependent proton pumps that function to acidify intracellular compartments in eukaryotic cells. This acidification is essential for such processes as receptor-mediated endocytosis, intracellular targeting of lysosomal enzymes, protein processing and degradation and the coupled transport of small molecules. V-ATPases in the plasma membrane of specialized cells also function in such processes as renal acidification, bone resorption and pH homeostasis. Work from our laboratory has focused on the V-ATPases from clathrin-coated vesicles and yeast vacuoles.Structurally, the V-ATPases are composed of two domains: a peripheral complex (V(1)) composed of eight different subunits (A-H) that is responsible for ATP hydrolysis and an integral complex (V(0)) composed of five different subunits (a, d, c, c' and c") that is responsible for proton translocation. Electron microscopy has revealed the presence of multiple stalks connecting the V(1) and V(0) domains, and crosslinking has been used to address the arrangement of subunits in the complex. Site-directed mutagenesis has been employed to identify residues involved in ATP hydrolysis and proton translocation and to study the topology of the 100 kDa a subunit. This subunit has been shown to control intracellular targeting of the V-ATPase and to influence reversible dissociation and coupling of proton transport and ATP hydrolysis.     

Division of divalent cations into two groups in relation to their effect on the coupling of the F0F1-ATPase of Rhodospirillum rubrum to the protonmotive force

Divalent cations are divided into two groups in relation to their ability to promote ATP synthase catalyzed reactions. In the presence of Mg2+, the following pattern rules: (i) uncoupler-stimulated ATP hydrolysis of Rhodospirillum rubrum chromatophores which shows an optimum concentration of the divalent cation; (ii) ATP-induced proton pumping in chromatophores; (iii) light-induced ATP synthesis in chromatophores; (iv) no or very low ATPase activity of purified F1-ATPase unmasked by diethylstilbestrol or n-octyl beta-D-glucopyranoside. In the presence of Ca2+, the following pattern occurs: (i) no stimulation of the ATP hydrolysis in chromatophores by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; (ii) no ATP-induced proton pumping; (iii) no light-induced ATP synthesis; (iv) a high ATPase activity of the purified F1-ATPase which is inhibited by diethylstilbestrol and n-octyl beta-D-glucopyranoside. Co2+, Mn2+, and Zn2+ are members of the "Mg2+-group", whereas Cd2+ is suggested to fall between the two groups. Intrinsic uncoupling of the membrane-bound ATP synthase has been suggested to account for the effect caused by Ca2+ in chloroplasts [Pick, U., & Weiss, M. (1988) Eur. J. Biochem. 173, 623-628]. Such an interpretation is consistent with our results on chromatophores. The uncoupling cannot occur at the level of the membrane since neither light-induced nor Mg-ATP-induced proton pumping is affected by Ca2+. A conformational change is suggested to be the reason for this intrinsic uncoupling, and it is proposed to be controlled by the diameters of the divalent cations (Ca2+ greater than Cd2+ greater than Mn2+ greater than Co2+ greater than Zn2+ greater than Mg2+).(ABSTRACT TRUNCATED AT 250 WORDS)     

The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function

Vacuolar-type H+-ATPase (V-ATPase)-driven proton pumping and organellar acidification is essential for vesicular trafficking along both the exocytotic and endocytotic pathways of eukaryotic cells. Deficient function of V-ATPase and defects of vesicular acidification have been recently recognized as important mechanisms in a variety of human diseases and are emerging as potential therapeutic targets. In the past few years, significant progress has been made in our understanding of function, regulation, and the cell biological role of V-ATPase. Here, we will review these studies with emphasis on novel direct roles of V-ATPase in the regulation of vesicular trafficking events.     

Vacuolar H+-ATPase meets glycosylation in patients with cutis laxa

Glycosylation of proteins is one of the most important post-translational modifications. Defects in the glycan biosynthesis result in congenital malformation syndromes, also known as congenital disorders of glycosylation (CDG). Based on the iso-electric focusing patterns of plasma transferrin and apolipoprotein C-III a combined defect in N- and O-glycosylation was identified in patients with autosomal recessive cutis laxa type II (ARCL II). Disease-causing mutations were identified in the ATP6V0A2 gene, encoding the a2 subunit of the vacuolar H⁺-ATPase (V-ATPase). The V-ATPases are multi-subunit, ATP-dependent proton pumps located in membranes of cells and organels. In this article, we describe the structure, function and regulation of the V-ATPase and the phenotypes currently known to result from V-ATPase mutations. A clinical overview of cutis laxa syndromes is presented with a focus on ARCL II. Finally, the relationship between ATP6V0A2 mutations, the glycosylation defect and the ARCLII phenotype is discussed.     

Evidence for an indirect coupling mechanism for the nitrate-sensitive proton pump from corn root tonoplast membranes

The nitrate-sensitive proton-translocating adenosine triphosphatase (H⁺-ATPase) of tonoplast membranes plays an important role in regulating the flow of nutrients and metabolic waste between the cytoplasm and vacuole in the cells of plant roots. Relatively little information is available regarding the coupling between ATP hydrolysis and proton pumping by the nitrate-sensitive, tonoplast H⁺-ATPase. The coupling may be achieved either directly, i. e. the two reaction pathways share at least one common molecular step, or indirectly, i. e. the two reaction pathways do not share an intermediate step. These coupling mechanisms may be differentiated by the responses of the two events to external perturbation. The effects of the presence of nitrate in the assay medium on the rates of ATP hydrolysis and proton transport catalyzed by the tonoplast H⁺-ATPase from maize ( Zea mays L. cv. FRB 73) were investigated. The presence of nitrate inhibited proton transport activity of the tonoplast H⁺-ATPase to a much greater degree than ATP hydrolysis. This differential response of the two activities to nitrate is the basis for a proposed reaction model for the tonoplast H⁺-ATPase that features an indirect coupling mechanism between ATP hydrolysis and proton transport.