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J Biol Chem, Vol. 274, Issue 3, 1301-1305, January 15, 1999

The Vacuolar H⁺-ATPase of Clathrin-coated Vesicles Is Reversibly Inhibited by S-Nitrosoglutathione^*

Michael Forgac

From the Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

	ABSTRACT

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It has been previously demonstrated that the vacuolar H⁺-ATPase (V-ATPase) of clathrin-coated vesicles is reversibly inhibited by disulfide bond formation between conserved cysteine residues at the catalytic site on the A subunit (Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 13224-13230). Proton transport and ATPase activity of the purified, reconstituted V-ATPase are now shown to be inhibited by the nitric oxide-generating reagent S-nitrosoglutathione (SNG). The K_0.5 for inhibition by SNG following incubation for 30 min at 37 °C is 200-400 µM. As with disulfide bond formation at the catalytic site, inhibition by SNG is reversed upon treatment with 100 mM dithiothreitol and is partially protected in the presence of ATP. Also as with disulfide bond formation, treatment of the V-ATPase with SNG protects activity from subsequent inactivation by N-ethylmaleimide, as demonstrated by restoration of activity by dithiothreitol following sequential treatment of the V-ATPase with SNG and N-ethylmaleimide. Moreover, inhibition by SNG is readily reversed by dithiothreitol but not by the reduced form of glutathione, suggesting that the disulfide bond formed at the catalytic site of the V-ATPase may not be immediately reduced under intracellular conditions. These results suggest that SNG inhibits the V-ATPase through disulfide bond formation between cysteine residues at the catalytic site and that nitric oxide (or nitrosothiols) might act as a negative regulator of V-ATPase activity in vivo.

	INTRODUCTION

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The vacuolar H⁺-ATPases (or V-ATPases¹) are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells (1-8). V-ATPases play an important role in such processes as receptor-mediated endocytosis, intracellular membrane traffic, protein processing and degradation, and coupled transport. V-ATPases in the plasma membrane of certain specialized cells also function in such events as renal acidification (9), bone resorption (10), pH homeostasis (11), and potassium secretion (12).

The V-ATPases are multisubunit complexes composed of two functional domains (1-8). The peripheral V₁ domain is a 570-kDa complex composed of eight subunits (subunits A-H) of molecular weight 70-13 kDa that is responsible for ATP hydrolysis, whereas the V₀ domain is a 260-kDa integral complex composed of five subunits (subunits a, d, c, c', c'') of molecular weight 100-17 kDa that is responsible for proton translocation. The V-ATPases are thus structurally and evolutionarily related to the F-ATPases of mitochondria, chloroplasts, and bacteria (13-19). The 70-kDa A subunit of the V-ATPases has been shown to possess the catalytic nucleotide binding sites of the V-ATPase complex (20-26).

We have previously demonstrated that the V-ATPase of clathrin-coated vesicles can be reversibly inhibited by disulfide bond formation between two conserved cysteine residues (Cys-254 and Cys-532) located at the catalytic site on the 73-kDa A subunit (22). Moreover, we have presented evidence that at least 50% of the V-ATPase in native clathrin-coated vesicles exists in the reversibly inactivated, disulfide-bonded state (27). It had been reported that V-ATPase activity in rat renal tubules is sensitive to nitric oxide (28), but whether this effect was directly on the V-ATPase and the mechanism of inhibition is unknown. We now report that the nitric oxide donor S-nitrosoglutathione can directly and reversibly inhibit the purified, reconstituted V-ATPase from clathrin-coated vesicles and that the properties of this effect suggest that inhibition results from disulfide bond formation at the catalytic site of the enzyme.

	EXPERIMENTAL PROCEDURES

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Materials-- Calf brains were obtained from a local slaughterhouse. Phospholipids were obtained as chloroform solutions from Avanti Polar Lipids, Inc. Acridine orange was obtained from Molecular Probes. S-Nitrosoglutathione (SNG), N-ethylmaleimide (NEM), dithiothreitol (DTT), adenosine-5'-triphosphate (ATP), valinomycin, and most other chemicals were obtained from Sigma Chemical Co.

Isolation of Clathrin-coated Vesicles and Purification and Reconstitution of the Coated Vesicle V-ATPase-- Clathrin-coated vesicles were isolated from calf brain as described previously (20). Following dissociation of the clathrin coat by treatment with 0.5 M Tris (pH 7.0), the V-ATPase was solubilized with polyoxyethylene-9-lauryl ether and purified by density gradient sedimentation on 15-30% glycerol gradients as described previously (20). The purified V-ATPase had a specific activity of 10-12 µmol of ATP/min/mg of protein at 37 °C. Reconstitution of the purified V-ATPase into phospholipid vesicles containing phosphatidylcholine, phosphatidylserine, and cholesterol was carried out by cholate dialysis as described previously (20).

ATPase and Proton Transport Assays-- ATPase activity of the purified, reconstituted V-ATPase was measured using a coupled, spectrophotometric assay in solubilization buffer (50 mM NaCl, 30 mM KCl, 20 mM HEPES, 10% glycerol, 0.2 mM EGTA) containing 1 mM ATP, 2 mM MgSO₄, 0.25 mg/ml NADH, 8 units/ml pyruvate kinase, 10 units/ml lactic dehydrogenase, 2 µM valinomycin, and 2 µM carbonyl cyanide p-chlorophenylhydrazone at 37 °C as described previously (20). Proton transport in clathrin-coated vesicles and reconstituted vesicles was measured by acridine orange fluorescence quenching using a Perkin Elmer LS-5 spectrofluorimeter in solubilization buffer (see above) containing 0.5 mM ATP, 1.0 mM MgSO₄, and 2 µM acridine orange, as described previously (20). The assay buffer for measurement of proton transport in reconstituted vesicles also contained 2 µM valinomycin to dissipate any membrane potential generated.

Treatment of Coated Vesicles and Purified, Reconstituted V-ATPase with SNG, Hydrogen Peroxide, NEM, DTT, and Glutathione-- Where indicated, coated vesicles (1 mg of protein/ml) or purified, reconstituted V-ATPase (6 µg of protein/ml) were treated with SNG or hydrogen peroxide in solubilization buffer at the concentrations shown for 30 min at 37 °C. Treatment with NEM (100 µM), DTT (5-100 mM), and the reduced form of glutathione (5-20 mM) were carried out in solubilization buffer for 30 min at 4 °C.

	RESULTS AND DISCUSSION

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It was previously observed (22) that disulfide bond formation between Cys-254 and Cys-532 at the catalytic site of the V-ATPase A subunit results in reversible inactivation of both proton transport and ATPase activity. In addition, approximately 50% of the V-ATPase in native clathrin-coated vesicles exists in this disulfide-bonded state (27) despite the fact that the A subunit is located on the cytoplasmic face of the V-ATPase complex (29, 30). This led to the suggestion that V-ATPase activity in vivo might be regulated by disulfide-bond formation (27). Formation of this inhibitory disulfide bond by molecular oxygen in vitro required prolonged incubation times (22, 27), and we were therefore interested in whether any naturally occurring oxidants could induce its formation. Fig. 1 shows that proton transport in intact clathrin-coated vesicles is inactivated by incubation with either SNG (a nitric oxide-generating reagent) (31) or hydrogen peroxide. These results are consistent with previous reports that V-ATPase activity in renal tubules is sensitive to nitric oxide (28) and that V-ATPase activity in Neurospora (32) and yeast (25) is sensitive to hydrogen peroxide. The studies in Neurospora in fact indicated that the V-ATPase is sensitive to a number of oxidizing agents, including nitrate (32). In addition, the results in Fig. 1 demonstrate that the inactivation induced by SNG or hydrogen peroxide can be largely reversed by treatment with the reducing agent DTT, a property shared with the inhibitory disulfide bond formed between active site cysteine residues (22, 27).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Inhibition of proton transport in clathrin-coated vesicles by SNG and hydrogen peroxide and reversal by DTT. Clathrin-coated vesicles (1 mg of protein/ml) were incubated with the indicated concentrations of SNG (A) or H2O2 (B) in solubilization buffer for 30 min at 37 °C and then an aliquot (10 µg of protein) was assayed for proton transport as described under "Experimental Procedures" () or incubated for an additional 30 min at 4 °C with 100 mM DTT () and then assayed for proton transport. Activities correspond to ATP-dependent quenching of acridine orange fluorescence relative to samples incubated under identical conditions except in the absence of SNG or H2O2.

Because the experiments carried out in Fig. 1 measure proton transport in intact coated vesicles, it was possible that SNG might be exerting its inhibitory effect indirectly, for example, by inhibiting a chloride channel required for acidification. Such modulation of acidification in coated vesicles through alteration of chloride channel activity has in fact been demonstrated for protein kinase A-dependent changes in acidification (33). Similarly, because V-ATPase activity was previously measured in crude renal tubules of undefined integrity (28), the same explanation could apply to that study. To address this question, the effect of SNG on activity of the purified, reconstituted V-ATPase was tested. As shown in Fig. 2, SNG inhibits both proton transport and ATPase activity of the purified, reconstituted V-ATPase, with 50% inhibition observed following incubation for 30 min at 37 °C with 200-400 µM SNG. Moreover, in both cases, inhibition is reversed by subsequent incubation with DTT.² These results indicate that SNG is directly inhibiting the V-ATPase and that this inhibition is reversed by treatment with reducing agents.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Inhibition of proton transport and ATPase activity of purified, reconstituted V-ATPase by SNG and reversal by DTT. Reconstituted vesicles containing purified V-ATPase (6 µg of protein/ml) were incubated with the indicated concentrations of SNG in solubilization buffer for 30 min at 37 °C and then an aliquot (0.3 µg of protein) was assayed for either proton transport (A) or ATPase activity (B) as described under "Experimental Procedures" (). Aliquots were incubated for an additional 30 min at 4 °C with 100 mM DTT before assay of activity (). In all cases, activities are expressed relative to samples incubated under identical conditions but in the absence of SNG. The specific activity of the purified V-ATPase was 10 µmol of ATP/min/mg of protein.

Because the reversibility by DTT is a property shared with the inhibitory disulfide bond formed between cysteine residues at the catalytic site of the V-ATPase (22, 27), we wished to determine whether inhibition of the V-ATPase by SNG could be protected in the presence of ATP, as shown for disulfide bond formation (22). As shown in Fig. 3, partial protection from inhibition by SNG was observed in the presence of ATP, suggesting that SNG was inducing the formation of an inhibitory disulfide bond at a nucleotide protectable site.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Protection of proton transport activity by purified, reconstituted V-ATPase from inhibition by SNG in the presence of ATP. Reconstituted vesicles containing the purified V-ATPase (6 µg of protein/ml) were treated with the indicated concentrations of SNG in the absence () or presence () of 2.5 mM ATP as described in the legend to Fig. 2 and proton transport was assayed as described under "Experimental Procedures." Activities are expressed relative to control samples incubated under identical conditions but in the absence of SNG.

To further test the relationship between inhibition by SNG and the inhibitory disulfide bond formed at the catalytic site of the V-ATPase, we tested the ability of treatment with SNG to protect V-ATPase activity from inhibition by NEM. Previously shown, disulfide bond formation between Cys-254 and Cys-532 causes partial protection of V-ATPase activity from inhibition by NEM (22, 27). The purified, reconstituted V-ATPase was first incubated in the presence or absence of 2 mM SNG for 30 min at 37 °C followed by incubation in the presence or absence of 100 µM NEM for 30 min at 4 °C. Reconstituted vesicles were then directly assayed for proton transport or incubated for an additional 30 min at 4 °C in the presence of 100 mM DTT. Fig. 4 shows that whereas reconstituted vesicles treated directly with NEM showed inhibition of proton transport that was not reversed by subsequent treatment with DTT (Fig. 4, bars 5 and 7), pretreatment of vesicles with SNG caused a partial protection from the inhibitory effects of NEM, as indicated by the partial restoration of activity by subsequent treatment with DTT (Fig. 4, bars 6 and 8). These results suggest that SNG inhibits V-ATPase activity through disulfide bond formation between cysteine residues at the catalytic site, although they do not rule out the possibility that inhibition occurs by S-nitrosylation of the active site cysteine residue, a process that has been shown to occur in other proteins (34).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Protection of proton transport activity of purified, reconstituted V-ATPase from inhibition by NEM by treatment with SNG. Reconstituted vesicles containing the purified V-ATPase (6 µg of protein/ml) were treated in the absence or presence of 2 mM SNG for 30 min at 37 °C as indicated in the legend to Fig. 2. Vesicles were then incubated an additional 30 min at 4 °C in the absence or presence of 100 µM NEM. Vesicles were then incubated for an additional 30 min at 4 °C in the absence or presence of 100 mM DTT and assayed for proton transport as described under "Experimental Procedures." Activities are expressed relative to a sample incubated under the identical conditions but in the absence of SNG, NEM, or DTT.

If an inhibitory disulfide bond is formed at the catalytic site of the V-ATPase in vivo, why is it not immediately reduced at the high concentrations of reduced glutathione found in the cytoplasm of cells? One possibility is that this regulatory disulfide bond is protected from reduction by one or more accessory polypeptides that are lost during purification of the V-ATPase. Alternatively, this disulfide bond may not be readily reduced by glutathione. To test this, purified, reconstituted V-ATPase, which had been inactivated by treatment with 2 mM SNG, was subsequently incubated with various concentrations of a reducing agent. As shown in Fig. 5, DTT was considerably more efficient at reversal of the inhibition induced by SNG than glutathione. This result suggests that once formed in vivo, the inhibitory disulfide bond formed at the catalytic site of the V-ATPase may not be rapidly reduced by cytosolic glutathione, but rather may be cleaved by an internal thio-disulfide exchange, as previously suggested (22).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Reversal of SNG inhibition of proton transport by the purified reconstituted V-ATPase in the presence of DTT or reduced glutathione. Reconstituted vesicles containing the purified V-ATPase (6 µg of protein) were incubated with 2 mM SNG for 30 in at 37 °C and then for an additional 30 min at 4 °C with the indicated concentrations of DTT () or reduced glutathione () before assay of proton transport as described under "Experimental Procedures." Activities are expressed relative to a sample incubated under identical conditions but in the absence of SNG or reductant.

What are the possible functions for nitric oxide inhibition of V-ATPase activity in vivo? Nitric oxide has been suggested to regulate proton flux across the apical membrane of intercalated cells in the kidney, which play an important role in acid base balance (28). Nitric oxide has also been shown to inhibit cytoplasmic pH regulation by V-ATPases in rat peritoneal macrophages (35), although the observed similar effects of a cGMP analog suggest that at least part of this effect may be through the more conventional effects of nitric oxide on guanyl cyclase. It is possible that nitric oxide synthase associated with the plasma membrane may serve to keep plasma membrane-associated V-ATPases in an inactivated state, so they only become activated by reduction following internalization and delivery to endocytic compartments. In fact, some isoforms of nitric oxide synthase have been shown to localize to the plasma membrane (36). Finally, nitric oxide has been implicated as an important modulator of long term depression of synaptic transmission (37), and it is possible that inhibition of the V-ATPase in synaptic vesicles (which is required for neurotransmitter uptake into these organelles) may contribute to this effect. It should be noted that, because some effects of nitric oxide (such as stimulation of guanyl cyclase) can be observed at submicromolar concentrations of nitric oxide (34, 37), it is unlikely that nitric oxide levels ever reach concentrations necessary to globally inhibit V-ATPases. Rather, V-ATPases may be inhibited by locally high concentrations of nitric oxide or nitrosothiols generated by nearby nitric oxide synthases.

Interestingly, it has been found that a mutation in the pathway for cysteine biosynthesis in yeast gives a phenotype characteristic of a V-ATPase knockout, and that this phenotype can be suppressed by a mutation in one of the A subunit cysteine residues participating in disulfide bond formation at the catalytic site (38). This study suggests that V-ATPase activity may be sensitive to the oxidation state of the cell and supports the model that vacuolar acidification may be regulated by disulfide bond formation in vivo (27). Additional work will be required, however, to elucidate the role that nitric oxide might play in controlling the oxidation state of the V-ATPase in the cell.

	ACKNOWLEDGEMENTS

I thank Dr. Elena Vasilyeva for isolation of clathrin-coated vesicles and Drs. Ting Xu, Tsuyoshi Nishi, and Amy Simon for helpful discussions.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM 34478. Fluorescence facilities were provided by NIH Grant DK34928.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.

To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6939; Fax: 617-636-0445.

¹ V-ATPase, vacuolar proton-translocating adenosine-5'-triphosphatase; SNG, S-nitrosoglutathione; NEM, N-ethylmaleimide; DTT, dithiothreitol.

² Proton transport by the purified, reconstituted V-ATPase was also inhibited by the nitric oxide-generating reagent 3-morpholino-sydnonimine (SIN-1), with 50% inhibition observed at 300-400 µM. Reversal of inhibition by DTT was less complete than with SNG, however, suggesting that secondary effects (besides disulfide-bond formation) may occur with this reagent.

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