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J. Biol. Chem., Vol. 275, Issue 28, 21761-21767, July 14, 2000

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The H Subunit (Vma13p) of the Yeast V-ATPase Inhibits the ATPase Activity of Cytosolic V₁ Complexes^*

Karlett J. Parra, Kelly L. Keenan, and Patricia M. Kane

From the Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210

Received for publication, March 20, 2000, and in revised form, April 9, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

V-ATPases are composed of a peripheral complex containing the ATP-binding sites, the V₁ sector, attached to a membrane complex containing the proton pore, the V_o sector. In vivo, free, inactive V₁ and V_o sectors exist in dynamic equilibrium with fully assembled, active V₁ V_o complexes, and this equilibrium can be perturbed by changes in carbon source. Free V₁ complexes were isolated from the cytosol of wild-type yeast cells and mutant strains lacking V_o subunit c (Vma3p) or V₁ subunit H (Vma13p). V₁ complexes from wild-type or vma3 mutant cells were very similar, and contained all previously identified yeast V₁ subunits except subunit C (Vma5p). These V₁ complexes hydrolyzed CaATP but not MgATP, and CaATP hydrolysis rapidly decelerated with time. V₁ complexes from vma13 cells contained all V₁ subunits except C and H, and had markedly different catalytic properties. The initial rate of CaATP hydrolysis was maintained for much longer. The complexes also hydrolyzed MgATP, but showed a rapid deceleration in hydrolysis. These results indicate that the H subunit plays an important role in silencing unproductive ATP hydrolysis by cytosolic V₁ complexes, but suggest that other mechanisms, such as product inhibition, may also play a role in silencing in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

V-ATPases¹ are highly conserved proton pumps distributed throughout the vacuolar network in all eukaryotic cells. V-ATPases maintain organelle acidification and affect cytosolic pH and ion balance, and their activity has been linked to a diverse array of cellular processes ranging from zymogen activation to protein sorting to viral membrane fusion events (1, 2). V-ATPases are comprised of two structural domains, the V₁ domain, which consists of a complex of peripheral subunits containing the nucleotide-binding sites attached to the cytoplasmic face of membrane, and the V_o domain, which is comprised of several integral membrane and tightly associated peripheral proteins that contain the proton pore (1, 2). The yeast V-ATPase has at least eight V₁ subunits (designated A, B, C, D, E, F, G, and H) and five V_o subunits (designated a, c, c', c", and d) (1, 3, 4). Genetic and biochemical approaches have converged to show that all of the subunits are required for function of V₁V_o complexes (3, 4). Other eukaryotic V-ATPases have very similar subunit compositions.

Functional interdependence of V₁ and V_o has been clearly established. Only fully assembled V₁V_o complexes can couple ATP hydrolysis to H⁺ translocation, and in vitro experiments indicate that upon V₁ dissociation, the V_o domain does not conduct protons and the V₁ domain does not perform MgATP hydrolysis (5-8). Nevertheless, many different cells have been shown to contain free V₁ and free V_o sectors in addition to fully assembled V₁V_o complexes (9-12). Independent experiments in yeast and Manduca sexta have indicated that the disassembled V₁ and V_o sectors exist in a dynamic equilibrium with fully assembled complexes and that this equilibrium can be shifted in response to changes in extracellular conditions (10, 13, 14). Starvation appears to stimulate disassembly of V₁ from V_o, but this disassembly is fully reversible upon refeeding (13-15). This reversible association between V₁ and V_o is believed to regulate V-ATPase function in vivo: disassembly of V-ATPase complexes may conserve ATP when energy reserves are low and reassembly of the enzyme may provide the renewed proton pumping capacity necessary to prevent cytosolic acidification when active metabolism resumes (16, 17).

A constitutively active free V₁ in the cytosol could quickly become lethal to the cell by hydrolyzing cytosolic reserves of ATP. Graf et al. (14) have isolated cytosolic V₁ complexes from M. sexta and shown that these complexes exhibit Ca²⁺-dependent ATP hydrolysis at nonphysiological Ca²⁺ concentrations but hydrolyze MgATP only in the presence of methanol. The properties of V₁ complexes have also been examined by reconstitution of expressed subunits and biochemically isolated subcomplexes of the bovine clathrin-coated vesicle ATPase (18-21). These studies have also revealed a shift from Mg²⁺-dependent to Ca²⁺-dependent ATPase activity in V₁ complexes detached from the membrane subunits and have suggested that CaATPase activity is a partial reaction characteristic of dissociated V₁ sectors that is functionally related to the MgATPase activity of the fully assembled proton pump (18).

In an attempt to gain more insight into the cellular mechanisms of V₁-ATPase silencing, we have purified and characterized native yeast cytosolic V₁ complexes. Cytosolic V₁ were isolated from wild-type cells and from two vma mutant strains. We found that V₁ subunit C was not present in any of the isolated complexes. All of the isolated V₁ complexes hydrolyzed ATP in the presence Ca²⁺, but only V₁ complexes lacking subunit H had MgATPase activity. The current study also indicates that product inhibition of ATPase activity may occur in cytosolic V₁ complexes but cannot fully account for the inactivation of these complexes. In addition, structural changes within the V₁ complex itself, such as lost of an activator subunit (C subunit) and presence of at least one inhibitory subunit (H subunit) may be critical for silencing the MgATPase activity in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials and Strains-- Zymolyase 100T was purchased from ICN. Concanamycin A was obtained from Wako Biochemicals. Prestained molecular mass markers (high range) were obtained from Life Technologies. ATP bioluminescence assay kit HS II and anti-Myc monoclonal antibody 9E10 were purchased from Roche Molecular Biochemicals. All other reagents were purchased from Sigma.

The wild-type yeast strain used in these experiments was SF838-1D (MAT ade6 leu2-3,112 ura3-52 pep4-3 gal2 (22)). The vma3 strain was congenic with the wild-type strain except for the vma3::URA3 mutation (23). A congenic vma13 strain was constructed by excising a BamHI-SacII fragment containing the vma13::LEU2 allele from the deletion plasmid described below, and integrating into the VMA13 locus by a one-step gene disruption (24). Replacement of the wild-type allele by the deletion allele was confirmed by PCR of chromosomal DNA prepared from yeast cells.

VMA13 Plasmid Constructions-- VMA13 in pRS315 was a gift from R. Hirata. VMA13 was tagged with the Myc epitope immediately following the methionine start codon by employing PCR and subcloning techniques. Two separate PCR reactions were performed using VMA13 as a template. Reaction A utilized primer 1: 5'-AGAAATAAGCTTTGTTCCATTGTTCCTGAAATCGC, and primer 2: 5'-GACGAAGGAATTTGAAAGAG, this reaction generated a 564-base product that encompassed 546 5'-untranslated region bases and 18 5' bases of the Myc epitope including the HindIII site that is found within the Myc sequence. Reaction B utilized primer 3: 5'-CAAAGCTTATTTCTGAAAGACTTGGGAGCACGAAGATATT and primer 4: 5'-GATCACGCATAACC generating a 1912-base product which contained 27 bases of the Myc epitope including the HindIII site, 1433 bases of VMA13 open reading frame, and 452 bases of 3'-untranslated region. PCR products were ligated into pCR2.1^TM (Invitrogen). Orientation of products was determined based on restriction digests. Reaction A was subcloned into pRS316 using BamHI and HindIII sites, Reaction B product was subcloned into the newly constructed pRS316 vector containing the Reaction A product using KpnI and HindIII sites. Sequencing confirmed the presence of the Myc epitope within wild-type VMA13. N-Myc VMA13 is able to complement the growth phenotypes of a vma13 strain, and vacuolar vesicles isolated from this strain possess 80% wild type ATPase activity.²

VMA13 was cloned into the yeast shuttle vector pRS316 at the BamHI and NotI sites. A deletion plasmid was constructed by replacing the 1084-base pair BglII fragment within the ORF of VMA13 with a 2.2-kilobase Leu2 fragment.

Purification of Cytosolic V₁ Complexes-- Cells were grown overnight to mid-log phase (3 A₆₀₀/ml) in YEPD (1% yeast extract, 2% peptone, 2% glucose) medium adjusted to pH 5. 6000 A₆₀₀ units of cells (approximately 6 × 10¹⁰ cells) were harvested by centrifugation at 2500 × g for 10 min and resuspended in 300 ml of 0.05 M Tris-HCl, pH 9.4, containing 10 mM dithiothreitol. Cells were rocked for 5 min at 30 °C, pelleted by centrifugation for 5 min at 2200 × g, and the pellet resuspended in 300 ml of 0.05 M Tris-HCl, pH 7.5, 1.2 M sorbitol, 2% glucose. Cells were converted to spheroplasts by adding 1500 units of zymolase 100T to the suspension and gently shaking at 30 °C for 20 min. Spheroplasts were washed twice with 300 ml of YEPD medium containing 1.2 M sorbitol. In certain experiments, spheroplasts were briefly deprived of glucose by incubating 5 min at 30 °C in 200 ml of YEP (1% yeast extract, 2% peptone) plus 1.2 M sorbitol. Otherwise, incubation was performed in 200 ml of YEPD plus 1.2 M sorbitol. Finally, spheroplasts were collected by centrifugation and lysed on ice in 15 ml of buffer A (0.05 M Tris-HCl, pH 7.5, 30 mM NaCl, 30 mM KCl, 0.3 mM EDTA) containing a protease inhibitor mixture (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, 5 µg/ml aprotinin A, 1 µg/ml leupeptin) in a Dounce homogenizer. Homogenate was centrifuged at 275,000 × g for 1.25 h in a Ti-75 rotor and the supernatant (15-20 mg of protein/ml) precipitated with 50% ammonium sulfate. Precipitation was performed by dropwise addition of a cold saturated solution of ammonium sulfate, pH 7, in three steps of 0-20, 20-35, and 35-50% (v/v) with 15 min incubation between additions and constant stirring on ice. After the final addition, the mixture was incubated on ice for 30 min and the protein pelleted at 9,000 × g for 11 min. The precipitated protein was resuspended in buffer A and desalted on a Centricon Plus-20 filter (100,000 dalton cutoff; Amicon), then filtered and applied to a Mono-Q2 column (Bio-Rad) equilibrated in buffer A containing 9.6 mM -mercaptoethanol. The column was washed with 10 ml of equilibration buffer and the bound protein eluted with three sequential linear gradients: 5 ml of 0-30% buffer B (0.05 M Tris-HCl, pH 7.5, 0.2 M NaCl, 0.2 M KCl, 0.3 mM EDTA, 9.6 mM -mercaptoethanol) followed by a 20-ml isocratic flow of 70% buffer A, 30% buffer B, 6 ml of 30-40% buffer B followed by a 20-ml isocratic flow in 60% buffer A, 40% buffer B, and 5 ml of 40-100% buffer B followed by a 5-ml isocratic flow in 100% buffer B. 1-ml fractions were collected. Fractions were analyzed for the presence of V₁ subunits by Western blotting, and fractions containing V₁ subunits were immunoprecipitated under nondenaturing conditions with monoclonal antibodies 8B1 or 13D11 (against the 69- and 60-kDa V₁ subunits, respectively) to identify those containing V₁ complexes (11). Fractions containing V₁ complexes (fractions 43-54) eluted at 40% buffer B (0.1 M NaCl, 0.1 M KCl) and were pooled and concentrated on a Centricon Plus-20 filter (100,000 dalton cut-off). Pooled Q-2 fractions were applied to a Bio-Rad Sec 400 gel filtration column equilibrated with 30% buffer A, 70% buffer B. Purified V₁ complexes (0.05-0.5 mg) were collected in a single fraction. Chromatography was performed on a Bio-Rad BioLogic system.

Enzyme Assays-- Hydrolysis of ATP quantitated colorimetrically as the phosphate released based on the Taussky and Schorr method (25). Briefly, reaction was started by addition of 1.5-15 µg of purified V₁ to 500 µl of ATPase assay medium (0.05 mM Tris-HCl, pH 6.8, containing 4 mM ATP or GTP and 1.6 mM CaCl₂ or MgCl₂). The final metal-nucleotide complex concentration in the medium was calculated for each condition by the Bound and Determined computer program (26). Incubations were performed at 37 °C for the indicated times (0.5-30 min). Reactions were stopped by addition of an equal volume of 10% (w/v) SDS. Phosphate released was determined by measuring the absorbance at 700 nm immediately after addition of 0.5 ml of Taussky and Schorr reagent (10% FeSO₄, 1.2 N sulfuric acid, 1.2% ammonium molybdate). A blank containing only assay medium was measured for each reaction. A standard calibration curve for P_i was used to calculate the micromoles of P_i formed. Data were analyzed using the Sigma Plot curve-fitting application program.

The amount of ATP and ADP bound to the purified V₁ preparation was determined as follows. V₁ (20-60 µg) was precipitated by addition of perchloric acid to a final concentration of 0.44 M, and after 15 min on ice, the mixture was neutralized by addition of equal volume of ice-cold fresh 0.8 M potassium bicarbonate, then incubated for an additional 20 min on ice. Soluble nucleotides were recovered in the supernatant after centrifugation. ATP and ADP concentrations were measured using the luciferin-luciferase assay in an Autolunat LB953 luminometer. ADP was converted to ATP by addition of 8.3 mM phosphoenolpyruvate and 48 µg of pyruvate kinase in buffer containing 50 mM HEPES-KOH, pH 7.5, 5 mM MgCl₂, and 20 mM KCl. Protein concentrations were determined by Lowry assay (27).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of Cytosolic V₁ Complexes-- In order to better understand how cytosolic V₁ sectors are inactivated and possibly to gain insights into how V₁ dissociation is triggered, we isolated and characterized cytosolic V₁ sectors from yeast. Cytosolic V₁ complexes from wild-type yeast cells and vma3 mutant cells, treated both with and without a brief glucose deprivation, were purified by a variation of the methods reported by Graf et al. (14). In wild-type cells, the population of V₁ complexes in the cytosol was increased by depriving the cells of glucose for 5 min. This treatment has been shown to trigger dissociation of approximately 75% of assembled V₁V_o complexes (13, 15). vma3 mutant cells lack the gene encoding the proteolipid subunit c of the V_o sector (28) and provided an alternative source of V₁. vma3 mutants do not form stable V_o sectors, but assemble stable V₁ complexes constitutively present in the cytosol (11, 23). Wild-type and vma3 cells were converted to spheroplasts and osmotically lysed. The purification procedure consisted of four fractionation steps and yielded ~0.05-0.1 and ~0.4-0.5 mg of V₁ from 2 liters of log-phase culture of wild-type and vma3 cells, respectively. Glucose deprivation improved the yield of V₁ sectors from wild-type cells, but did not significantly affect the yield of V₁ sectors from the vma3 cells. Briefly, the purification consisted of isolation of a soluble fraction by high speed centrifugation, followed by protein precipitation with 50% ammonium sulfate and two sequential chromatographic columns: ion exchange on a Mono-Q column and gel filtration on a Biosilect Sec-400 column. Fig. 1A shows the protein elution profile from the ion exchange column, and Fig. 1B is a Western blot analysis of the elution pattern of A, B, and C V₁ subunits. The C subunit failed to bind to the column even at the lowest salt concentration and fractionated away from the rest of the cytosolic V₁ subunits. Because the A and B subunits were detected throughout the gradient, we assessed whether they were assembled with other subunits by nondenaturing immunoprecipitation of selected pooled fractions (not shown). Assembled V₁ complexes were present only in fractions eluted with 0.2 M salts (0.1 M NaCl, 0.1 M KCl); A and B subunits that eluted elsewhere from the column were either partially or fully dissociated from the remaining V₁ subunits. The assembled V₁ complexes eluted from the Mono-Q column (Fig. 1B, fractions 43-54) were concentrated and subjected to gel filtration chromatography. The elution profile for the gel filtration column is shown in Fig. 1C. The V₁ complexes eluted in a single peak (Fig. 1D) with an estimated molecular mass of 445 kDa.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Purification of cytosolic V1 sectors. A and B, ion exchange chromatography of yeast cytosol. A, supernatant protein obtained after high speed centrifugation of a yeast cell lysate was precipitated with 50% ammonium sulfate, desalted, and applied to a Mono-Q2 ion exchange column as described under "Experimental Procedures." After an initial wash, proteins were eluted from the column with three sequential linear gradients. Protein concentration was monitored by measuring absorbance at 280 nm (A280). A profile of the stepwise salt gradient used is superimposed on the protein elution profile and is described in more detail under "Experimental Procedures." B, the indicated fractions collected from the chromatogram shown in A were precipitated with 10% trichloroacetic acid, solubilized, and separated by SDS-PAGE, and blotted to nitrocellulose. The blot was probed with mouse monoclonal antibodies 7A2, which recognizes the C subunit, 13D11, which recognizes the B subunit, and 8B1, which recognizes the A subunit, followed by alkaline-phosphatase-conjugated goat anti-mouse antibodies (11). The fractions containing assembled V1 complexes were identified by nondenaturing immunoprecipitation with monoclonal antibody 8B1 as described under "Experimental Procedures." C and D, isolation of cytosolic V1 complexes by gel filtration. C, fractions 43-54 from the ion exchange chromatography column shown in A were pooled, concentrated, and loaded on a Bio-Rad Sec400 gel filtration column. Protein concentration in fractions eluted from the column was monitored by measuring the A280. D, V1 subunits eluted from the gel filtration column in a single peak, centered at fraction 20. The indicated fractions were subjected to SDS-PAGE and immunoblotting. The A and B subunits were recognized with monoclonal antibodies 8B1 and 13D11 as described above, and the E subunit was recognized by polyclonal antiserum raised against the yeast E subunit (generously provided by Dr. Tom Stevens).

Purified V₁ complexes from wild-type and vma3 cells, either with or without glucose deprivation, showed a similar subunit composition. The presence of the 27-kDa E subunit in the V₁ complexes was confirmed by Western blotting (Fig. 1D). Silver staining of the peak eluted from gel filtration column (Fig. 2) showed additional bands of 32, 16, and 14 kDa that correspond in molecular mass to the previously identified D, G, and F subunits, respectively (4). These data indicate that the V₁ complexes obtained from both strains contained the A, B, D, E, F, and G V₁ subunits (Figs. 1D and 2). Both the E and G subunit had a somewhat smeared appearance in the V₁ preparations. In addition to the previously characterized V₁ subunits, bands of approximately 25 and 80 kDa and several high molecular mass bands were consistently present in the fractions containing the V₁ complexes. We have not yet determined whether these proteins are associated with cytosolic V₁ complexes.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Subunit composition of cytosolic V1 complexes isolated from vma3 cells. The combined fractions containing assembled V1 complexes from the ion exchange column (Q2 Pool) and fraction 20 from the gel filtration column (Sec 400 Frac.) were precipitated, solubilized, and subjected to SDS-PAGE. The gels were then silver stained (23). The positions of known V1 subunits are indicated; the identities of the A, B, H, and E subunits were confirmed by immunoblotting.

We were particularly interested in determining whether the H subunit, encoded by the VMA13 gene in yeast (29), was associated with the cytosolic V₁ complexes. This protein has a molecular mass of 54 kDa and is often masked by the 60-kDa subunit, so the VMA13 gene was tagged with a Myc epitope to allow it to be clearly identified. The tagged protein was expressed in a vma13 yeast strain and shown to fully complement the growth defects of the strain. Co-purification of subunit H with cytosolic V₁ complexes was confirmed using anti-Myc antibodies against V₁ complexes purified from vma13 cells expressing the Myc-tagged VMA13 gene (Fig. 3). Therefore, the cytosolic V₁ sectors appear to contain all the previously characterized V₁ subunits except subunit C. 

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Cytosolic V1 complexes contain the H subunit. V1 complexes were isolated from vma13 mutant cells bearing a Myc epitope-tagged VMA13 gene on a low copy plasmid as described in the legend to Fig. 1 and under "Experimental Procedures." The indicated fractions from the gel filtration column were separated by SDS-PAGE and subjected to immunoblotting. The blot was probed with monoclonal antibodies 8B1 against the A subunit and 9E10 against the myc epitope attached to the H subunit.

Enzymatic Activities of Cytosolic V₁ Complexes-- The isolated V₁ domain of the M. sexta V-ATPase is not active as a MgATPase except in the presence of organic solvents (14). Similarly, the isolated yeast V₁ complexes did not hydrolyze ATP if the divalent cation supplied was Mg²⁺. In an attempt to activate the yeast V₁ ATPase activity, the purified V₁ was treated with 25% methanol, 30 mM octylglucoside, 5-50 mM sodium sulfite, 0.5% N,N-dimethyldodecylamine-N-oxide, and 5-10 mM dithiothreitol, treatments which had effectively activated the MgATPase activity of the Manduca V₁ (14) or F₁-ATPases from various sources (30-33). None of these treatments elicited any MgATPase activity in the yeast V₁ complexes.

Cytosolic V₁ complexes from both wild-type and vma3 cells did hydrolyze ATP in a Ca²⁺-dependent manner at nonphysiological (mM) Ca²⁺ concentrations, however. CaATPase activity has been described in purified V₁ complexes from M. sexta (14), isolated chloroplast and Bacillus firmus F₁ complexes (30, 31), and reconstituted mixtures of bovine V₁ subunits (18-21). The enzymatic properties of complexes purified from glucose-deprived wild-type cells or vma3 cells with or without glucose deprivation were very similar. Because vma3 cells provided a more abundant source of cytosolic V₁ than wild-type cells, the kinetic analysis described below was performed on cytosolic V₁ complexes isolated from vma3 mutant cells.

CaATP hydrolysis was first examined as a function of the time at a constant CaATP concentration (1.4 mM). When the incubation time was varied from 0.5 to 20 min, the plot of micromole of P_i formed versus time had a hyperbolic shape showing a rapid initial rate that decayed until there was little further ATP hydrolysis after 3 min (Fig. 4A). The initial activity, detected at 1 min, was 1.7 µmol of P_i/min/mg. At a lower CaATP concentration (0.3 mM), it took longer for the activity to decay, but the activity was gone by 20 min. An apparent K_m of 0.183 mM for CaATP, which is similar to the K_m of yeast V₁V_o (0.210 mM; Ref. 34) was estimated from 1-min reactions performed at a larger range of concentrations. Based on this information, 1.4 mM CaATP should nearly saturate the enzyme, and the loss of activity over time seen in Fig. 4A cannot be attributed to substrate depletion.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   CaATPase and CaGTPase activity of cytosolic V1 complexes. A, cytosolic V1 complexes were isolated from vma3 cells, and 11.2 µg of the isolated complexes were incubated with 1.4 mM CaATP (open circles) in a 500-µl total volume for the indicated times. Pi release was monitored by colorimetric assay (25). CaATPase activity was also measured after a 1-min preincubation of the complexes with 1.1 mM CaADP followed by a 30-fold dilution into assay buffer (500 µl final volume) containing 1.4 mM CaATP (closed circles). B, cytosolic V1 complexes (11.2 µg) were incubated with 1.4 mM CaGTP (closed circles) in a 500-µl total volume for the indicated times (closed circles), and phosphate release was monitored as described above. CaATPase activity from A (open circles) is shown for comparison.

The substrate specificity of the yeast V₁ complexes was examined. Ca²⁺-dependent hydrolysis of GTP was observed (Fig. 4B). Interestingly, CaGTP hydrolysis was linear for at least 20 min under conditions where ATP hydrolysis had ceased after 3 min. The V₁ complexes exhibited a specific activity for GTP hydrolysis of 0.47 µmol/min/mg of protein. Once again the hydrolysis was Ca²⁺-dependent; Mg²⁺ did not support any GTPase activity in the cytosolic V₁ complexes.

Loss of activity over time could be an indication of product inhibition of the ATPase activity. Product inhibition has been studied in considerable detail in F₁-ATPases, and appears to be specific to ADP in many cases (35-37). Thus, although GTP is a substrate for F₁, GDP is much less efficient in product inhibition (36). To further explore the possibility that the ATPase activity of the cytosolic V₁ complexes was inhibited by ADP, V₁ complexes were preincubated in the presence of CaADP before measurement of the CaATPase activity. Isolated V₁ complexes were preincubated with 1.1 mM CaADP for 1 min, then the V₁-CaADP mixture was diluted 30-fold into assay medium and the CaATPase activity measured in the presence of 1.4 mM CaATP (Fig. 4A). The CaATPase activity of the V₁ complexes was not fully inhibited by CaADP preincubation. The initial ATPase activity was 51% that in the absence of ADP, and a decay in ATPase activity similar to that seen in the absence of ADP preincubation was observed over the next 3 min. We also determined whether the cytosolic V₁ preparation contained tightly bound nucleotides after isolation that might be involved in inhibition of either Mg²⁺-dependent or Ca²⁺-dependent ATPase activity. Only substoichiometric amounts of ADP (0.02 mol/mol V₁) and ATP (0.005 mol/mol V₁) were detected in the isolated cytosolic V₁ complexes. Pyrophosphate was shown to enhance MgATPase activity of F₁-ATPases by removing tightly bound nucleotides (36). However, addition of 4.8-9.4 mM PPi did not activate the ATPase activity of cytosolic yeast V₁.

The sensitivity of the yeast cytosolic V₁ to a variety of inhibitors was examined. The CaATPase activity of the cytosolic V₁ was not affected by addition of the specific P- and F-type ATPases inhibitors sodium orthovanadate (1 mM) and sodium azide (10 mM), respectively. Concanamycin A, a specific V-type ATPase inhibitor believed to interact with the V_o domain at the membrane (38) had no effect on the ATPase activity of purified V₁. V-ATPases contain a set of three conserved cysteine residues that are essential for activity and render the enzyme sensitive to low concentrations of N-ethylmaleimide (39). Measuring N-ethylmaleimide sensitivity of the cytosolic V₁ sectors was difficult because the presence of reducing agent (-mercaptoethanol) appeared to be essential for purification of an active V₁ and maintenance of its activity. However, addition of N-ethylmaleimide in excess to the concentration of -mercaptoethanol in the isolated V₁ preparation allowed us to estimate an IC₅₀ of 0.5 mM for N-ethylmaleimide.

Purification and Characterization of Cytosolic V₁ Complexes Lacking the H Subunit-- To address the function of the H subunit in cytosolic V₁ complexes, we isolated the V₁ complex from a vma13 mutant strain. vma13 cells assemble unstable and inactive V₁V_o complexes at the vacuolar membrane (29). vma13 cells were briefly deprived of glucose (5 min in YEP) and the cytosolic V₁ complexes purified as described previously. As shown in Fig. 5, V₁ complexes isolated from vma13 cells showed the same subunit composition as V₁ from wild-type and vma3 cells with the exception of the loss of the H subunit, which runs just below the 60-kDa B subunit.

View larger version (54K): [in this window] [in a new window]   Fig. 5.   Subunit composition of cytosolic V1 complexes isolated from vma13 cells. Cytosolic V1 complexes were isolated from wild-type and vma13 mutant cells as described in the legend to Fig. 1 and under "Experimental Procedures." The V1 peak fraction after gel filtration chromatography was subjected to SDS-PAGE and stained with Coomassie Blue. The positions of known V1 subunits are indicated; the identities of the A, B, and E subunits were confirmed by immunoblotting.

Overall CaATPase activity of V₁ complexes missing H subunit was higher than that of V₁ complexes from wild-type and vma3 cells, primarily because the kinetics of ATP hydrolysis were linear for almost 20 min. The initial specific activity was 1.8-4.0 µmol of P_i/min/mg in two different preparations (Fig. 6), only slightly higher than that seen in the V₁ complexes isolated from vma3 cells. Linearity of the reaction over as much as 20 min suggested little or no product inhibition occurred during hydrolysis by the cytosolic V₁ complexes lacking subunit H. To better understand the lack of decay in the catalytic activity, we repeated the ADP preincubation experiment shown in Fig. 4A with the complexes from vma13 cells. The cytosolic V₁ complex from vma13 cells was preincubated with 1.1 mM CaADP and its ATPase activity measured as described above (Fig. 6B). After preincubation with ADP, the enzymes initial activity was 66% that without preincubation. V₁ complexes isolated from vma3 cells retained 51% of the initial activity, so complexes from vma13 cells were only slightly less sensitive to ADP inhibition. The kinetics of hydrolysis remained nearly linear over a 30-min period, however. These results indicate that in the absence of subunit H, the cytosolic V₁ complexes can still be inhibited by preincubation with ADP, but they still do not experience the decay of activity over time that may be an additional effect of product ADP in the complexes containing subunit H. 

View larger version (12K): [in this window] [in a new window]   Fig. 6.   CaATPase activity of cytosolic V1 complexes from vma13 cells. A, cytosolic V1 complexes were isolated from vma13 cells, and 10.3 µg of the isolated complexes were incubated with 1.4 mM CaATP in a 500-µl total volume for the indicated times (closed circles). The activity from 11.2 µg of V1 complexes isolated from vma3 cells and assayed under identical conditions is shown for comparison (open circles). Pi release was monitored by colorimetric assay (25). B, CaATPase activity was also measured after a 1-min preincubation of the complexes with 1.1 mM CaADP followed by a 30-fold dilution into assay buffer (500-µl final volume) containing 1.4 mM CaATP (closed circles), and compared with the activity in the absence of CaADP preincubation (open circles). For this set of experiments, 1.5 µg of protein was used.

The cytosolic V₁ complexes lacking subunit H also exhibited some MgATPase activity, even in the absence of activating agents. Kinetics of MgATP hydrolysis revealed an initial specific activity of 1.2 µmol of P_i/min/mg measured at 1 min (Fig. 7). Activity drastically decreased after the first 5 min. An apparent specific activity of 0.265 µmol of P_i/min/mg was measured after 15 min in assay medium containing an initial concentration of 1.4 mM MgATP. Potential activators were added during a 15-min incubation, and their effects on the MgATPase activity are shown in Table I. Methanol (25% v/v) gave an almost 2-fold increase in the apparent specific activity, but octylglucoside and sodium sulfite proved to be somewhat inhibitory.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   MgATPase activity of cytosolic V1 complexes from vma13 cells. Cytosolic V1 complexes were isolated from vma13 cells, and 1.5 µg of the isolated complexes were incubated with 1.4 mM MgATP in a 500-µl total volume for the indicated times. Pi release was monitored by colorimetric assay (25).

View this table: [in this window] [in a new window]   Table I Effect of activators on the MgATPase activity of cytosolic V1 complexes isolated from vma13 cells Cytosolic V1 complexes were isolated from vma13 cells, and 1.5 µg of the isolated complexes were incubated in the presence of 1.4 mM MgATP for 15 min in 500 µl of assay buffer either without additions or with the indicated concentrations of potential activators present throughout the incubation. Pi release was determined colorimetrically after 15 min and used to calculate the apparent specific activity.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Subunit Composition and Enzymatic Activities of Cytosolic V₁ Complexes-- The yeast cytosolic V₁ complexes contain established V₁ subunits A, B, D, E, F, G, and H. Subunit C was the only V₁ subunit not associated with yeast cytosolic V₁ complexes; this subunit was present in a high speed supernatant, but fractionated away from the other V₁ subunits in ion exchange chromatography. Earlier immunoprecipitation experiments had indicated that the C subunit dissociated from both the V₁ and V_o sectors during V-ATPase disassembly (13). The subunit composition of the yeast cytosolic V₁ complexes closely resembles that of the cytosolic V₁-ATPase complexes from M. sexta (14, 40). The M. sexta complexes appear to contain subunits A, B, D, E, F, and G, along with substoichiometric amounts of the C subunit (40). Subunit H has only recently been identified in M. sexta (17), so it is unclear whether this subunit is really not present in the insect cytosolic V₁ complexes, or is hidden by the B subunit, which generally runs very close to the H subunit on SDS-PAGE.

Both the insect and yeast cytosolic V₁ complexes are active as CaATPases, indicating that this activity does not require the presence of the C subunit. There were other striking similarities in the activities of the two enzyme preparations. Both showed a loss of CaATPase activity over time and a lower level of CaGTPase activity that did not decay over time. As noted by Graf et al. (14), these features are also shared by isolated F₁ sectors from chloroplasts and B. firmus (30, 31). One difference between the insect and yeast V₁ preparations is the activation of MgATPase activity in the insect enzyme in the presence of 25% methanol; no MgATPase activity was observed for the wild-type yeast V₁ preparation, even in the presence of a wide variety of potential activating agents. A shift from Mg²⁺-dependent to Ca²⁺-dependent ATP hydrolysis has been observed in V₁ subunit reconstitution experiments of the bovine clathrin-coated vesicle V-ATPase, as well. These subunit reconstitution experiments had indicated an essential role for the C subunit in CaATP hydrolysis (19), however, and this appears to conflict with results from the native cytosolic V₁ preparations.

We had anticipated that cytosolic V₁ sectors isolated from wild-type yeast cells and vma3 mutant cells before and after glucose deprivation might show differences in subunit composition that reflected their different histories and provided indications as to how glucose deprivation signals V₁ dissociation. This did not prove to be the case, at least at the level of analysis reported here. Both the subunit composition and basic enzymatic properties of the cytosolic V₁ sectors from different sources were very similar. It may still be that there are subtle differences in post-translational modifications of the subunits that we have not yet identified, and we plan to look at the different preparations in more detail in the future. It is also possible, however, that cytosolic V₁ sectors from wild-type cells before and after glucose deprivation have the same structure, and that different amounts of V₁ are present because the equilibrium of an ongoing dissociation and reassociation is shifted when glucose becomes limiting. Along the same lines, the cytosolic V₁ sectors that are formed in vma3 cells but never attach to the membrane may be stable because they resemble the cytosolic wild-type V₁ sectors that are normally cycling on and off the membrane.

How Is Mg²⁺-dependent ATP Hydrolysis by Cytosolic V₁ Sectors Silenced?-- Reversible disassembly of V-ATPases has been proposed to be a mechanism of down-regulating V-ATPase activity when growth conditions are unfavorable (16, 17). Underlying this proposal is the assumption, consistent with in vitro data (6, 8), that the cytosolic V₁ sectors are inactive in ATP hydrolysis. As expected, native cytosolic V₁ complexes purified from wild-type yeast cells could not hydrolyze ATP when Mg²⁺ was provided as the divalent cation, indicating that under physiological conditions, the yeast V₁ complexes are catalytically inactive. The results reported here suggest several potential reasons cytosolic V₁ sectors are not active in vivo.

Characterization of V₁ ATPase activity from a vma13 mutant suggests that the H subunit may play an important role in inhibiting both Mg²⁺- and Ca²⁺-dependent ATP hydrolysis by cytosolic V₁ sectors. An inhibitory role for the H subunit was not expected from previous data. The yeast vma13 mutant, which lacks the H subunit, assembles V₁V_o complexes in the membrane (29), but the complexes are unstable and inactive. Addition of sub-57-kDa dimer, which consists of two isoforms of the H subunit, to a V₁ complex reconstituted from bovine clathrin-coated vesicle subunits enhances CaATPase activity of the complexes, and sub-57-kDa dimer or either of the individual H subunit isoforms appears to be essential for MgATPase activity and proton pumping by the fully assembled bovine clathrin-coated vesicle pump (21, 41). Taken together, these data have suggested that the H subunit may act as an activator, not an inhibitor, of V-ATPase activity, but these experiments have focused predominantly on the intact V₁V_o complex, not isolated V₁ sectors. The experiments presented here suggest that the H subunit may play a role more similar to that of the  subunit of the E. coli F-ATPase, which inhibits the F₁-ATPase when it is detached from the membrane (33), but may be critical for proper structural and function coupling of F₁ and F_o (42, 43).

Comparison of CaATP hydrolysis by cytosolic V₁ sectors with and without the H subunit (Fig. 6A) indicates that the H subunit may be particularly critical for the decay in ATP hydrolysis rate after the first few minutes of turnover. Cytosolic V₁ complexes from vma13 cells showed only a slightly higher initial rate than those from vma3 cells, but they were able to maintain this rate for at least 20 min, under conditions where complexes from vma3 cells were almost completely inactive after less than 5 min. The higher activity of the complexes from vma13 cells could not be attributed to loss of another V₁ subunit, because all of the subunits except subunit H appeared to be present in these complexes. It is even more intriguing that cytosolic V₁ complexes lacking the H subunit appear to exhibit some Mg²⁺-dependent ATP hydrolysis that could be further activated in the presence of methanol. As described above, cytosolic V₁ sectors from M. sexta, which may or may not contain an equivalent of the H subunit, exhibited methanol-activated MgATPase activity in the cytosolic V₁ sectors. However, it is notable that the M. sexta enzyme appeared to lose Ca²⁺-dependent activity under conditions where it gained Mg²⁺-dependent activity, but both Ca²⁺ and Mg²⁺-dependent activities appear to be activated in the cytosolic V₁ complexes from vma13 cells. This result suggests that the methanol does not act on the M. sexta enzyme simply through release of the H subunit or a functional equivalent. The MgATPase activity of yeast vma13 complexes showed a loss of activity with time similar to that seen for the CaATPase activity of cytosolic V₁ sectors from wild-type cells, indicating that cytosolic V₁ sectors are still prevented from exhibiting high levels of unproductive ATP hydrolysis in vma13 cells in vivo. These data suggest that the H subunit is important in inactivating cytosolic V₁-ATPase activity, but there are probably other silencing mechanisms that act in combination, as described below.

One of these other mechanisms may be inhibition by ADP. The data presented here suggest that ADP could play a rather complex role in inhibiting the activity of cytosolic V₁ complexes. The loss of CaATPase activity in the wild-type complexes or MgATPase activity in the vma13 complexes over time could have at least two explanations. First, enzyme activity may destabilize the complex so that one or more subunits is lost, inactivating the enzyme. We cannot eliminate this possibility at present, but it should be possible to address it by careful determination of the subunit composition before and after catalysis. Alternatively, the loss of activity is suggestive of product inhibition, which could also be an effective means of minimizing unproductive ATP hydrolysis in vivo. Similar behavior of the M. sexta cytosolic V₁ has been attributed to product inhibition (14), and a more detailed analysis of the V₁-ATPase of Thermus thermophilus (44) has clearly demonstrated that this enzyme can be inactivated during ATP hydrolysis by entrapping an inhibitory MgADP at the catalytic site. In both of these cases, the similarity to entrapping of MgADP by F₁-ATPases has been noted, but there are also some important differences between the behavior of the yeast cytosolic V₁ complexes and F₁-ATPases (32, 35-37). First, briefly preincubating the yeast cytosolic V₁ with 1 mM CaADP gave only a partial inhibition of the initial CaATPase activity and did not appear to accelerate its decay. The extent of inhibition due to CaADP preincubation was similar in cytosolic V₁ complexes with or without the H subunit, even though the complexes without the H subunit showed much less inactivation during hydrolysis. Second, a number of activating agents that are believed to act by stimulating release of MgADP entrapped at a catalytic site of F₁-ATPases, for example, sulfite (32), do not have any effect on the Ca²⁺-dependent activity of the yeast cytosolic V₁. Perhaps most importantly, entrapment of a tightly bound MgADP or MgATP that persists through our purification protocol cannot account for the lack of MgATPase activity in cytosolic V₁ complexes from wild-type cells because the complexes as isolated are almost completely devoid of ADP and ATP. These data indicate that there may be at least two inhibitory effects of ADP: one type of inhibition depends on the formation of ADP during turnover and does not occur in complexes lacking the H subunit and the second type can be seen after a brief preincubation with ADP and occurs in complexes with and without the H subunit. The switch from MgATPase activity to CaATPase activity in the cytosolic V₁ complexes cannot be easily accounted for by the tighter binding of an inhibitory MgADP, unless this binding is so rapid and so tight that it occurs before significant MgATP hydrolysis can be observed. Complex effects of ADP on the V-ATPase of bovine clathrin-coated vesicles have been reported previously (45). Further experiments will be necessary to characterize the mechanisms of ADP inhibition of the yeast cytosolic V₁ complexes and fully assess their physiological significance.

The data presented here suggest that the inhibitory H subunit and inhibition by product ADP may play important roles is silencing unproductive hydrolysis by cytosolic V₁ complexes in yeast, but it is important to emphasize that they do not exclude other mechanisms of silencing. We have demonstrated release of the C subunit from cytosolic V₁ sectors, but have not yet determined whether this release plays a functional role. We have not yet assessed whether there are post-translational modifications of any of the V₁ subunits when they are released from the membrane, but with the purification protocol developed here, we are poised to determine both whether there are reversible modifications and whether these modifications affect activity. Silencing cytosolic V₁ complexes in vivo is likely to be a synergistic effect rather than a simple event. Considering that inhibition of cytosolic V₁ complexes is vital, it would not be surprising if cells had more than one mechanism to lock the catalytic conformation of the complex and prevent futile ATP hydrolysis.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant R01-GM50322 (to P. M. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

American Heart Association Established Investigator. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams St., Syracuse, NY 13210. Tel.: 315-464-8742; Fax: 315-464-8736; E-mail: kanepm@mail.upstate.edu.

Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M002305200

² K. L. Keenan and P. M. Kane, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; V₁, peripheral sector of V-ATPase; V_o, membrane sector of V-ATPase; F-ATPase, F₁F_o-ATP synthase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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