Tissue Specificity of E Subunit Isoforms of Plant Vacuolar H 1 -ATPase and Existence of Isotype Enzymes*

Immunoblot analyses and partial amino acid sequenc-ings revealed that both the 40- (E1) and 37-kDa (E2) subunits of V-ATPase in the pea epicotyl were E subunit isoforms. Similarly, both the 35- (D1) and 29-kDa (D2) subunits were D subunit isoforms, although the similarity of the amino acid sequences is still unknown. In immunoblot analyses, two or three E subunit isoforms with molecular masses ranging from 29 to 40 kDa were detected in other plants. Two isotypes of V-ATPase from the pea epicotyl were separated by ion exchange chromatography and had subunit compositions differing only in the ratio of E1 and E2. There was a difference in the V max and K m of ATP hydrolysis between the two isotypes. E1 was scarcely detected in crude membrane fractions from the leaf and cotyledon, while E2 was detected in fractions from all of the tissues examined. The compositions of D subunit isoforms in the leaf and epicotyl were different, and the vacuolar membrane in the leaf did not contain D2. The efficiency of H 1 pumping activity in the vacuolar membrane of the leaf was higher than that of the epicotyl. The results suggest that the presence of the isoforms of D and E subunits is charac-teristic to plants and that the isoforms are closely related to the enzymatic properties.

Vacuoles of plant cells play a fundamental role in the regulation of cell turgor and in the transport and storage of ions and metabolites (1). Vacuolar H ϩ -ATPase (V-ATPase), 1 together with H ϩ -pyrophosphatase, generates an electrochemical gradient across the membrane and provides a driving force for secondary active transporters. V-ATPase is composed of two general domains, a peripheral catalytic domain (V 1 ) and a transmembranous proton channel (V 0 ) (2,3). The V 1 domain of animal and yeast enzymes consists of eight subunits: three copies of A and B subunits and one copy of several other accessory subunits of C to H (4,5). The V 0 domain consists of five subunits: six copies of the c subunit and one copy of other accessory subunits of a, cЈ, cЉ, and d (5). In the coated vesicle V-ATPase, the stoichiometry was determined to be A 3 B 3 C 1 D 1 E 1 a 1 c 6 (6). The tertiary structure of V-ATPase is similar to that of F-ATPase (7). High resolution analysis of the structure of the mitochondrial F 1 domain (peripheral domain) revealed that the central cavity of the F 1 domain is formed by alternating the ␣ and ␤ subunits (␣ 3 ␤ 3 ), which correspond to the B and A subunits in V-ATPase, respectively, and is occupied with ␥ subunit (8). The ␥ subunit has recently been demonstrated to rotate against the ␣ 3 ␤ 3 complex during ATP hydrolysis (9,10). The rotation of the ␥ subunit is thought to be physicochemically coupled with H ϩ translocation in the F 0 domain (membrane domain). In V-ATPase, the homologue of the ␥ subunit has not yet been identified, although some candidates have been recommended, such as the D subunit (11) or a pair of D and E subunits (12). On the other hand, Xie reported that at least the A, B, C, E, and G subunits, but not the D or F subunits, were essential for ATP hydrolysis (4).
We have been studying V-ATPase properties in the mung bean (chilling-sensitive) and pea (chilling-tolerant) from the viewpoint of cold inactivation of the enzyme (13)(14)(15). In this study, we purified and characterized V-ATPase in the pea. We found that the 40-and 37-kDa subunits were both E subunit isoforms. The existence of isoforms of the catalytic A and B subunits in the V 1 domain has been established in the enzymes of plants and mammals (16 -25). There are a few reports on the isoforms of the accessory subunits in the V 1 domain. V-ATPases in the plasma and microsomal membranes of the kidney brush border have the heterogeneity of the 31-kDa subunit (E subunit), although the E subunit was encoded by a single gene (26). Crider et al. (27) reported an organ-specific isoform of the G subunit (ϳ14 kDa) in animal enzymes and demonstrated that the enzymatic activity of the reconstituted complex with A, B, C, E, and G1 subunits was different from that with A, B, C, E, and G2 subunits. In addition to these works, there have been some studies on the V-ATPase isotypes purified from different tissues. V-ATPases in the kidney cortex brush border and cortex microsome were different in pH optimum, K m for ATP, phospholipid dependence, and the structural microheterogeneity of the B and E subunits (28,29). The lemon V-ATPase is a good example for plant systems. V-ATPases purified from the fruit and epicotyl of the lemon have different features such as subunit composition, shape in electron micrography, efficiency of H ϩ pumping activity, and sensitivity to vanadate (30).
An investigation of the isotypes of V-ATPase with a different subunit isoform and the tissue specificity of isoforms may help us to understand both the structure-function relationship and the physiological significance of tissue-specific expression of the subunit isoforms. In this work, we mainly investigated the isoforms of V-ATPase subunits, the isotypes of V-ATPase, and the tissue-specific distribution of the subunit isoforms. We also investigated the relationship between the E subunit of the pea and the corresponding subunits of other organisms. To our knowledge, this is the first report on the E subunit isoforms of V-ATPase at the protein level. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
Plant Materials-Seeds of the pea (Pisum sativum L. cv. Kinusaya) were sown in moist vermiculite in the dark at 20°C and left to grow for 7 days. The epicotyls, leaves, cotyledons, and roots were then removed from the etiolated seedlings and used for membrane preparation. Mature leaves and flower petals were excised from pea plants that had been growing for 2 months in the field. Seeds of the mung bean (Vigna radiata L. cv. Wilczek) were imbibed in 1 mM CaSO 4 and sown in the dark at 26°C. Hypocotyls from the etiolated 3.5-day-old seedlings were used to prepare vacuolar membranes. Seeds of Arabidopsis thaliana were germinated and grown on moist rock wool in the light at 22°C for 1 month, and green leaves were used for membrane preparation. Leaves of spinach (Spinacia oleracea) were purchased at a local market and used for membrane preparation.
Vacuolar Membrane Preparation and V-ATPase Purification-The total crude membrane and vacuolar membrane fractions were prepared from epicotyls and other tissues of the pea and mung bean by differential and floating centrifugation methods as described previously (13,31). Vacuolar membranes (3 mg of protein/ml) were treated with 5% (w/v) Triton X-100 in 0.1 M KCl, and then V-ATPase was solubilized from the membrane with 2 mg/ml lysophosphatidylcholine (Sigma, egg yolk, type I) (13). The enzyme was purified by ion exchange chromatography using a column of QAE-Toyopearl 550C (Tosoh, Tokyo, Japan) pre-equilibrated with a running buffer containing 20 mM Tris acetate (pH 7.5), 1 mM EGTA, 1 mM dithiothreitol, 2 mM MgCl 2 , 20% (w/v) glycerol, and 0.1% (v/v) Tween 20 (buffer A). The column (bed volume: 4 ml) was washed with 20 ml of buffer A containing 0.2 M NaCl, and then the ATPase was eluted from the column with 20 ml of buffer A containing 0.4 M NaCl (pea enzyme) or 0.3 M NaCl (mung bean enzyme). The enzyme solution from QAE-Toyopearl chromatography was desalted and concentrated using an Ultrafree UFC3 LTK 00 (Millipore) at 0°C. The concentrated fraction (0.4 ml) was layered onto 4 ml of linear glycerol density gradient of 20 -40% (w/v) glycerol in 20 mM Tris acetate (pH 7.5), 1 mM EGTA, 1 mM dithiothreitol, 2 mM MgCl 2 , and 0.1% (v/v) Tween 20 and centrifuged at 200,000 ϫ g (45,000 rpm) in a swinging rotor (Hitachi, RPS-56 rotor) for 10 h at 4°C. The gradient after centrifugation was fractionated into 0.25-ml aliquots, and the fractions were subjected to ATPase assay. The major peak fractions of ATPase activity were further purified by ion exchange chromatography of Mono Q (Amersham Pharmacia Biotech) with an FPLC system (Amersham Pharmacia Biotech). The column (bed volume: 1 ml) was pre-equilibrated with buffer A, and then the V-ATPase was eluted with 60 ml of a shallow linear gradient of 0.2-0.4 M NaCl in buffer A at a flow rate of 0.15 ml/min and fractionated into 0.5-ml aliquots at room temperature.
Analytical Measurements-ATPase activity was assayed at 30°C in 0.25 ml of medium containing 3 mM ATP, 3 mM MgSO 4 , 50 mM KCl, 1 mM sodium molybdate, 20 g of phosphatidylcholine (Sigma, soybean, type IV-S), and 25 mM Hepes-BTP (pH 7.2) (13). Released P i was colorimetrically measured by the modified method of Hoges and Leonard (32). Protein concentration was determined by the method of Bradford using ␥-globulin as the standard (33).
H ϩ pumping activity and ATPase activity were simultaneously measured by the modified method of Palmgren (34) or Mü ller et al. (35). The assay medium consisted of 25 mM Hepes-BTP, pH 7.2, 250 mM sorbitol, 3 mM ATP (Roche Molecular Biochemicals), 50 mM KCl, 0.5 M valinomycin (Sigma), 0.25 mM very freshly prepared NADH (Roche Molecular Biochemicals), 1 mM phosphoenolpyruvate (Sigma), 20 M acridine orange, and 25 l/ml of a mixture of pyruvate kinase and lactate dehydrogenase (Sigma) and was equilibrated at 25°C. An aliquot (2.955 ml) of the assay medium was transferred to a quartz cuvette, and 30 l of the vacuolar membrane was added. After incubation for 3 min, the reaction was started by the addition of 15 l of MgSO 4 to a final concentration of 3 mM, and the absorbance at 340 and 495 nm was recorded in 10-s intervals with a spectrophotometer (Shimadzu, model UV-160) that can measure the absorbance at two different wavelengths by automatic switching between them. In this assay, H ϩ pumping activity was measured by the absorbance decrease of acridine orange at 495 nm, and ATPase activity was simultaneously measured by coupling the appearance of ADP to the oxidation of NADH, which was followed by the absorbance decrease at 340 nm.
PAGE, Two-dimensional Electrophoresis, and Immunoblot Analysis-SDS-PAGE on 13% polyacrylamide gel containing 0.1% SDS was carried out according to Laemmli (36), and then proteins in the gels were visualized with silver staining (37). Two-dimensional PAGE was performed by isoelectric focusing (IEF) as the first dimension and SDS-PAGE as the second dimension. IEF was carried out according to the method of O'Farrel et al. (37).
The electrotransfer of proteins in a gel onto a PVDF membrane (Millipore) and immunostaining with the antibodies were performed according to the standard procedure (39). The antigens on PVDF membranes were visualized with alkaline phosphatase-linked anti-rabbit goat IgG (40) using bromochloroindolyl phosphate and nitro blue tetrazolium as color development reagents.
Preparation of Antibodies-To prepare the antibodies, we thoroughly purified the 40-and 37-kDa subunits of V-ATPase by SDS-PAGE of the purified enzyme and electroelution with an Electroeluter (Bio-Rad, model 422). Before immunization, we confirmed that each purified fraction contained a single polypeptide band in SDS-PAGE and silver staining. Antibodies against the subunits were raised in rabbits by injection of each subunit (total amount, 80 g) in 50% adjuvant (Titer Max, CytRx Co.) solution. The immunoglobulin G fraction was prepared from each antiserum by fractionation with ammonium sulfate and affinity chromatography on an Ampure PA column (Amersham Pharmacia Biotech). The antibodies against the 40-and 37-kDa subunits were designated anti-40 and anti-37. The antibodies against the A, B, and 32-kDa subunits of mung bean enzyme were prepared as described previously (14) and were designated anti-A, anti-B, and anti-32, respectively.
Peptide Mapping and Sequencing-The 40-, 37-, and 35-kDa subunits of V-ATPase from the pea epicotyl were isolated by SDS-PAGE and electroelution. Proteins after elution were precipitated by cold acetone and then dried. A 100-l aliquot of digestion buffer containing 0.2 M ammonium bicarbonate (pH 7.8), 2 M urea, and 40 pmol of V8 protease (Wako) was added to the precipitates of the 40-and 37-kDa subunits, and a 100-l aliquot of digestion buffer containing 0.1 M Tris-HCl (pH 9.0), 0.1% SDS, 4 M urea, and 40 pmol of lysylendopeptidase (Wako) was added to the precipitate of the 35-kDa subunit. The resulting peptides were separated by SDS-PAGE on a 16% polyacrylamide gel and electrotransferred onto a PVDF membrane. The peptides on PVDF membrane were visualized by Coomassie Brilliant Blue R-250 staining, and the bands were excised from the membrane for amino acid sequencing. Amino acid sequences of the NH 2 termini of the proteolytic fragments were determined by Edman degradation in a gas phase sequencer (Shimadzu, model PPSQ-10).

RESULTS
Partial Amino Acid Sequence of 40-, 37-, and 35-kDa Subunits of Pea V-ATPase-After V-ATPase was solubilized by lysophosphatidylcholine from vacuolar membranes, the enzyme in a detergent-soluble fraction was purified by ion exchange chromatography of a QAE-Toyopearl column. At this step, the proteins in the eluted fraction were mostly V-ATPase complexes (13). Mung bean V-ATPase is composed of nine subunits: 68-(A subunit), 57-(B subunit), 44-, 38-, 37-, 32-, 16-(proteolipid subunit), 13-, and 12-kDa subunits, as reported previously (13). The subunit composition of pea V-ATPase was compared with that of the mung bean enzyme using QAE-Toyopearl fractions (Fig. 1). In the pea V-ATPase, the 68-and 57-kDa polypeptides correspond to the A and B subunits of V-ATPase, judging from the immunoreactivity with antibodies to the A and B subunits of mung bean enzyme (for example, Fig. 7). The two species had different subunits with molecular masses ranging from 40 to 30 kDa, although the molecular masses of the other subunits, including the 100-and 51-kDa subunits, were almost identical. We therefore determined the internal, partial amino acid sequences of the 35-kDa subunit of pea enzyme after treatment with lysylendopeptidase and the 40-and 37-kDa subunits of pea enzyme after treatment with V8 protease (Fig. 2). A homology search showed that one peptide sequence (Pea-40k-1) from the 40-kDa subunit and three (Pea-37k-1, -2, and -3) from the 37-kDa subunit were homologous to the E subunits of V-ATPase in several organisms ( Fig.  2A). The partial sequence of 40k-1 was identical to the portion of 37k-2. The partial sequences of 37k-3, 37k-4, 40k-2, and 40k-3 were very similar, but some amino acid residues were different from those of other species. We concluded that the 40and 37-kDa subunits were isoforms of the E subunit and designated them E1 and E2 subunits, respectively. The partial sequence of pea 35-kDa subunit corresponded to the D subunit sequences of other organisms, such as A. thaliana (41), Saccharomyces cerevisiae (42), bovine (11), and Caenorhabditis elegans (National Center for Biotechnology (NCB) accession no. P34462) (Fig. 2B). Because the antibody specific to the 32-kDa subunit of mung bean enzyme (anti-32) reacted with the pea 35-kDa subunit, the 32-kDa subunit of the mung bean was also concluded to be a D subunit (see, for example, Fig. 7). Immunoblot analysis with anti-32 showed that the 29-kDa subunits of the pea and mung bean were immunochemically similar to the D subunit.
Immunoblot Analysis of E Subunit Isoforms of Plant V-ATPase-We prepared the antibodies, anti-40 and anti-37, against the isolated E1 and E2 subunits of pea enzyme, respectively. Anti-40 reacted preferentially with the E1 subunit and also with the E2 subunit of pea V-ATPase (Fig. 3, lane 1). Anti-37 strongly reacted with the E2 subunit and slightly with the E1 subunit of pea enzyme (Fig. 3, lane 2). Both 40-and 37-kDa polypeptides in the microsome fraction as well as in the purified V-ATPase were detected by using either anti-40 or anti-37 (see, for example, Fig. 9), indicating that the E1 and E2 subunits of pea V-ATPase are immunologically similar. Immunoblot analysis of mung bean enzyme revealed that both anti-40 and anti-37 cross-reacted equally with 38-and 37-kDa subunits of mung bean enzyme (Fig. 3, lanes 3 and 4), suggesting that these two polypeptides are also closely related to the 40-and 37-kDa subunits of the pea. The 38-and 37-kDa subunits of mung bean enzymes were also thought to be isoforms of the E subunit.
The cDNAs for the E subunit cloned from A. thaliana (Ref.  7 and 8). Thus, there is variation in the molecular mass of the E subunits among plant species.
To obtain more information about E subunit isoforms, we performed two-dimensional PAGE and immunoblot analysis of pea and mung bean enzymes. As shown in Fig. 4A, the E1 and E2 subunits of pea enzyme gave a single spot with pI values of 6.4 and 5.5, respectively. The 38-kDa subunit of mung bean enzyme also gave a single spot with a pI value of 5.9, while the 37-kDa subunit gave two spots with pI values of 6.5 and 6.3, respectively (Fig. 4B).
Separation of Isotypes of V-ATPase-The existence of isoforms suggests the presence of isotypes of V-ATPase. For detailed studies of the V-ATPase isotypes, the enzyme fraction of the pea epicotyls that was purified by QAE-Toyopearls was further subjected to glycerol density gradient centrifugation. As shown in Fig. 5A, the profile of ATPase activity showed one major peak around fraction 9 (fractions 8 -12) and a small A, the 40-and 37-kDa subunits isolated from the pea enzyme were treated with V8 protease and were then subjected to internal amino acid sequence analyses. The sequences of three and four fragments from 40-and 37-kDa subunits, respectively, were determined (40k-1 to -3, 37k-1 to -4). The internal sequences of the 40-and 37-kDa subunits were compared with the homologous sequences deduced from the cDNA for spinach (S. oleracea) (43), M. crystallinum (46), barley (H. vulgare) (44), upland cotton (G. hirsutum) (45), and A. thaliana1 (43) and A. thaliana2 (NCB accession no. AAC35545) E subunits. B, the 35-kDa subunit isolated from the pea enzyme was treated with lysylendopeptidase and then the internal sequences were determined. The internal sequence of the 35-kDa subunit was compared with the homologous sequences deduced from the cDNA for A. thaliana (41), S. cerevisiae (42), B. gauras (11), and C. elegans (NCB accession no. P34462) D subunits. Multiple sequence alignment was performed using clustalW. Identical amino acids are indicated by asterisks, high similarities are indicated by colons, and low similarities are indicated by dots. The numbers beside amino acid sequences represent the sequence position of the amino acids from the amino terminus.
increase in fraction 15. The fractions were subjected to SDS-PAGE to compare the polypeptide composition (Fig. 5B). The major peak fractions were further purified by ion exchange chromatography of a Mono Q column with a shallow linear gradient of NaCl. The elution profile of the enzyme activity showed two peaks (Fig. 6A) at fractions 31 (0.3 M NaCl) and 36 (0.32 M NaCl). Fig. 6B shows the protein profile after SDS-PAGE of these fractions. The small peak (fraction 31) contained 13 polypeptides of 100, 68, 57, 51, 45, 40, 37, 35, 29, 16, 15.5, 13, and 12 kDa. The polypeptide composition of the large peak (fraction 36) was the same as that of fraction 31 except for depletion of the E1 subunit (40 kDa). The E1 subunit was distributed only around fraction 31.
To discriminate between the two peaks of V-ATPase activity separated by Mono Q column chromatography, we designated the mixture of fractions 31 and 32 as P1 and the mixture of fractions 36 and 37 as P2. The total activities of P1 and P2 were 3.52 and 5.39 mol h Ϫ1 , respectively. The SDS-PAGE profile obtained with the same amounts of protein revealed different levels of E1 (40 kDa) and E2 (37 kDa) subunits in P1 and P2 (Fig. 7). Two fractions of P1 and P2 were subjected to immunoblot analysis to compare the amounts of E1 and E2 subunits with those of A (68 kDa), B (57 kDa), D1 (35 kDa), and D2 (29 kDa) subunits between the two fractions (Fig. 7A). There was no difference in the amounts of A, B, D1, and D2 subunits between the P1 and P2 fractions (Fig. 7, B and D). On the other hand, the intensities of the E1 and E2 subunits were markedly different in the P1 and P2 fractions (Fig. 7C). The ratio of the E1 subunit intensity in P1 to that in P2 was 5.9, and the ratio of the E2 subunit was 0.71. The E1 subunit was predominant in the isotype of V-ATPase in the P1 fraction. We then analyzed the enzymatic properties of V-ATPase in the fractions of the P1 and P2 (Fig. 8A). The K m values for ATP of P1 and P2 fractions were 0.94 Ϯ 0.08 and 1.56 Ϯ 0.06 mM, respectively. The V max values of P1 and P2 were 36.0 Ϯ 1.2 and 51.5 Ϯ 1.1 mol mg Ϫ1 h Ϫ1 , respectively. This showed that the P2 fraction contained a relatively high capacity and high K m enzyme. These results suggest that the pea epicotyl contains isotypes of V-ATPase with different kinetic properties and different subunit compositions.
The same experiment was performed for mung bean enzyme from hypocotyl. We observed four peaks, P1 (0.23 M NaCl), P2 (0.24 M NaCl), P3 (0.25 M NaCl), and P4 (0.265 M NaCl), of ATPase activity after Mono Q column chromatography of a shallow linear gradient of NaCl. The SDS-PAGE profile obtained with the same amounts of protein in each peak fraction revealed the same levels of 100-, 68-, 57-, 51-, 44-, 16-, 13-, and 12-kDa subunits and different levels of 38-, 37-, 32-, and 29-kDa subunits among the four fractions (Fig. 7E). These four fractions were subjected to immunoblot analysis to investigate the distribution of E1 (38 kDa), E2 (37 kDa), D1 (32 kDa), and D2 (29 kDa) subunits, containing A (68 kDa) and B (57 kDa) subunits. There were no differences in the amounts of A and B subunits among the four fractions (Fig. 7F). The E1 subunit intensity increased from the P1 to P4 fraction, and, reversely, the E2 subunit intensity decreased and almost disappeared in the P4 fraction (Fig. 7G). Similarly, the D1 subunit intensity increased, and the D2 subunit intensity decreased from the P1 FIG. 3. Immunochemical detection of E subunits in the pea,  mung bean, A. thaliana, and spinach. In the lanes of pea and mung bean, proteins (4 g) of the purified enzymes were analyzed after QAE-Toyopearl chromatography. In the lanes of A. thaliana, proteins (40 g) of the total crude membrane fraction from mature leaf were analyzed. In the lanes of spinach, proteins (10 g) of the vacuolar membrane from mature leaf were analyzed. The E subunit was detected by immunoblot analyses with anti-40 (lanes 1, 3, 5, and 7) and anti-37 (lanes 2, 4, 6, and 8). to P4 fraction (Fig. 7H). The D2 subunit completely disappeared in the P4 fraction. When we analyzed the enzymatic properties of mung bean V-ATPase in the four fractions (Fig.  8B), the K m values for ATP of P1 to P4 fractions were 1.11 Ϯ 0.06, 1.27 Ϯ 0.04, 1.27 Ϯ 0.09, and 0.83 Ϯ 0.08 mM, respectively, and the V max values of P1 to P4 were 32.1 Ϯ 0.6, 34.8 Ϯ 0.5, 36.2 Ϯ 1.2, and 22.8 Ϯ 0.7 mol mg Ϫ1 h Ϫ1 , respectively. In enzymatic properties of ATPase activity, the fractions of P1, P2, and P3 contained similar enzymes, but only the P4 fraction was different from other fractions and contained a relatively low capacity and low K m enzyme.
Tissue Specificity of the E Subunit Isoforms-We investigated the distribution of the two isoforms of the E subunit in various tissues of the pea by immunoblot analysis with anti-40 and anti-37 (Fig. 9A). In this experiment, we used total crude membranes prepared from leaves, cotyledons, epicotyls, and roots of etiolated seedlings and leaves and flower petals of mature plants. The E2 subunit was detected in all of the tissues examined. The E1 subunit was clearly detected in the root and epicotyl (Fig. 9A, lanes 5-7) and weakly detected in the flower petal (lane 4). However, the E1 subunit was not detected in the leaf or cotyledon of etiolated seedling (lanes 1 and 2) and was hardly detected in the green leaf of a mature plant (lane 3). These results indicate that the E1 subunit is a tissue-specific isoform and that the E2 subunit is a common isoform. We also examined the distribution of E subunit isoforms in the crude microsomal fractions prepared from tissues of mung bean and tomato. Two E subunit isoforms were immunochemically detected in the mung bean and tomato. The isoform with the larger molecular mass was found in smaller amounts in the leaf than in the hypocotyl and root of the mung bean (Fig. 9B). Additionally, the tomato E subunit with a larger molecular mass was less abundant in the petiole than in the leaf (Fig. 9C).
We then prepared vacuolar membrane fractions from the pea leaf and epicotyl and compared the ATPase activities and H ϩ pumping activities in the two tissues. These fractions were subjected to immunoblot analysis to compare the amounts of B (57 kDa), E1 (40 kDa), E2 (37 kDa), D1 (35 kDa), and D2 (29 kDa) subunits on the basis of the A subunit (68 kDa) in the leaf and epicotyl (Table I). As was the case using total crude membrane fractions, the amount of the E1 subunit in the leaf was less than that in the epicotyl. The amounts of D1 and D2  subunits were also different in the leaf and epicotyl. The amount of the D1 subunit in the leaf was higher than that in the epicotyl, but the D2 subunit was not detected in the leaf. The ATPase activities determined on the basis of proteins were almost the same in the leaf and epicotyl, but the ATPase activities determined on the basis of the A subunit were clearly different (Table II). Then, to determine the efficiency of H ϩ pumping activity on the basis of ATP hydrolysis, H ϩ pumping and ATPase activities in vacuolar membrane fractions were simultaneously measured by using the spectrophotometric assay. After adjusting the ATPase activities in the leaf and epicotyl to almost same levels, we compared the H ϩ pumping activities in the two tissues. When the protein concentrations of vacuolar membranes from the leaf and epicotyl were adjusted to 22 and 43 g/ml, respectively, the total ATPase activities and nitrate-insensitive ATPase activities were almost the same in the leaf and epicotyl (Fig. 10, A and B). The nitrate-sensitive ATPase activities of leaves and epicotyls were calculated to be 0.048 Ϯ 0.001 and 0.048 Ϯ 0.002 absorbance/min, respectively. Because the H ϩ pumping activity was completely inhibited by 200 mM nitrate (Fig. 10D), it was thought that this proton gradient was formed by V-ATPase. As shown in Fig. 10C, the efficiency of H ϩ pumping activity differed in vacuolar membranes from leaf and epicotyl. The initial rates in the leaf and epicotyl were 0.156 Ϯ 0.001 and 0.113 Ϯ 0.006 absorbance/min, respectively, and ⌬pH maximums were 0.308 Ϯ 0.006 and 0.214 Ϯ 0.010 absorbance, respectively.

V-ATPase Isotypes and Tissue Specificity of the Subunit Iso-
forms-In the present study, we focused on the isoforms of the subunits of plant V-ATPase. V-ATPase from the pea epicotyl gave 13 subunits on SDS-PAGE: 100, 68, 57, 51, 44, 40, 37, 35, 29, 16, 15.5, 13, and 12 kDa (Fig. 7). Among these subunits, both the 40-kDa (E1) and 37-kDa (E2) subunits were demonstrated to be isoforms of the E subunit, from which cDNAs were cloned from various organisms, including S. cerevisiae and A. thaliana. The presence of E subunit isoforms was also demonstrated in other plants, such as the mung bean and A. thaliana, by immunoblot analysis with the antibody to the pea E subunit (Fig. 3). Similarly, both the 35-kDa (D1) and 29-kDa (D2) subunits were found to be D subunit isoforms, which also existed in the enzyme from the mung bean hypocotyl (Fig. 7), although the similarity of the amino acid sequences is still unknown. This is the first demonstration of the presence of isoforms of the accessory subunits of plant V-ATPase.
Since it has been reported that the coated vesicle V-ATPase contained only one molecule of the D and E subunits in one enzyme complex (6), it would be expected that there were some V-ATPase isotypes on the plant vacuolar membrane of the same tissue. By MonoQ column chromatography, we separated two purified enzyme fractions (P1 and P2) from pea epicotyl that had subunit compositions differing only in the ratio of E subunit isoforms and four purified enzyme fractions (P1-P4) from mung bean hypocotyl that had subunit compositions differing in the ratio of D and E subunit isoforms (Fig. 8). At present, it is not clear whether different isotypes with specific subunit isoforms exist in the same vacuolar membrane or in different vacuoles in the same cell. Although the V-ATPase isotypes were not completely separated, the P1 and P2 fractions of the pea epicotyl contained equal amounts of all subunits except for the E subunit isoforms (Fig. 7), P2 showed higher V max and higher K m than did P1 (Fig. 8). We therefore    speculated that the properties of two E subunit isoforms reflected the different enzymatic properties of the P1 and P2 fractions. The E subunit is not the main catalytic subunit of V-ATPase, but it was reported that the E subunit was essential for ATP hydrolysis (4) and might neighbor the A 3 B 3 complex of the main catalytic domain (12). The exact position of the E subunit in the tertiary structure of V-ATPase and the role of the subunit in enzymatic function remain to be clarified.
Interestingly, we found that the E subunit isoforms of the pea, mung bean, and tomato showed tissue-specific distributions that were different in leaf tissues and in the other tissues ( Fig. 9). Further, the compositions of D subunit isoforms in the pea leaf and epicotyl were different, and the vacuolar membrane from the leaf did not contain the D2 (29 kDa) subunit ( Table I). The tissue-specific localization of these subunit isoforms suggested that V-ATPase isotypes with different kinetic properties may function in an organ-specific manner. Actually, the H ϩ pumping activities by V-ATPase differed in vacuolar membrane fractions from the leaf and epicotyl, and it was expected that leaf vacuolar membrane contained enzymes with highly efficient H ϩ pumping activity (Fig. 10). These differences in H ϩ pumping activity might be responsible for not only enzymatic properties but also membrane properties in each tissue. With regard to the enzymatic properties, it is possible that the D subunit, E subunit, or a combination of the two subunits is related to the efficiency of H ϩ pumping. In all cases, the clear differences in isoform distribution among plant tissues raises the question of whether or not the tissue-specific detection of the isoforms reflects their cell-specific expression.
There are a few reports about E subunit isoforms in other plants. Ratajczak et al. (47) demonstrated that V-ATPase activity increased and the subunit composition partially changed in M. crystallinum when the carbon metabolism was converted from C3-photosynthesis (C3) type to Crassulacean acid metabolism type. After conversion from the C3 state into the Crassulacean acid metabolism state, the 31-and 27-kDa subunits were found to be attached to the V-ATPase of the C3 state that consisted of 69-, 55-, 41-, 32-, and 16-kDa subunits (48). Dietz and Arbinger confirmed that the 31-kDa subunit of M. crystallinum was immunochemically related to the barley E subunit (46), and they suggested that the 32-kDa subunit was also an E subunit because of similarities in the molecular mass. Thus, these reports and our results led us to hypothesize that the E subunit isoforms in plants are encoded by a gene family and expressed so as to be physiologically suitable for each tissue.
Multiplicity of the E Subunits in Plants-To detect E subunit isoforms in various plants, we performed immunoblot analyses of anti-37 and anti-40 in 10 different plants (pea, mung bean, adzuki bean, kidney bean, A. thaliana, cucumber, Jerusalem artichoke, pumpkin, spinach, and tomato). The results revealed two or three immunostained bands at 29 -40 kDa in all plants except in the spinach leaf (Fig. 3) and in cucumber fruit (data not shown), in which a single band was detected. However, this does not exclude the possibility of the existence of isoforms of the same size in these plants. The E subunits of leguminous plants showed relatively large molecular masses (37-40 kDa), although the cDNAs for the E subunit from five plants, which are shown in Fig. 3, were homologous and their molecular masses were calculated to be about 26 kDa. It is possible that the increase in size is a result of post-translational modification. Results of motif research suggested that the cDNAs for the E subunit in five plant species possess the motifs of an N-glycosylation site and myristoylation site in the polypeptide. We investigated the glycosylation of the E subunit by the method of Kondo et al. (49), but glycosylation of the subunits was not found in the pea, mung bean, or cucumber (data not shown). Furthermore, post-translational myristoylation of the E subunit may cause only a small change in size of the protein because of the low molecular mass of myristic acid (M r 228.38), and it is therefore not likely that myristoylation is the main cause of the difference in molecular size. Therefore, the slow migration of E subunit isoforms in SDS-PAGE may be due to the primary sequences. Another possibility is that the large isoforms detected in immunoblots were encoded by genes different from that for the small isoforms.
Because the 29-kDa polypeptide in the A. thaliana leaf was cross-reacted by anti-40 but not by anti-37, it is categorized as a 40-kDa type of E subunit (Fig. 3). The 32-kDa polypeptide was cross-reacted by both anti-37 and anti-40 (Fig. 8). Thus, the 29-kDa polypeptide of the E subunit in A. thaliana may differ in primary structure from the 32-kDa polypeptide. Two cDNAs of the E subunit have been reported in A. thaliana (Ref. 43 and NCB accession no. AAC35545), and three different sequences are also listed in the A. thaliana EST data base. Their deduced amino acid sequences are significantly different. Thus, there are more than five isoforms of the E subunit in A. thaliana. Rice and M. crystallinum have at least six and three isoforms of the E subunit, respectively, judging from their nucleotide sequences listed in the EST data base. Similarly, A. thaliana and G. hirsutum have at least three and four isoforms of the D subunit, respectively, judging from their nucleotide sequences in the EST data base, and this is consistent with the existence of two D subunit isoforms in the pea and mung bean, although Southern analysis of A. thaliana genomic DNA showed that the D subunit was encoded by a single copy gene (41). Thus, there may be a gene family encoding the D or E subunits in plants.
Correspondence of Subunits of Pea V-ATPase to Yeast Enzyme-The E subunit isoforms were detected with apparent molecular masses ranging from 29 to 40 kDa in many plants, and two D subunit isoforms were found in the pea epicotyl and mung bean hypocotyl. Furthermore, cDNAs for the A (  The molecular mass of each subunit was calculated from the sequence of each cDNA sequence. EST: The C, a and d subunits of plant V-ATPase have been found in A. thaliana and rice EST databases.
b Parentheses indicate subunits for which we did not examine the correspondence to yeast subunits. c ND, not determined. d -, no homologous sequences in the plant EST data base.
V-ATPase in the pea, as well as in the mung bean and other plants, to those of yeast enzyme (Table III). The cDNAs for the eight subunits from A to H of the V 1 sector and the five subunits from a to d of the V 0 sector of animal and yeast V-ATPases have been reported (5). Homologous sequences of the subunits, except for the cЈ and cЉ subunits, have also been found in A. thaliana and rice EST data bases. As shown in Table III, we identified A, B, D, E, and c subunits in pea and mung bean enzymes in the present and previous studies (13). The proteolipid c subunits in plants are highly conserved in the size of 16 kDa as estimated by SDS-PAGE, and the molecular mass of the c subunit has been calculated to be 16 kDa from its nucleotide sequence. Isoforms (cЈ and cЉ) of the proteolipid subunit exist in yeast V-ATPase (54) and have been calculated to be 17 and 23 kDa, respectively. As shown in Fig. 6B, the 16-kDa subunit of the purified pea enzyme was detected as a double band (16 and 15.5 kDa). At present, although it is unclear whether subunits corresponding to the cЈ and cЉ subunits exist in plants, 16-and 15.5-kDa polypeptides might be isoforms of the c subunit. Li and Sze reported that the 100-kDa polypeptide of the oat V 0 sector reacted with the antibodies against the a subunit of yeast and bovine V-ATPase (55). The 51-and 45-kDa polypeptides were detected in purified enzymes from the pea and mung bean in this study, and they have also been detected in other plant species, such as red beet (56), lemon (30), and cucumber. 2 Lu et al. reported that the cDNA for the 52-kDa subunit of VATPase in red beet encoded a 54-kDa polypeptide and was homologous to the sequence of the yeast H subunit, the VMA13 gene product (52). The 44-kDa polypeptide of red beet V-ATPase was cross-reacted by the polyclonal antibodies against the VMA6 gene product of yeast (d subunit) (57). As mentioned above, we assigned the subunits of pea enzyme to subunits of animal or yeast enzymes, except for the C subunit which has been found in A. thaliana EST data base (Table III).
In conclusion, we found that the isoforms of the accessory subunits of plant V-ATPase and the isotypes of the enzyme are different in catalytic properties and tissue distribution. Further studies on their primary sequences and the spatial relationship of the subunits in the enzyme complex may provide more detailed information on the biochemical and physiological significance of the presence of the isoforms of the V-ATPase subunits.