High purity preparations of higher plant vacuolar H+-ATPase reveal additional subunits. Revised subunit composition.

A fast protein liquid chromatography procedure for purification of the V-type H+-ATPase from higher plant vacuolar membrane to yield near-homogeneous enzyme with a specific activity of 20-25 mumol/mg.min is described. When precautions are taken to ensure the quantitative recovery of protein before sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the preparation is found to be constituted of seven major polypeptides of 100, 67, 55, 52, 44, 32, and 16 kDa, respectively, and two minor components of 42 and 29 kDa. The 52-, 44-, and 32-kDa polypeptides do not cross-react with antisera raised to the 67- and 55-kDa subunits of the enzyme, and two independent sample preparation procedures yield the same apparent subunit composition. The additional polypeptides are not breakdown products or aggregates of the previously identified subunits of the ATPase. The ATPase of tonoplast vesicles is subject to MgATP-dependent cold inactivation, and the conditions for inactivation are identical to those for the bovine chromaffin granule H+-ATPase (Moriyama, Y., and Nelson, N. (1989) J. Biol. Chem. 264, 3577-3582). Cold inactivation is accompanied by the detachment of five major polypeptides of 67, 55, 52, 44, and 32 kDa from the membrane, and all five components co-migrate with the corresponding polypeptides of the purified ATPase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 100- and 16-kDa polypeptides of the ATPase are not removed from the membrane during cold inactivation, but the latter can be purified to homogeneity by chloroform:methanol extraction of the fast protein liquid chromatography-purified enzyme. It is concluded that the tonoplast H+-ATPase is constituted of 6-7 major polypeptides organized into a peripheral sector comprising the 67-, 55-, 52-, 44-, and 32-kDa components and an integral sector consisting of the 100- and 16-kDa polypeptides. The V-type H+-ATPase from animal endomembranes and higher plant vacuolar membranes therefore have remarkably similar subunit compositions and gross topographies.

A fast protein liquid chromatography procedure for purification of the V-type H+-ATPase from higher plant vacuolar membrane to yield near-homogeneous enzyme with a specific activity of 20-25 ~mol/mg*min is described. When precautions are taken to ensure the quantitative recovery of protein before sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the preparation is found to be constituted of seven major polypeptides of 100, 67, 55, 52, 44, 32, and 16 kDa, respectively, and two minor components of 42 and 29 kDa. The 52-, 44-, and 32-kDa polypeptides do not cross-react with antisera raised to the 67-and 55-kDa subunits of the enzyme, and two independent sample preparation procedures yield the same apparent subunit composition. The additional polypeptides are not breakdown products or aggregates of the previously identified subunits of the ATPase.
The ATPase of tonoplast vesicles is subject to MgATP-dependent cold inactivation, and the conditions for inactivation are identical to those for the bovine chromaffin granule H+-ATPase (Moriyama, Y.,  J. Biol. Chem. 264,[3577][3578][3579][3580][3581][3582]. Cold inactivation is accompanied by the detachment of five major polypeptides of 67,55,52,44, and 32 kDa from the membrane, and all five components co-migrate with the corresponding polypeptides of the purified ATPase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 100-and 16-kDa polypeptides of the ATPase are not removed from the membrane during cold inactivation, but the latter can be purified to homogeneity by ch1oroform:methanol extraction of the fast protein liquid chromatographypurified enzyme.
It is concluded that the tonoplast H+-ATPase is constituted of 6-7 major polypeptides organized into a peripheral sector comprising the 67-, 55-, 52-, 44-, and 32-kDa components and an integral sector consisting of the 100-and 16-kDa polypeptides. The Vtype H+-ATPases from animal endomembranes and higher plant vacuolar membranes therefore have remarkably similar subunit compositions and gross topographies.
The endomembrane or vacuolar (V-type) H+-ATPases of * This work was supported by the Agriculture and Food Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ T o whom correspondence should be addressed. Tel.: 44-5827-both animal and plant cells constitute a third category of H+translocating phosphohydrolase distinct from the F-type (FoF1-type) H+-ATPases of mitochondria, chloroplasts, and eubacterial plasma membranes and the P-type ("plasma membrane-type") H+-ATPases of the plasma membranes of plants and fungi (Pedersen and Carafoli, 1987;Rea and Sanders, 1987).
V-type H'-ATPases have an apparent functional mass of 400,000-600,000 and comprise three to nine different subunits, of which the nucleotide-binding, 67-73-and 55-62-kDa polypeptides and a 16-kDa proteolipid are universal components (e.g. Pederson and Carafoli, 1987;Rea and Sanders, 1987). Immunological cross-reactivity between the nucleotidebinding subunits of the enzymes from plant and fungal vacuolar membranes and (animal) clathrin-coated vesicles and chromaffin granules is demonstrable (Manolson et al., 1987), and all of the enzymes concerned are inhibited by nitrate. Genomic and cDNA clones of the structural genes for the 67-73-kDa subunits of the enzymes from Neurospora and Daucus, respectively, yield deduced amino acid sequences with more than 60% identity (Bowman et al., 1988a;Zimniak et al., 1988), whereas the 55-62 kDa subunits from Arabidopsis (Manolson et al., 1988), Neurospora (Bowman et al. 1988b), and Saccharomyces  show greater than 70% sequence identity. Conservation of primary structure within the V-type category is therefore pronounced. A major but as yet unaddressed issue, however, is the extent of conservation of subunit composition within the category.
The reason for the differences between the enzymes from animal endomembranes and plant and fungal vacuolar membranes is not known. On the one hand, it has been suggested that the enzymes in membranes bounding catabolic intracellular compartments, such as lysosomes and plant vacuoles, may differ from those participating in receptor-mediated endocytosis and those present in secretory granules (Nelson, 1988), in that the latter may require "accessory" subunits for intracellular sorting. On the other hand, it should be appreciated that while the H+-ATPases from chromaffin granules

Revised Subunit Composition
for Tonoplast ATPase and clathrin-coated vesicles have been purified exhaustively from isolated membranes (Arai et al., 187;Cidon and Nelson, 1986;Xie and Stone, 1986), the enzymes from plant and fungal vacuolar membranes have only been purified partially, SO necessitating affinity labeling in parallel with purification for the ascription of specific polypeptides to the enzyme complex (e.g. Bowman et at., 1986;Manolson et al., 1985;Randall and Sze, 1986). Subunits without reactivity toward the affinity probes tested might consequently have been overlooked. Thus, in this investigation we present methods for the purification of the H+-ATPase of higher plant vacuolar membrane to near-homogeneity to test the notion of subgroups. The purification data in combination with studies of the mechanism of cold inactivation of the enzyme of native membranes provide the first clear evidence that the V-type AT-Pases from animal endomembranes and higher plant cells have identical subunit organizations.

MATERIALS AND METHODS
Preparation of Tonoplast Vesicles-Tonoplast vesicles were isolated from storage root of fresh, greenhouse-grown red beet (Beta vulgaris L.) by differential and sucrose density gradient centrifugation .
Chromatography-Solubilized tonoplast ATPase was purified by two successive chromatographic steps: gel filtration on Sephacryl S-400 and anion-exchange FPLC on Mono-Q. A 100 X 1-cm diameter column packed with Sephacryl s-400 was equilibrated with running buffer (10% (v/v) glycerol, 0.3% (w/v) Triton X-100, 0.05 mg/ml Type IV-S phospholipid, 5 mM dithiothreitol, 1 mM Tris-EDTA, 4 mM MgC12, 5 mM Tris-Mes (pH 8.0)). Triton X-100-solubilized membrane (3-5 mg of protein) was applied and the column was run at a flow rate of 3-4 ml/h and a temperature of 4 "C. Fractions of 1.2 ml were collected and assayed for protein and ATPase activity.
The peak ATPase fractions from chromatography on Sephacryl S-400 were then subjected to FPLC on an HR 5/5 Mono-Q column equilibrated with running buffer (0.1% (w/v) polyoxyethylene 10tridecyl ether (C12E,J, 20% (v/v) glycerol, 5 mM Tris-C1 (pH 6.01, 1 mM Tris-EDTA, 4 mM MgC12, 0.05 mg/ml Type IV-S phospholipid, 2 mM dithiothreitol). The sample (8-10 ml) was applied to the column with a Superloop (Pharmacia Biotechnology Inc., Milton Keynes, Great Britain) and eluted at a flow rate of 0.5 ml/min with a 5-phase salt gradient of 0-1 M KC1 in running buffer (see the legend to Fig.  1B for gradient conditions). One-ml fractions were collected and aliquots were assayed for ATPase activity.
Cold Inactivation of Tonoplast ATPase-The method of Moriyama and Nelson (1989) was employed for cold inactivation of the tonoplast ATPase and examination of its gross topography. Tonoplast vesicles were resuspended in medium consisting of 15 mM Tris-Mes (pH 8.01, 0.5 mM dithiothreitol, and 0.15 M NaCl in the presence or absence of 5 mM MgATP. The samples were incubated on ice and aliquots were taken for the measurement of ATPase activity at the times indicated. For the detection of polypeptides released from the membranes to the bulk medium during cold inactivation, tonoplast vesicles (3 mg/ ml) were incubated in inactivation buffer for 1 h at 4 "C. The mixture was then centrifuged at 200,000 X g for 1 h before removal of the supernatant and lyophilization for SDS-PAGE.
Ch1oroform:Methanol Extraction of Purified ATPase-A method based on that developed for the preparation of crude proteolipid from The abbreviations used are: Mes, 2-(N-morpholino)ethanesulfonic acid C,,E,, polyoxyethylene 10-tridecyl ether; FPLC, fast protein liquid chromatography; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis.
tonoplast (Rea et al., 1987a) was used. The peak ATPase fractions from FPLC on Mono-Q were lyophilized, resuspended in 1 volume of distilled water and 50 volumes of acetone:ethanol (1:l; -20 "C) were added. The samples were incubated for 3-4 h at -20 "C and centrifuged at 10,000 X g for 10-15 min. The supernatants were discarded and residual solvent dried under a stream of nitrogen. The dry pellet was resuspended in 1 volume of distilled water, and 25 volumes of ice-cold ch1oroform:methanol (2:l) were added. The mixture was stirred gently for 4-5 h at 4 "C and centrifuged at 12,000 X g for 10 min. The supernatant was carefully decanted, taken to dryness under nitrogen, and prepared for SDS-PAGE.
Zmmunobbts-The component polypeptides of the purified ATPase were transferred from 11% (w/v) polyacrylamide mini-gels to 0.45-pm nitrocellulose filters at 60 V (150-200 mA) for l h a t 4 "C in a Mini Trans-Blot transfer cell (Bio-Rad Laboratories Ltd., Watford, United Kingdom) in transfer buffer (Burnette, 1981). The nitrocellulose blots were blocked by incubation in "Blotto" (Johnson et al., 1984) for 1 h before overnight incubation in Blotto containing antisera raised against the 67-or 55-kDa subunits of the tonoplast ATPase from Beta (Manolson et al., 1987). The blots were washed with five changes of phosphate-buffered saline (Burnette, 1981) and incubated for 2 h in 50 ml of Blotto containing 5 pg of protein A-alkaline phosphatase conjugate. The filters were washed exhaustively with 1% (v/v) Triton X-100 in phosphate-buffered saline and then rinsed with 100 mM diethanolamine-HC1 buffer (pH 9.8). Alkaline phosphatase activity was detected using 5-bromo-4-chloro-3-indoyl phosphate and nitro blue tetrazolium (Blake et al., 1984).
Delipidatwn of Chromatographic Fractions-Two methods were used for the removal of detergent and lipid from the column fractions before SDS-PAGE. In the first, detergent and lipid were removed by sequential extraction with ethanol and diethyl ether (Piccioni et al., 1982). The samples were made 10% (w/v) with trichloroacetic acid, left on ice for 10 min, and centrifuged for 10 min in an Eppendorf microcentrifuge. The supernatants were aspirated, the pellets extracted with 1 ml of ice-cold 90% (v/v) ethanol, and the centrifugation step repeated. The pellets from ethanol extraction were resuspended in 50 p1 of 3 mM Tris-Mes (pH 8.0) containing 0.1% (w/v) SDS and extracted with 1 ml of diethyl ether at room temperature for 10 min. The two phases were resolved by centrifugation, the upper phase aspirated, and the residual ether removed by evaporation under nitrogen. The final aqueous phase was frozen, lyophilized, and denatured for 15 min at 60 "C in denaturation buffer (5% (w/v) SDS, 5% (w/v) 2-mercaptoethanol, 10 mM Tris-C1, pH 8.0) before SDS-PAGE.
In the second method, 1-ml volumes of the fractions were lyophilized, resuspended in l volume of distilled water, and 50 volumes of a 1:1 (v/v) mixture of acetone:ethanol (-20 'C) were added. The solutions were thoroughly mixed and incubated at -20 "C for 3-4 h, after which time they were centrifuged at 10,000 X g for 10 min. The supernatants were aspirated and the pellets were resuspended in denaturation buffer before SDS-PAGE.
SDS-PAGE-One-dimensional SDS-PAGE was performed routinely as described by Laemmli (1970) on concave exponential gradient gels (O'Farrell, 1975). The molecular weight markers were: The gels were stained with silver (Bio-Rad Laboratories Ltd., Watford, United Kingdom) and/or Coomassie Blue. Double-staining was by the method of Dzandu et al. (1984). Quantitative densitometry was performed by scanning the stained gels with an LKB Bromma Ultroscan XL enhanced laser densitometer at 633 nm.
ATPase Assays-ATPase activity was determined by measuring the rate of liberation of Pi from ATP at 37 "C. The reaction medium contained 30 mM Tris-Mes (pH 8.0), 50 mM KCl, 5 p M gramicidin D, 3 mM MgSO,, and 3 mM Tris-ATP. The reaction was initiated by the addition of membrane protein. The reaction was stopped (and, if present, C,ZEs or Triton X-100 and added phospholipid were precipitated) by the addition of 1 volume of ice-cold 10% (w/v) trichloroacetic acid, 4% (w/v) perchloric acid. The samples were left on ice for 2 min, centrifuged for 3 min in an Eppendorf microcentrifuge, and the supernatants were assayed for Pi by the method of Ames (1966).
To maximize the activity of the solubilized membranes and the fractions from chromatography, the reaction media were supplemented with 1.33 mg/ml sonicated Type IV-S phospholipid (Rea and Poole, 1986).
Protein-Protein was estimated routinely by the dye-binding method of Bradford (1976). Protein in the column fractions was measured by a modification of the Lowry method (Peterson, 1979).
Chemicals-Sephacryl S-400 and Mono-Q were from Pharmacia Biotechnology, Inc. (Milton Keynes, United Kingdom). Triton X-100 CI2Ea, Type IV-S phospholipid (partially purified soybean L-a-phosphatidylcholine), and protein A-alkaline phosphatase conjugate were purchased from Sigma Chemical Co. Ltd. (Poole, United Kingdom). All of the general laboratory reagents were from Sigma, British Drug House (Poole, United Kingdom), or FSA Laboratory Supplies (Loughborough, United Kingdom).

RESULTS AND DISCUSSION
Solubilization and Chromatography-Chromatography of Triton X-100-solubilized tonoplast on Sephacryl S-400, equilibrated and eluted with running buffer containing 0.3% (w/v) Triton X-100 and 0.05 mg/ml phospholipid (Fig. lA), resulted in an approximately 7-fold enrichment of the ATPase relative to native tonoplast and an approximately 9-fold purification relative to solubilized tonoplast ( Table I). Subsequent FPLC of the peak ATPase fractions from the Sephacryl S-400 column on Mono-Q (Fig. 1B) enabled a further 8-9-fold enrichment of the ATPase to yield enzyme with a specific activity of 20-22 pmol/mg.min. The overall purifications were 61and 74-fold relative to native and solubilized membranes, respectively, with an overall recovery of 26% (Table I).
Exogenous phospholipid in the running buffers and additional sonicated phospholipid in the assay media were neces-sary for the quantitative recovery of activity. Supplementation with phospholipid after chromatography in the absence of added phospholipid did not restore activity, indicating irreversible denaturation of the enzyme. Chromatography in the presence of phospholipid but assay without further supplementation, on the other hand, underestimated ATPase activity by a factor of 6-8. Consequently, phospholipid (0.05 mg/ml) was routinely included in the running buffers and all assays were performed in reaction medium containing 1.33 mg/ml sonicated phospholipid.
Nitrate Inhibitability-The identity of the purified ATPase was confirmed by its nitrate inhibitability (Table 11). The activity of the purified enzyme was 88% inhibited by 50 mM KNOs (in the presence of 50 mM KCl), whereas native tonoplast vesicles and the peak fractions from Sephacryl S-400 chromatography were inhibited by 55 and 69%, respectively.
Polypeptide Composition-Crucial to determining the polypeptide composition of the purified ATPase was the method employed for the preparation of the chromatographic fractions for SDS-PAGE (Fig. 2). Precipitation of the fractions

TABLE I Purification of tonoplast H+-ATPase
Tonoplast vesicles were solubilized with 4% (w/v) Triton X-100 (TX-100) (see "Materials and Methods") and chromatographed on Sephacryl S-400. The peak ATPase fractions (total volume 8 ml) from gel filtration chromatography were then applied to a Mono-Q column and eluted with KC1 as described in the legend to Fig. 1B. Native membranes were assayed in the presence of 5 ,.LM gramicidin D to ensure H+/cation equilibration. The solubilized membranes and chromatographic fractions were assayed in the presence of 1.33 mg/ ml sonicated phospholipid to ensure maximum phospholipid activation. "Peak ATPase'' refers to the column fractions containing the highest ATPase activity. The values shown are the mean from three separate purifications.
Step  Mono-Q with trichloroacetic acid and sequential extraction of the pellet with 90% (v/v) ethanol and diethyl ether to remove detergent and lipid (Piccioni et al., 1982) gave rise to a polypeptide composition similar to that reported previously by ourselves (Manolson et al., 1985) and others (e.g. Mandala and Taiz, 1986;Randall and Sze, 1986). The purified enzyme was almost wholly constituted of two polypeptides of 67 and 55 kDa (Fig. 2 A ) . Polypeptides with apparent molecular masses of 100, 44, and 32 kDa could be seen in the peak ATPase fractions, but their contribution to the staining reaction was small. Delipidation with acetone:ethanol (1:l) a t -20 "C, on the other hand, yielded a very different pattern (Fig. 2B). The purified enzyme was not only enriched for the 67-and 55-kDa polypeptides but also contained equally prominent components of 100, 52, 44, 32, and 16 kDa, and two minor components of 42 and 29 kDa after double staining with silver stain and Coomassie Blue. Direct comparison of the two delipidation procedures on the same chromatographic fractions confirmed that the differences were attributable to the method employed for preparing the samples for SDS-PAGE (Fig. 2C). Differential Solubility of ATPase Subunits-SDS-PAGE analysis of the successive solvent washes from delipidation by the method of Piccioni et al. (1982) revealed preferential dissolution of the 52-, 44-, 32-, and 16-kDa polypeptides of the purified ATPase in the 90% ethanol wash (Fig. 3A). Trichloroacetic acid precipitation without delipidation resulted in a polypeptide composition identical to that obtained by delipidation with acetone:ethanol but subsequent washing of the trichloroacetic acid pellet with 90% ethanol quantitatively extracted the 52-, 44-, 33-, and 16-kDa polypeptides to yield a polypeptide composition greatly enriched for the 67and 55-kDa subunits. The 52-, 44-, 32-, and 16-kDa polypeptides are apparently sufficiently hydrophobic to partition into 90% ethanol and so be overlooked, or at least be identified as only minor, potentially contaminating, components, when delipidation is by the method of Piccioni et al. (1982). Delipidation with acetone:ethanol was therefore employed routinely. Immunological Cross-reactiuity-Generation of the additional low molecular mass polypeptides by proteolysis and/or aggregation is refuted by the finding that antisera raised to the 67-and 55-kDa subunits of the ATPase were not crossreactive with the 52-, 44-, 42-, or 32-kDa polypeptides of the purified ATPase (Fig. 3B). Whether aggregation might account for the 100-kDa band, on the other hand, is less clear. Aggregated material of high molecular mass was evident on the blots probed with anti-67-kDa serum. While the most prominent high molecular mass, cross-reactive component did not comigrate with the 100-kDa band, a low intensity aggregate of the appropriate mass was discernible in the blots. The diffuse band at 100 kDa in the purified enzyme is therefore only tentatively identified as a discrete polypeptide.
Proportionality between Polypeptides-The component polypeptides of the ATPase-containing fractions copurified proportionately and behaved as if subunits of the same macromolecular complex. Quantitative densitometry of the fractions from FPLC on Mono-Q after SDS-PAGE demonstrated that the loo-, 55-, 52-, 44-, 32-, and 16-kDa polypeptides were enriched in direct proportion to the 67-kDa subunit to yield correlation coefficients of 0.96, 0.98,0.99, 0.98, 0.95, and 0.94, respectively (Table 111).
Cold Inactiuation-Recent investigations by Moriyama and Nelson (1989) demonstrate that the V-type H+-ATPases from a wide range of sources are subject to MgATP-dependent cold inactivation. The conditions for cold inactivation are well defined and the abolition of ATPase activity is associated with detachment of the 72-, 57-, 41-, 34-, and 33-kDa subunits of the chromaffin granule H+-ATPase from the membrane. Thus, in order to test our deductions concerning the subunit composition of the tonoplast ATPase, experiments were performed to determine if cold inactivation of the tonoplast enzyme has the same requirements as those defined by Moriyama and  and is associated with the selective detachment of polypeptides identical to those constituting the purified enzyme. The results are summarized in Figs. 4 and 5.
Incubation of tonoplast vesicles at 4 "C in the presence of 5 mM M8+, 5 mM ATP, and 0.15 M NaCl caused a rapid and irreversible abolition of ATPase activity. Inactivation was maximal after 20 min and resulted in a 65% loss of activity relative to controls (Fig. 4). ATP, alone, elicited some inactivation but vesicles incubated at 4 "C in M$+, alone, or in the absence of both M e and ATP exhibited negligible loss of activity over the course of the 60-min incubation period. While incubation at 4 "C for 5-10 min was sufficient to cause 50% inhibition, more than 60 min were required for similar inhibitions at room temperature. The requirements for cold inactivation of the tonoplast ATPase therefore closely ap- proximate those elucidated by Moriyama and Nelson (1989) for the chromaffin granule ATPase. Incubation of tonoplast vesicles with MgATP and NaCl at 4 "C for 60 min before centrifugation at 200,000 X g resulted in the release of 5 prominent polypeptides from the membranes (Fig. 5A)  The Mono-Q fractions containing ATPase activity were electrophoresed on 7-1276 (w/v) or 8-14% (w/v) concave exponential gels and double-stained. Individual bands were quantitated relative to the M, 67,000 band by integrating laser densitometry. The lines of best fit for plots of peak area for the M, 67,000 subunit (y) against the peak areas of the other polypeptides in the preparation ( x ) were computed using the algorithm of Marquadt (1963). Six fractions from five different chromatographic runs were quantitated (n = 30). The values for the correlation coefficient ( r ) were estimated by the least squares method.  tivation. The 16-kDa component could, however, be extracted from the purified ATPase with ch1oroform:methanol (2:l) to yield homogeneous polypeptide (Fig. 5B).
Our findings appear to invalidate this proposal. When precautions are taken to ensure quantitative recovery of all the component polypeptides of the chromatographic fractions, high purity ATPase preparations from higher plant vacuolar membrane have a polypeptide composition which closely corresponds with the enzymes from clathrin-coated vesicles and chromaffin granules (Table IV).
Lack of purity, proteolysis, and/or aggregation cannot explain the presence of the 52-, 44-, or 32-kDa polypeptides in the high purity tonoplast ATPase preparation. First, the specific activity of the purified ATPase (20-25 pmol/mg . min) is the highest achieved for any plant V-type enzyme and is closely comparable to the values of 15-18 pmol/mg.min reported for the 100-200-fold purified enzymes from clathrincoated vesicles (Xie and Stone, 1986) and chromaffin granules (Cidon and Nelson, 1986). Second, the 52-, 44-, and 32-kDa