Interaction of inhibitors of the vacuolar H(+)-ATPase with the transmembrane Vo-sector

The macrolide antibiotic concanamycin A and a designed derivative of 5-(2-indolyl)-2,4-pentadienamide (INDOL0) are potent inhibitors of vacuolar H(+)-ATPases, with IC(50) values in the low and medium nanomolar range, respectively. Interaction of these V-ATPase inhibitors with spin-labeled subunit c in the transmembrane V(o)-sector of the ATPase was studied by using the transport-active 16-kDa proteolipid analogue of subunit c from the hepatopancreas of Nephrops norvegicus. Analogous experiments were also performed with vacuolar membranes from Saccharomyces cerevisiae. Membranous preparations of the Nephrops 16-kDa proteolipid were spin-labeled either on the unique cysteine C54, with a nitroxyl maleimide, or on the functionally essential glutamate E140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). These residues were previously demonstrated to be accessible to lipid. Interaction of the inhibitors with these lipid-exposed residues was studied by using both conventional and saturation transfer EPR spectroscopy. Immobilization of the spin-labeled residues by the inhibitors was observed on both the nanosecond and microsecond time scales. The perturbation by INDOL0 was mostly greater than that by concanamycin A. Qualitatively similar but quantitatively greater effects were obtained with the same spin-label reagents and vacuolar membranes in which the Nephrops 16-kDa proteolipid was expressed in place of the native vma3p proteolipid of yeast. The spin-label immobilization corresponds to a direct interaction of the inhibitors with these intramembranous sites on the protein. A mutational analysis on transmembrane segment 4 known to give resistance to concanamycin A also gave partial resistance to INDOL0. The results are consistent with transmembrane segments 2 and 4 of the 16-kDa putative four-helix bundle, and particularly the functionally essential protonation locus, being involved in the inhibitor binding sites. Inhibition of proton transport may also involve immobilization of the overall rotation of the proteolipid subunit assembly.     

Interaction of spin-labeled inhibitors of the vacuolar H+-ATPase with the transmembrane Vo-sector

The osteoclast variant of the vacuolar H⁺-ATPase (V-ATPase) is a potential therapeutic target for combating the excessive bone resorption that is involved in osteoporosis. The most potent in a series of synthetic inhibitors based on 5-(5,6-dichloro-2-indolyl)-2-methoxy-2,4-pentadienamide (INDOL0) has demonstrated specificity for the osteoclast enzyme, over other V-ATPases. Interaction of two nitroxide spin-labeled derivatives (INDOL6 and INDOL5) with the V-ATPase is studied here by using the transport-active 16-kDa proteolipid analog of subunit c from the hepatopancreas of Nephrops norvegicus, in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Analogous experiments are also performed with vacuolar membranes from Saccharomyces cerevisiae, in which subunit c of the V-ATPase is replaced functionally by the Nephrops 16-kDa proteolipid. The INDOL5 derivative is designed to optimize detection of interaction with the V-ATPase by EPR. In membranous preparations of the Nephrops 16-kDa proteolipid, the EPR spectra of INDOL5 contain a motionally restricted component that arises from direct association of the indolyl inhibitor with the transmembrane domain of the proteolipid subunit c. A similar, but considerably smaller, motionally restricted population is detected in the EPR spectra of the INDOL6 derivative in vacuolar membranes, in addition to the larger population from INDOL6 in the fluid bilayer regions of the membrane. The potent classical V-ATPase inhibitor concanamycin A at high concentrations induces motional restriction of INDOL5, which masks the spectral effects of displacement at lower concentrations of concanamycin A. The INDOL6 derivative, which is closest to the parent INDOL0 inhibitor, displays limited subtype specificity for the osteoclast V-ATPase, with an IC₅₀ in the 10-nanomolar range.     

Interaction between vacuolar H(+)-ATPase and microfilaments during osteoclast activation.

Vacuolar H(+)-ATPases (V-ATPases) are multisubunit enzymes that acidify compartments of the vacuolar system of all eukaryotic cells. In osteoclasts, the cells that degrade bone, V-ATPases, are recruited from intracellular membrane compartments to the ruffled membrane, a specialized domain of the plasma membrane, where they are maintained at high densities, serving to acidify the resorption bay at the osteoclast attachment site on bone (Blair, H. C., Teitelbaum, S. L., Ghiselli, R., and Gluck, S. L. (1989) Science 249, 855-857). Here, we describe a new mechanism involved in controlling the activity of the bone-resorptive cell. V-ATPase in osteoclasts cultured in vitro was found to form a detergent-insoluble complex with actin and myosin II through direct binding of V-ATPase to actin filaments. Plating bone marrow cells onto dentine slices, a physiologic stimulus that activates osteoclast resorption, produced a profound change in the association of the V-ATPase with actin, assayed by coimmunoprecipitation and immunocytochemical colocalization of actin filaments and V-ATPase in osteoclasts. Mouse marrow and bovine kidney V-ATPase bound rabbit muscle F-actin directly with a maximum stoichiometry of 1 mol of V-ATPase per 8 mol of F-actin and an apparent affinity of 0.05 microM. Electron microscopy of negatively stained samples confirmed the binding interaction. These findings link transport of V-ATPase to reorganization of the actin cytoskeleton during osteoclast activation.     

Biochemical characterization of the yeast vacuolar H(+)-ATPase

The yeast vacuolar proton-translocating ATPase was isolated by two different methods. A previously reported purification of the enzyme (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095) was repeated. This procedure consisted of isolation of vacuoles, solubilization with the zwitterionic detergent ZW3-14, and glycerol gradient centrifugation of the solubilized vacuoles. The fraction with the highest specific activity (11 mumol of ATP hydrolyzed mg-1 min-1) included eight polypeptides of apparent molecular masses of 100, 69, 60, 42, 36, 32, 27, and 17 kDa, suggesting that the enzyme may be more complex than the three-subunit composition proposed from the original purification. The 69-kDa polypeptide was recognized by antisera against the catalytic subunits of two other vacuolar ATPases and labeled with the ATP analog 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, indicating that it contains all or part of the catalytic site. A monoclonal antibody was prepared against this subunit. Under nondenaturing conditions, the antibody immunoprecipitated eight polypeptides, of the same molecular masses as those seen in the glycerol gradient fraction, from solubilized vacuolar vesicles. All eight of these polypeptides are therefore good candidates for being genuine subunits of the enzyme. The structure and function of the yeast vacuolar H+-ATPase were further characterized by examining the inhibition of ATPase activity by KNO3. In the presence of 5 mM MgATP, 100 mM KNO3 inhibited 71% of the ATPase activity of vacuolar vesicles, and the 69- and 60-kDa subunits (and possibly the 42-kDa subunit) were removed from the vacuolar membrane to a similar extent. At concentrations of less than 200 mM KNO3, the stripping of the ATPase subunits and the inhibition of ATPase activity were dependent on the presence of MgATP, suggesting that this is a conformation-specific disassembly of the enzyme. The yeast vacuolar H+-ATPase is a multisubunit enzyme, consisting of a combination of peripheral and integral membrane subunits. Its structure and subunit composition are very similar to other vacuolar ATPase, and it shares some characteristics with the F1F0-ATPases.     

Regulation and reversibility of vacuolar H(+)-ATPase

Arabidopsis thaliana vacuolar H(+)-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H(+)-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (DeltamuH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (DeltapH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (DeltaPsi). Chimeric vacuolar membranes showed ATP synthesis coupled with DeltamuH established by V-PPase. The ATP synthesis was sensitive to bafilomycin A(1) and exhibited two apparent K(m) values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates DeltapH but not DeltaPsi, suggesting that DeltapH is essential for ATP synthesis.     

Spectroscopic and physicochemical studies on the interactions of reversible H+/K(+)-ATPase inhibitors with phospholipid bilayers

Three complementary techniques, differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, have been used to characterise the interactions between dimyristoylphosphatidylcholine (DMPC) model biological membranes and two non-covalent inhibitors of the gastric (H+, K+)-ATPase. DSC, FT-IR and deuterium NMR studies of side-chain perdeuterated DMPC (DMPC-d54) support the prediction, based on physical property measurements, that SK&F 96079 partitions readily into phospholipid bilayers, resulting in a slight but measurable disordering of the lipid hydrocarbon side-chain motion and a concomitant reduction in the co-operativity and onset temperature of the gel to liquid crystalline phase transition. However, FT-IR and deuterium NMR studies show that the bilayer structure remains intact even at high (1:4) compound to lipid molar ratios. Proton (1H) NMR nuclear Overhauser effect determinations in sonicated codispersions reveal details of the membrane bound conformations of SK&F 96079. The structurally related analogue SK&F 96464, also studied by 1H-NMR, can be shown, by interpreting the effects of nitroxide-labelled fatty acid relaxation probes, to adopt a well-defined orientation relative to the bilayer, in contrast to SK&F 96079. This orientation directs the proton at the 5-position of the quinoline ring towards the hydrophobic centre of the bilayer, and the quinoline 8-methoxy group towards the surface and hence the aqueous phase. Molecular modelling has been used to rationalise this orientation in terms of hydrogen bonds between the amino NH group of SK&F 96464 and the sn-1 carbonyl group of DMPC, and between the NH group of the protonated quinoline ring of SK&F 96464 and the DMPC phosphodiester group.     

Stimulus-induced phosphorylation of vacuolar H(+)-ATPase by protein kinase A

Eukaryotic vacuolar-type H(+)-ATPases (V-ATPases) are regulated by the reversible disassembly of the active V(1)V(0) holoenzyme into a cytosolic V(1) complex and a membrane-bound V(0) complex. The signaling cascades that trigger these events in response to changing cellular conditions are largely unknown. We report that the V(1) subunit C of the tobacco hornworm Manduca sexta interacts with protein kinase A and is the only V-ATPase subunit that is phosphorylated by protein kinase A. Subunit C can be phosphorylated as single polypeptide as well as a part of the V(1) complex but not as a part of the V(1)V(0) holoenzyme. Both the phosphorylated and the unphosphorylated form of subunit C are able to reassociate with the V(1) complex from which subunit C had been removed before. Using salivary glands of the blowfly Calliphora vicina in which V-ATPase reassembly and activity is regulated by the neurohormone serotonin via protein kinase A, we show that the membrane-permeable cAMP analog 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate (8-CPT-cAMP) causes phosphorylation of subunit C in a tissue homogenate and that phosphorylation is reduced by incubation with antibodies against subunit C. Similarly, incubation of intact salivary glands with 8-CPT-cAMP or serotonin leads to the phosphorylation of subunit C, but this is abolished by H-89, an inhibitor of protein kinase A. These data suggest that subunit C binds to and serves as a substrate for protein kinase A and that this phosphorylation may be a regulatory switch for the formation of the active V(1)V(0) holoenzyme.     

Subunit composition of vacuolar membrane H(+)-ATPase from mung bean

The vacuolar H(+)-ATPase from mung bean hypocotyls was solubilized from the membrane with lysophosphatidycholine and purified by QAE-Toyopearl column chromatography. The purified ATPase was active only in the presence of exogenous phospholipid and was inhibited by nitrate, dicyclohexyl carbodiimide and Triton X-100, but not by vanadate or azide. Dodecyl sulfate/polyacrylamide gel electrophoresis of the purified ATPase yielded ten polypeptides of molecular masses of 68 kDa, 57 kDa, 44 kDa, 43 kDa, 38 kDa, 37 kDa 32 kDa, 16 kDa, 13 kDa and 12 kDa. All polypeptides remained in the peak activity fraction after glycerol density gradient centrifugation. Nine of them, excluding the 43-kDa polypeptide, comigrated in a polyacrylamide gradient gel in the presence of 0.1% Triton X-100. The 16-kDa polypeptide could be labeled with [14C]dicyclohexylcarbodiimide. The amino-terminal amino acid sequence of the isolated 68-kDa polypeptide generally agreed with that deduced from the cDNA for the carrot 69-kDa subunit [Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H. & Taiz, L. (1988) J. Biol. Chem. 263, 9102-9112]. Thus, mung bean vacuolar H(+)-ATPase seems to consist of nine distinct subunits.     

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.     

Association of L-type calcium channels with a vacuolar H(+)-ATPase G2 subunit

The class C L-type calcium (Ca(2+)) channels have been implicated in many important physiological processes. Here, we have identified a mouse vacuolar H(+)-ATPase (V-ATPase) G2 subunit protein that bound to the C-terminal domain of the pore-forming alpha(1C) subunit using a yeast two-hybrid screen. Protein-protein interaction between the V-ATPase G subunit and the alpha(1C) subunit was confirmed using in vitro GST pull-down assays and coimmunoprecipitation from intact cells. Moreover, treatment of cells expressing L-type Ca(2+) channels with a specific inhibitor of the V-ATPase blocked proper targeting of the channels to the plasma membrane.     

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.     

A malaria parasite-encoded vacuolar H(+)-ATPase is targeted to the host erythrocyte

The asexual development of malaria parasites inside the erythrocyte is accompanied by changes in the composition, structure, and function of the host cell membrane and cytoplasm. The parasite exports a membrane network into the host cytoplasm and several proteins that are inserted into the erythrocyte membrane, although none of these proteins has been shown to have enzymatic activity. We report here that a functional malaria parasite-encoded vacuolar (V)-H(+)-ATPase is exported to the erythrocyte and localized in membranous structures and in the plasma membrane of the infected erythrocyte. This localization was determined by separation of parasite and erythrocyte membranes and determination of enzyme marker activities and by immunofluorescence microscopy assays using antibodies against the B subunit of the malarial V-H(+)-ATPase and erythrocyte (spectrins) and parasite (merozoite surface protein 1) markers. Our results suggest that this pump has a role in the maintenance of the intracellular pH (pH(i)) of the infected erythrocyte. Our results also indicate that although the pH(i) maintained by the V-H(+)-ATPase is important for maximum uptake of small metabolites at equilibrium, it does not appear to affect transport across the erythrocyte membrane and is, therefore, not involved in the previously described phenomenon of increased permeability of infected erythrocytes that is sensitive to chloride channel inhibitors (new permeation pathway). This constitutes the first report of the presence of a functional enzyme of parasite origin in the plasma membrane of its host.     

Conformation of a peptide encompassing the proton translocation channel of vacuolar H(+)-ATPase

The structural properties of a crucial transmembrane helix for proton translocation in vacuolar ATPase are studied using double site-directed spin-labeling combined with electron spin resonance (ESR) (or electron paramagnetic resonance) and circular dichroism spectroscopy in sodium dodecyl sulfate micelles. For this purpose, we use a synthetic peptide derived from transmembrane helix 7 of subunit a from the yeast Saccharomyces cerevisiae vacuolar proton-translocating ATPase that contains two natural cysteine residues suitable for spin-labeling. The interspin distance is calculated using a second-moment analysis of the methanethiosulfonate spin-label ESR spectra at 150 K. Molecular dynamics simulation is used to study the effect of the side-chain dynamics and backbone dynamics on the interspin distance. Based on the combined results from ESR, circular dichroism, and molecular dynamics simulation we conclude that the peptide forms a dynamic alpha-helix. We discuss this finding in the light of current models for proton translocation. A novel role for a buried charged residue (H729) is proposed.     

A functional arginine residue in the vacuolar H(+)-ATPase of higher plants.

The arginine-specific reagent phenylglyoxal inactivated the vacuolar H(+)-ATPase of red beet. Inactivation by phenylglyoxal followed pseudo-first-order kinetics and a double log plot of the t1/2 of inactivation versus phenylglyoxal concentration yielded a slope of 1.18. Neither inorganic anions nor DIDS protected from phenylglyoxal-mediated inactivation of the H(+)-ATPase. Indeed, Cl- stimulated the rate of phenylglyoxal-mediated H(+)-ATPase inactivation relative to SO4(2-). ATP, but not MgATP or ADP, protected from phenylglyoxal-mediated inactivation and inactivation resulted in a decrease in the Vmax of the H(+)-ATPase with little effect on the Km. Collectively, these results are consistent with phenylglyoxal-mediated inactivation of the vacuolar H(+)-ATPase resulting from modification of a single arginine residue in the catalytic nucleotide binding site of the vacuolar H(+)-ATPase. Stimulation of phenylglyoxal-mediated inactivation by Cl- indicates that exposure of the phenylglyoxal-sensitive functional arginine residue is enhanced in the presence of Cl-. The failure of MgATP to protect from phenylglyoxal inactivation suggests that ATP, rather than MgATP, binds directly to the catalytic site and that Mg2+ may act to promote catalysis subsequent to ATP binding.     

RAVE is essential for the efficient assembly of the C subunit with the vacuolar H(+)-ATPase

The RAVE complex is required for stable assembly of the yeast vacuolar proton-translocating ATPase (V-ATPase) during both biosynthesis of the enzyme and regulated reassembly of disassembled V(1) and V(0) sectors. It is not yet known how RAVE effects V-ATPase assembly. Previous work has shown that V(1) peripheral or stator stalk subunits E and G are critical for binding of RAVE to cytosolic V(1) complexes, suggesting that RAVE may play a role in docking of the V(1) peripheral stalk to the V(0) complex at the membrane. Here we provide evidence for an interaction between the RAVE complex and V(1) subunit C, another subunit that has been assigned to the peripheral stalk. The C subunit is unique in that it is released from both V(1) and V(0) sectors during disassembly, suggesting that subunit C may control the regulated assembly of the V-ATPase. Mutants lacking subunit C have assembly phenotypes resembling that of RAVE mutants. Both are able to assemble V(1)/V(0) complexes in vivo, but these complexes are highly unstable in vitro, and V-ATPase activity is extremely low. We show that in the absence of the RAVE complex, subunit C is not able to stably assemble with the vacuolar ATPase. Our data support a model where RAVE, through its interaction with subunit C, is facilitating V(1) peripheral stalk subunit interactions with V(0) during V-ATPase assembly.     

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.     

Activation of melanogenesis by vacuolar type H(+)-ATPase inhibitors in amelanotic, tyrosinase positive human and mouse melanoma cells

In this study, we describe the activation of melanogenesis by selective vacuolar type H(+)-ATPase inhibitors (bafilomycin A1 and concanamycin A) in amelanotic human and mouse melanoma cells which express tyrosinase but show no melanogenesis. Addition of the inhibitors activated tyrosinase within 4 h, and by 24 h the cells contained measurable amounts of melanin. These effects were not inhibited by cycloheximide (2 microgram/ml) which is consistent with a post-translational mechanism of activation. Our findings suggest that melanosomal pH could be an important and dynamic factor in the control of melanogenesis in mammalian cells.     

Energy coupling of L-glutamate transport and vacuolar H(+)-ATPase in brain synaptic vesicles

Energy coupling of L-glutamate transport in brain synaptic vesicles has been studied. ATP-dependent acidification of the bovine brain synaptic vesicles was shown to require CI-, to be accelerated by valinomycin and to be abolished by ammonium sulfate, nigericin or CCCP plus valinomycin, and K+. On the other hand, ATP-driven formation of a membrane potential (positive inside) was found to be stimulated by ammonium sulfate, not to be affected by nigericin and to be abolished by CCCP plus valinomycin and K+. Like formation of a membrane potential, ATP-dependent L-[3H]glutamate uptake into vesicles was stimulated by ammonium sulfate, not affected by nigericin and abolished by CCCP plus valinomycin and K+. The L-[3H]glutamate uptake differed in specificity from the transport system in synaptic plasma membranes. Both ATP-dependent H+ pump activity and L-glutamate uptake were inhibited by bafilomycin and cold treatment (common properties of vacuolar H(+)-ATPase). ATP-dependent acidification in the presence of L-glutamate was also observed, suggesting that L-glutamate uptake lowered the membrane potential to drive further entry of H+. These results were consistent with the notion that the vacuolar H(+)-ATPase of synpatic vesicles formed a membrane potential to drive L-glutamate uptake. ATPase activity of the vesicles was not affected by the addition of Cl-, glutamate or nigericin, indicating that an electrochemical H+ gradient had no effect on the ATPase activity.