From the 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
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ABSTRACT |
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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.
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.
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.
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 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A list of the pairs of mutagenic primer and antisense standard primer
used for amino acid substitution of mung bean V-PPase
80 °C until use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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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).
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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.
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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.
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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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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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.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. K. Nakamura, A. Morikami, K. Matsuoka, and H. Ueoka-Nakanishi of Nagoya University for stimulating discussions.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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The abbreviations used are: V-PPase, vacuolar H+-translocating inorganic pyrophosphatase; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiothreitol; Mes, 4-morpholineethanesulfonic acid.
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