Mutagenic Analysis of Functional Residues in Putative Substrate-binding Site and Acidic Domains of Vacuolar H+-Pyrophosphatase*

Yoichi NakanishiDagger §, Takanori SaijoDagger , Yoh Wada, and Masayoshi MaeshimaDagger ||

From the Dagger  Laboratory of Biochemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan and the  Division of Biological Science, Institute of Science and Industrial Research, Osaka University, Osaka 567-0047, Japan

Received for publication, October 25, 2000, and in revised form, December 11, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase) uses PPi as an energy donor and requires free Mg2+ for enzyme activity and stability. To determine the catalytic domain, we analyzed charged residues (Asp253, Lys261, Glu263, Asp279, Asp283, Asp287, Asp723, Asp727, and Asp731) in the putative PPi-binding site and two conserved acidic regions of mung bean V-PPase by site-directed mutagenesis and heterologous expression in yeast. Amino acid substitution of the residues with alanine and conservative residues resulted in a marked decrease in PPi hydrolysis activity and a complete loss of H+ transport activity. The conformational change of V-PPase induced by the binding of the substrate was reflected in the susceptibility to trypsin. Wild-type V-PPase was completely digested by trypsin but not in the presence of Mg-PPi, while two V-PPase mutants, K261A and E263A, became sensitive to trypsin even in the presence of the substrate. These results suggest that the second acidic region is also implicated in the substrate hydrolysis and that at least two residues, Lys261 and Glu263, are essential for the substrate-binding function. From the observation that the conservative mutants K261R and E263D showed partial activity of PPi hydrolysis but no proton pump activity, we estimated that two residues, Lys261 and Glu263, might be related to the energy conversion from PPi hydrolysis to H+ transport. The importance of two residues, Asp253 and Glu263, in the Mg2+-binding function was also suggested from the trypsin susceptibility in the presence of Mg2+. Furthermore, it was found that the two acidic regions include essential common motifs shared among the P-type ATPases.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vacuolar H+-pyrophosphatase (V-PPase)1 belongs to the fourth class of electrogenic proton pump in addition to the P-, F-, and V-type ATPases. The proton pumping reaction couples with the hydrolysis of PPi. V-PPase acidifies vacuoles together with vacuolar H+-ATPase in the plant cell and actively exports protons from the cytosol in the bacterial plasma membrane (1-3). V-PPase has the simplest structure among the proton pumps except for bacteriorhodopsin, a light-driven proton pump. The molecular mass calculated from the cDNA sequences range from 80 to 81 kDa for V-PPases of land plants and algae (for a review, see Refs. 3 and 4), while V-PPases in photosynthetic bacteria Rhodospirillum rubrum (5) and archaebacteria Pyrobaculum aerophilum (6) are relatively small. The simplicity of the enzyme structure and its substrate is an advantage to analyze the structure-function relationship. The enzyme activity is stimulated by K+ at relatively high concentrations. Also, Mg2+ is essential to form a Mg-PPi complex and to keep the active conformation of V-PPase (1, 7, 8). Ca2+ prevents formation of a Mg-PPi complex (8) and directly inhibits V-PPase (9). Thus, V-PPase should have a specific binding site for its substrate (Mg-PPi), Mg2+, K+, and Ca2+ in addition to a H+ transport channel.

Multiple amino acid sequence alignment of V-PPases of various organisms revealed highly conserved regions (2, 3, 10). There is a putative substrate-binding motif of DXXXXXXXKXE in the cytoplasmic loop (1, 11). This was supported by immunochemical study with an antibody specific to this sequence (DVGADLVGKVE) (12). This sequence is common among V-PPases not only from land plants but also from Chara corallina (10), Acetabularia acetabulum (13), R. rubrum (5), Thermotoga maritima (GenBankTM accession number AE001702), and P. aerophilum (6). Studies using substrate analogs, such as aminomethylenebisphosphonate, have also provided information on the catalytic domain (14-16). Furthermore, the N-ethylmaleimide-binding cysteine residue (Cys634) (17) and the N,N'-dicyclohexylcarbodiimde-binding residues (Glu305 and Asp504) (18) have been identified by a combination of site-directed mutagenesis of Arabidopsis V-PPase and heterologous expression in yeast.

The aim of this study is to clarify the substrate-binding site of V-PPase by the method of site-directed and random mutagenesis. We prepared a line of constructs, in which charged residues in a putative substrate-binding site were replaced, expressed in Saccharomyces cerevisiae, and then examined for enzymatic properties. V-PPase has been proposed to have three conserved regions (3, 10). In addition to a putative PPi-binding site in the first conserved region, we investigated the two acidic motifs in the first and third conserved regions. Each aspartic acid residue in the two acidic regions was substituted and examined for enzymatic properties. Here, the functional roles of these residues on the substrate hydrolysis, binding of free Mg2+, and a coupling reaction between PPi hydrolysis and proton transport were examined. The similarity of the conserved functional motifs of V-PPase with the P-type ATPase is also discussed.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heterologous Expression of Mung Bean V-PPase in Yeast Cell-- A EcoRI-SalI fragment of VVP2 cDNA encoding mung bean V-PPase (Ref. 19; DDBJ accession number AB009077) was inserted into a URA3-marked, high copy (2 µm) yeast expression vector pKT10 (20, 21). The obtained pKVVP2 plasmid was introduced into a S. cerevisiae strain BJ5458, which was deficient in major vacuolar proteinases (22), by the lithium accetate/single-stranded DNA/polyethylene glycol transformation method (23). Positive Ura+ colonies were selected, and the expression of V-PPase was confirmed by immunoblotting with the anti-V-PPase antibodies previously prepared (24).

Plasmid Preparation for V-PPase Mutants-- Site-directed mutagenesis was performed using a QuickChange site-directed mutagenesis kit (Stratagene) by the method of Kirsch and Joly (25). Mutagenic and antisense standard primers used in this study are listed in Table I. The DNA sequences of at least two independent plasmids for each mutant were determined to confirm the mutation points.

Preparation of a Random Mutation Library and Screening of V-PPase Mutant-- For convenience of genetic manipulation, the AatII site of pKVVP2 plasmid in pKT10 vector was removed, and a SacI site at position 715 in the plasmid was introduced by substitution with a synonymous codon. The resulting pKVVP2/S plasmid was used as a seed for hypermutagenic polymerase chain reaction (26). The reaction mixture contained 40 pg/ml pKVVP2/S plasmid, 250 nM primers (Table I, F668 and R1251), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.001% gelatin, 2.5 mM MgCl2, 0.5 mM MnCl2, 0.1 mM dATP, 0.1 mM dCTP, 1 mM dGTP, 1 mM dTTP, and 0.08 units/ml Taq DNA polymerase. Amplification was done using 20 cycles of a set of 30 s at 95 °C, 45 s at 55 °C, and 5 min at 72 °C. The obtained polymerase chain reaction fragments were digested and inserted into the SacI-AatII site of the pKVVP2/S plasmid. This library was amplified in E. coli and introduced into yeast BJ5458. A region that was mutagenized in each mutant was amplified by polymerase chain reaction using F668 and R1251 primers. Mutation points were defined by DNA sequencing. In most mutants, plural sites were mutated.


                              
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Table I
A list of the pairs of mutagenic primer and antisense standard primer used for amino acid substitution of mung bean V-PPase
The nucleotides changed for the substitution are underlined.

Preparation of V-PPase Enriched Membrane Fraction-- Crude membrane fractions were prepared from yeast cells by the method of Kim et al. (27) with a few modifications. Yeast cells were precultured at 30 °C for 2 days in AHCW/Glc medium that contained 50 mM potassium phosphate buffer, pH 5.5, 0.002% (w/v) adenine sulfate, 0.002% tryptophan, 2% glucose, 1% casamino acids (Nihon Pharmaceutical Co.), and 0.67% yeast nitrogen base without amino acids (Difco). The cell culture was diluted 64-fold and then grown for 12 h to reach an exponential phase. After being washed with 0.1 M Tris-HCl, pH 9.4, 50 mM 2- mercaptoethanol, and 0.1 M glucose at 30 °C for 10 min, cells were treated with a zymolyase medium at 30 °C for 1 h with gentle agitation. The medium contained 0.05% zymolyase 20T (Seikagaku Kogyo Co.), 0.9 M sorbitol, 0.1 M glucose, 50 mM Tris-Mes, pH 7.6, 5 mM DTT, 0.043% yeast nitrogen base without amino acids and ammonium sulfate, and 0.25× dropout solution composed of all amino acids and adenines (28). Spheroplasts were collected from the suspension by centrifugation at 3,000 × g for 10 min and washed with 1 M sorbitol.

The spheroplasts were resuspended in 50 mM Tris-ascorbate, pH 7.6, 1.1 M glycerol, 1.5% polyvinylpyrrolidone (Mr 40,000), 5 mM EGTA-Tris, 1 mM DTT, 0.2% bovine serum albumin, 1 mM PMSF, and 1 mg/liter leupeptin and then homogenized with a motor-driven Teflon homogenizer. After centrifugation at 2,000 × g for 10 min, the precipitate was suspended in the same buffer and centrifuged again. All of the supernatant fractions were pooled and centrifuged at 120,000 × g for 30 min. The precipitate (membranes) was suspended in 15% (w/w) sucrose and layered on a 35% (w/w) sucrose solution. Both sucrose solutions contained 10 mM Tris-Mes, pH 7.6, 1 mM EGTA-Tris, 2 mM DTT, 25 mM KCl, 1.1 M glycerol, 0.2% bovine serum albumin, 1 mM PMSF, and 1 mg/liter leupeptin. After centrifugation at 150,000 × g for 30 min, the interface portion was collected and diluted with 5 mM Tris-Mes, pH 7.6, 0.3 M sorbitol, 1 mM DTT, 1 mM EGTA-Tris, 0.1 M KCl, 1 mM PMSF, 1 mg/liter leupeptin, and 5 mM MgCl2. Most of the V-PPase activity was recovered in this fraction. The precipitate after centrifugation at 150,000 × g for 30 min was resuspended in 5 mM Tris-Mes, pH 7.6, 0.3 M sorbitol, 1 mM DTT, 1 mM EGTA-Tris, 1 mM PMSF, 1 mg/liter leupeptin, and 1.5 mM MgCl2. The suspension (V-PPase-enriched membrane fraction) was stored at -80 °C until use.

Protein and Enzyme Assays-- Protein content was determined by the method of Bradford (29). PPi hydrolysis activity was measured in a reaction medium supplemented with 0.5 mM KF (24). PPi-dependent H+ transport activity was determined as described previously (24) with a few modifications. Membrane preparations were preincubated with a reaction buffer containing 0.3 M sorbitol, 5 mM Tris-Mes, pH 7.6, 0.1 M KCl, 0.5 mM EGTA-Tris, 1.5 mM MgCl2, 0.2% bovine serum albumin, and 1 µM acridine orange, and then the reaction was initiated by the addition of 1 mM Na4PPi.

Immunoblotting-- Proteins were separated by SDS-polyacrylamide gel electrophoresis on 10% gels and transferred to a polyvinylidene difluoride membrane by the standard procedure. Immunoblotting was carried out using polyclonal antibodies against V-PPase purified from mung bean (24) and an ECL procedure (Amersham Pharmacia Biotech).

Trypsin Digestion Analysis-- To remove Mg2+ and PMSF, yeast membrane preparations were washed with 20 mM Tris-Mes, pH 7.6, 20% glycerol, 50 mM KCl, 0.05 mM MgCl2. The membrane fraction was mixed with an equal volume of 20 mM Tris-Mes, pH 7.6, 10 µg/ml trypsin, 20% glycerol, 50 mM KCl, 1 mM DTT, and 1% MOA Excellent (a mixture of alkylglucosides; Kao Co., Japan) and then incubated at 30 °C for 40 min. The reaction was stopped by the addition of an SDS-sample buffer containing 5 mM PMSF. The samples were subjected to SDS-polyacrylamide gel electrophoresis and subsequent immunoblotting with anti-V-PPase antibody.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of Mung Bean V-PPase in Yeast as a Functional Enzyme-- A yeast expression vector pKT10 was used to express a cDNA (VVP2) encoding mung bean V-PPase in yeast (Fig. 1A). For DNA manipulation, an EcoRI site in VVP2 was eliminated by substitution with synonymous codons. VVP2 was efficiently transcribed under the control of the promoter of glyceraldehyde-3-phosphate dehydrogenase in a medium supplemented with glucose (30). The V-PPase-enriched membrane fraction was prepared by a stepwise sucrose gradient centrifugation and then subjected to immunoblotting with the polyclonal antibodies specific to V-PPase. Transformants produced a 73-kDa protein that was reacted with the antibodies (Fig. 1B). The recombinant V-PPase accounted for 2 to 3% of the total membrane protein. The membranes showed the PPi hydrolysis activity that was insensitive to KF, an inhibitor of acid phosphatase (Fig. 1C). The activity was 3.5 times enhanced by 50 mM KCl but not by 50 mM NaCl. The membrane vesicles prepared from VVP2 transformant gave the PPi-dependent H+ transport activity (Fig. 1D). The pH gradient was collapsed by the addition of membrane-permeable ammonium ion, indicating the electrogenic H+ transport in the vesicles. The elimination of an EcoRI site from the original VPP2 sequence did not affect the expression level and the enzymatic activity. The results indicate that V-PPase expressed in yeast functions normally in the heterologous system.



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Fig. 1.   Heterologous expression of V-PPase in yeast. A, the mung bean V-PPase cDNA was linked with the GAP promoter (pKVVP2). A transformant containing the pKT10 vector was used as a negative control. B, both constructs of wild type (WT) and vector (V) were transformed and expressed in S. cerevisiae. The membrane fractions enriched with V-PPase were prepared and then subjected to SDS-polyacrylamide gel electrophoresis (left) and subsequent immunoblot analysis with the anti-V-PPase antibody (right). The arrowheads indicate the position of V-PPase. C, the membrane vesicles prepared from S. cerevisiae strains that expressed V-PPase (WT) and a vector (V) were assayed for the PPi hydrolysis activity. The reaction medium contained 1 mM Na4PPi, 1 mM MgSO4, 50 mM KCl, 1 mM sodium molybdate, 0.02% Triton, X-100, and 30 mM Tris-Mes, pH 7.2 in the presence (right) or absence (left) of 0.5 mM KF. In an experiment (Na), NaCl at 50 mM was used instead of KCl. Released Pi was measured colorimetrically. D, the membrane vesicles (200 µg) were also assayed for the PPi-dependent H+ transport activity with 1 µM acridine orange. Reaction was started by the addition of PPi (1 mM). The initial rate of fluorescence quenching was defined as the H+ transport activity.

A Series of V-PPase Mutants in Respect to Putative Functional Motifs-- The sequence DVGADLVGKVE is a putative substrate-binding site of V-PPase (2, 11, 31). This motif has been demonstrated to be exposed to the cytosol (12). To evaluate the functional significance of the charged residues in this motif, we generated a series of V-PPase mutants, in which the residues were replaced with alanine (D253A, K261A, E263A) or conservative residues (D253E, K261R, E263D).

A comparison of the primary structures of V-PPases of various organisms revealed that there are two consensus acidic regions, DNVGDNVGD (acidic region 1) and DTXGDPXKD (acidic region 2). The former is near the DVGADLVGKVE motif in loop e, and the latter is in loop m between the 13th and 14th transmembrane domains (Fig. 2). Both regions can be expressed as a common motif DXXXDXXXD. To test whether the aspartate residues in the two motifs are implicated in the interaction with the substrate and Mg2+, we generated a series of mutants substituted with alanine (D279A, D283A, D287A, D723A, D727A, and D731A) and glutamic acid (D279E, D283E, D287E, D723E, D727E, and D731E). These V-PPase mutants were assayed for the enzymatic activities.



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Fig. 2.   Transmembrane model of mung bean V-PPase. Fourteen transmembrane domains (white box, 1-14) and 13 hydrophilic loops (a--m) were predicted for mung bean V-PPase (19) by the TMpred program (36). Conserved amino acid residues among land plants, C. corallina, A. acetabulum, R. rubrum, and T. maritima, are highlighted in black circles. The predicted PPi-binding site and two acidic regions (positions 279-287 and 723-731) are marked by hatched ovals. The substituted amino acid residues in this study are shown as quadrilaterals (Asp253, Lys261, Glu263, Asp279, Asp283, Asp287, Asp723, Asp727, and Asp731).

Enzymatic Activities of V-PPase Mutants-- The V-PPase protein in each mutant was clearly detected as a 73-kDa protein in an immunoblot (Fig. 3A). PPi hydrolysis and PPi-dependent H+ transport activities of the mutants were assayed at 1 mM (Fig. 3, B and D) or 0.3 mM Mg2+ (Fig. 3C). The membrane preparation of the wild-type strain showed high activities compared with a low basal level in the control vector. All nine V-PPase mutants substituted with alanine (D253A, K261A, E263A, D279A, D283A, D287A, D723A, D727A, and D731A) lost the activity completely (Fig. 3, B-D). Conservative exchange with glutamate in seven mutants (D253E, D279E, D283E, D287E, D723E, D727E, and D731E) also caused a loss of function of H+ transport (Fig. 3, B and C). The PPi hydrolysis activity was decreased in these mutants. As a control, we obtained a V262A mutant from a random mutation library. This V262A mutant had the same V-PPase activity and protein level as the wild type. These results proved that aspartate residues in the putative PPi-binding site and the acidic motifs are involved in the binding and hydrolysis of the substrate. There was no difference in the protein level of V-PPase in the membranes among the mutants (Fig. 3A). Thus, the loss of activity was not due to the absence or low expression of V-PPase.



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Fig. 3.   Loss of activity in V-PPase mutants. Membrane fractions were prepared from yeast BJ5458 expressing wild type V-PPase (WT) and several mutants in which a certain residue of the enzyme was replaced with the indicated residue. A, V-PPase protein in the membrane fraction of each mutant was quantified by immunoblotting with the anti-V-PPase antibody. B, PPi hydrolysis activity was measured in the presence of 1 mM PPi, 1 mM MgCl2, 50 mM KCl, and 0.5 mM KF. C, PPi hydrolysis activity was determined at 0.3 mM Mg2+. D, PPi-dependent H+ transport activity of the membrane vesicle (200 µg) was assayed with acridine orange. All of the data are means ± S.D. for the duplicated assay of two independent lines. Horizontal lines in B, C, and D show the activity of the vector transformant as a background level.

Interestingly, the K261R and E263D mutants retained 25 and 50% of the original PPi hydrolytic activity, respectively, at 1 mM MgCl2, but they had no H+ transport activity. Thus, Lys261 and Glu263 may be involved in the energy transduction from PPi hydrolysis to H+ translocation. Furthermore, three other mutants (E263G, V259A, and C304R) with a single substitution of an amino acid were obtained by a random mutation technique. The E263G and C304R mutants gave no activity of PPi hydrolysis or H+ transport (Fig. 4). The V259A mutant had 60% of the original activity of PPi hydrolysis but no H+ transport activity as well as K261R and E263D.



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Fig. 4.   Activities of V-PPase mutants obtained by random mutation. Three V-PPase mutants with a single amino acid substitution were obtained by the random mutation method as described under "Experimental Procedures." The expressed protein level of V-PPase in yeast was determined by immunoblotting (A), and the activities of PPi hydrolysis (B) and PPi-dependent H+ transport (C) were assayed in the presence of 1 mM MgCl2.

Mg2+ and Substrate Binding Properties in V-PPase Mutants-- V-PPase requires Mg2+ for activation, structural stabilization, and protection from protease digestion (1, 7, 8). In this study, yeast vacuolar membranes containing V-PPase were treated with trypsin in the presence or absence of Mg2+, PPi, and Mg-PPi. Trypsin preferentially cleaves at the carboxyl sides of arginine and lysine residues. As shown in Fig. 5A, Mg2+ and Mg-PPi, but not PPi, at relatively high concentrations partially prevented digestion of V-PPase by trypsin. Thus, the trypsin susceptibility is a good marker for the structural change caused by the binding of Mg2+ and Mg-PPi.



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Fig. 5.   Trypsin digestion assay of V-PPase mutants in the presence of Mg2+ and PPi. A, yeast membrane preparation (25 µg) containing wild type V-PPase was mixed with 0.5% detergent and then incubated with 0.1 µg of trypsin in the presence of MgCl2 and PPi at the indicated concentrations. Samples (5 µg) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-V-PPase antibody. NT, nontreatment with trypsin. B, membrane preparations of V-PPase mutants were treated with trypsin in the presence of 1 mM PPi, 1 mM MgCl2, or 1 mM MgCl2 plus 1 mM PPi. The remaining V-PPase protein was determined by immunoblotting.

All V-PPase mutants were thoroughly digested by trypsin in the absence of Mg2+ (Fig. 5B, panels none and 1 mM PPi). Therefore, the amino acid substitution did not affect the trypsin cleavage site. It should be noted that Mg2+ protected the enzyme from trypsin digestion in mutants of K261A, D279A, D283A, D287A, D723A, D727A, D731A, E263D, and D727E as compared with the wild type V-PPase (Fig. 5B, 1 mM Mg). Neither Mg2+ nor PPi has a direct inhibitory effect on trypsin, since V-PPase in the E263A mutant was completely digested even in the presence of 1 mM Mg2+ and 1 mM PPi (Fig. 5B). This observation indicates that these amino acid substitutions increase the affinity of the enzyme for Mg2+. In other words, the charged residues of Lys261, Asp279, Asp283, Asp287, Asp723, Asp727, and Asp731 may have a negative effect on the Mg2+-binding property of V-PPase.

In the presence of both Mg2+ and PPi, certain mutants were resistant to trypsin as was the wild type V-PPase. However, the K261A, E263A, and K261R mutants were sensitive to trypsin, and the other alanine mutants (D279A, D723A) and the glutamate mutants (D253E, D279E, D283E, D287E, and D723E) were partially sensitive under the assay conditions. These results suggest that Lys261 and Glu263 are essential for the substrate-binding function. The other aspartate residues at 253, 279, 283, 287, and 723 may also be implicated in the substrate-binding function or located near the substrate-binding site of V-PPase.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

V-PPase has been estimated to possess the binding sites for its substrate (Mg-PPi), Mg2+, K+, and Ca2+, independently, and a H+ transport pathway. In the present study, we examined the functional role of charged residues in the putative substrate-binding site (DVGADLVGKVE) and two conservative acidic regions of V-PPase by site-directed and random mutagenesis in combination with a heterologous expression system in S. cerevisiae. The V-PPase expressed in yeast exhibited the PPi hydrolysis and proton pump activities, and the enzyme protein was detected by immunoblotting. V-PPase translated in yeast was considered to be localized mainly in vacuolar membranes judging from the following three observations: vacuolar type H+-ATPase was detected in the same membrane fraction; a PPi-dependent H+ current was detected in intact vacuoles prepared from the VVP2-transformed yeast cells by the patch clamp technique2; and the amount of the V-PPase protein in a yeast strain that lacked the major vacuolar proteases was higher than that in the normal strain.

Role of a DVGADLVGKVE Motif and Acidic Regions in Enzyme Activity-- The substitution of acidic residues in the putative substrate-binding motif and two acidic regions had a negative effect on the V-PPase activity (Fig. 3). This is a direct effect of amino acid substitution, since all V-PPase mutants expressed in yeast were accumulated in the membrane at equal levels. The PPi hydrolysis activity was markedly decreased even in the case of conservative substitution (D253E, K261R, and E263D), although the V262A mutant retained the original activity. Thus, these residues (Asp253, Lys261, and Glu263) are essential for V-PPase activity.

The present study showed that V-PPase might not interact with PPi in the absence of Mg2+, since PPi did not affect trypsin susceptibility even at 1 mM, but PPi plus Mg2+ makes V-PPase resistant to trypsin (Fig. 5A). Furthermore, the trypsin susceptibility assay revealed that Lys261 and Glu263 are essential for the binding of the substrate (Fig. 5). Fig. 6B shows a scheme of our model.



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Fig. 6.   Schematic model for substrate- and Mg2+-binding sites of V-PPase. A, nine amino acid residues of V-PPase were substituted to the indicated residues. The trypsin susceptibility of the mutants in the presence of Mg2+ (Mg) or Mg2+ plus PPi (Mg + PPi) were examined as shown in Fig. 5. The intensity of immunostained band of V-PPase was determined densitometrically and compared with the wild type V-PPase. The original residues are circled. The mutant with the indicated residue on the left of each column is more resistant to trypsin. By comparison, between wild type and mutant, the role of each original residue is evaluated to be positive (P) or negative (N) for activity to protect the enzyme from trypsin. B, the amino acid residues that are involved in binding of Mg2+ (left) and a Mg2+-PPi complex (right) are shown with solid lines. Some charged residues that have a negative effect on Mg2+ binding are shown with broken lines.

This work demonstrated the functional significance of acidic residues in the acidic regions (DNVGDNVGD287 and DTXGDPXKD731) (Fig. 2). The sequences are conserved among V-PPases of various organisms including R. rubrum (5) and T. maritima (GenBankTM accession number AE001702). Substitution of the aspartate residue at 279, 283, 287, 723, 727, and 731 with alanine or glutamate residue resulted in the complete loss of the activity (Fig. 3). The digestion assay with trypsin also supported the implication of aspartate residues at 279, 283, 287, and 723 in the binding of Mg-PPi (Fig. 5). These residues may be adjacent to the DVGADLVGKVE motif in the tertiary structure of V-PPase as discussed later.

The present study also suggested the residues involved in energy coupling of V-PPase. The K261R and E263D mutants partially retained the PPi hydrolysis activity, 25 and 50% of that of the wild-type enzyme, respectively, but the mutants did not exhibit the H+ transport activity (Fig. 3). The V259A mutant also retained partial activity (60%) of PPi hydrolysis but no proton pump activity (Fig. 4). These three residues are located in the putative PPi-binding site. Thus, the conserved motif including Val259, Lys261, and Glu263 may be implicated in the initial step of the energy transfer from PPi hydrolysis to the H+ translocation. In Arabidopsis V-PPase, Glu427 in the transmembrane domain has been demonstrated to be involved in the energy coupling by the site-directed mutagenesis (18). The role of several charged residues in the transmembrane domains remains to be investigated for their role in H+ translocation.

Residues Involved in Binding of Free Mg2+-- The presence of Mg2+ decreased the susceptibility of V-PPase to trypsin (Fig. 5A), which is consistent with a previous report (8). Interestingly, V-PPase became resistant to trypsin in several V-PPase mutants (K261A, D279A, D283A, D287A, D723A, D727A, D731A, E263D, and D727E) compared with the wild type V-PPase, while the D253A and E263A mutants were completely digested by trypsin (Fig. 5). This suggests that the two residues Asp253 and Glu263 are essential for Mg2+ binding (Fig. 6). On the other hand, Lys261 and other aspartate residues in the acidic regions seem to interfere with V-PPase in the binding of Mg2+ (Fig. 6B, broken lines). Probably, these aspartate residues weaken the interaction between Mg2+ and a binding site that includes Asp253 and Glu263 (Fig. 6B). Since Asp253 and Glu263 may be involved in the binding of a Mg-PPi complex as mentioned above, the two acidic residues may be located in the substrate-binding pocket and also have an ability to interact with free Mg2+. Probably, there is another, high affinity binding site for free Mg2+ independent of the substrate-binding site. However, those residues could not be detected in the present assay system of trypsin susceptibility in the presence of 1 mM Mg2+.

Common Motifs in Acidic Regions among V-PPase and P-type ATPase-- With respect to the conserved acidic regions, we found that V-PPases share common motifs with the P-type ATPases such as Ca2+-ATPase. In the P-type ATPase, the ATP-binding and ATP-hydrolyzing domain has been proposed to consist of several conserved motifs of DKTGT, DPPR (or DKVR), (T/S)GD(N/K), and GDGXNDA (32, 33). Among these motifs, the TGDN and GDGXNDA motifs are conserved in V-PPases as a GDN motif including Asp283 in the first acidic region and a GDTIGD motif including Asp723 and Asp727 in the second acidic region (Fig. 7). It has been proposed that the TGDN motif is involved in ATP hydrolysis together with a DPPR motif in the P-ATPases and that the adenosine moiety of ATP interacts with the other motifs such as a KGAP motif of the P-ATPase (32, 33). It has been recently reported that the amino acid residues that are involved in the binding of adenosine moiety and the hydrolysis of phosphoanhydride bond of ATP can be topologically distinguished in the crystal structure of sarcoplasmic reticulum Ca2+-ATPase (34). The DPPR (or DKVR) motif, which is located in the hydrolysis pocket, is present as DDRR272 in loop e and DNAK695 in loop m of V-PPases except for A. acetabulum V-PPase (3, 13), but an adenosine-binding KGAP motif is not conserved in the V-PPase. Aravind et al. (35) have proposed that two aspartate residues in the GDGXNDX motif of the P-ATPases have a role in hydrolysis of a phosphoanhydride bond of ATP. The present study revealed not only the importance of the two acidic regions in addition to the PPi-binding motif proposed previously but also the functional and sequence similarity of V-PPases to the P-type ATPases. However, it cannot be concluded that V-PPase is a member of the P-type ATPase family, since V-PPase lacks a phosphorylation domain (DKTGTLT) that is common to the P-ATPases including plasma membrane H+-ATPase, Ca2+-ATPase and other heavy metal-transporting ATPase (32). At present, it is unclear whether V-PPase forms an intermediate phosphorylation form during PPi hydrolysis.



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Fig. 7.   Amino acid sequence alignment of three consensus motifs among P-type ATPases and V-PPases. A, GDN; B, GDXXXD; C, DXX(R/K). The accession numbers for the amino acid sequences aligned are as follows: ECA1 (Arabidopsis thaliana Ca2+-ATPase, accession number U96455), ATC1 (rabbit sarcoplasmic reticulum Ca2+-ATPase, accession number P04191), ATN1 (sheep Na+/K+-ATPase, accession number P04074), PMA1 (S. cerevisiae H+-ATPase, accession number X03534), CCC2 (S. cerevisiae Cu2+-ATPase, accession number P38995), PAA1 (A. thaliana heavy metal transporter, accession number D89981), and VVP2 (Vigna radiata, accession number AB009077). The identical residues among V-PPases are underlined for VVP2. The identical residues among all sequences listed are marked by asterisks. Numbers on both sides of each sequence represent the position of the residues.

In summary, Lys261 and Glu263 of mung bean V-PPase are essential for the substrate-binding function, and Asp253 and Glu263 are essential for the Mg2+-binding function (Fig. 6). All aspartate residues (Asp253, Asp279, Asp283, Asp287, Asp723, Asp727, and Asp731) in the PPi-binding motif and two acidic regions may be involved in interaction with the substrate. It has also been reported that Glu305 and Asp504 of Arabidopsis V-PPase, Glu301 and Asp500 for mung bean V-PPase, respectively, are essential for PPi hydrolysis activity (18). Thus, we propose that these two acidic regions (279-287 and 723-731) and a common DXXADLVGKXE (253) motif form a core catalytic domain of the V-PPase together with a few other motifs. To verify our model, we need to perform a high resolution crystallographic study to determine the structure.


    ACKNOWLEDGEMENTS

We thank Drs. K. Nakamura, A. Morikami, K. Matsuoka, and H. Ueoka-Nakanishi of Nagoya University for stimulating discussions.


    FOOTNOTES

* This work was supported by Grants-in-Aid for Scientific Research 10219203 and 11163212 (to M. M.) from the Ministry of Education, Science, Sports and Culture of Japan.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.

§ Recipient of Japan Society for the Promotion of Science for Young Scientists Research Fellowship 11000510.

|| To whom correspondence should be addressed. Tel.: 81-52-789-4096; Fax: 81-52-789-4094; E-mail: maeshima@agr.nagoya-u.ac.jp.

Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M009743200

2 Y. Nakanishi, I. Yabe, and M. Maeshima, unpublished data.


    ABBREVIATIONS

The abbreviations used are: V-PPase, vacuolar H+-translocating inorganic pyrophosphatase; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiothreitol; Mes, 4-morpholineethanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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