From the Departments of Cellular and Molecular Physiology and
Biochemistry, Tufts University School of Medicine,
Boston, Massachusetts 02111
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ABSTRACT |
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To further define the structure of the nucleotide
binding sites on the vacuolar proton-translocating ATPase
(V-ATPase), the role of aromatic residues at the catalytic sites
was probed using site-directed mutagenesis of the VMA1 gene
that encodes the A subunit in yeast. Substitutions were made at three
positions (Phe452, Tyr532, and
Phe538) that correspond to residues observed in the crystal
structure of the homologous subunit of the bovine mitochondrial
F-ATPase to be in proximity to the adenine ring of bound ATP. Although conservative substitutions at these positions had relatively little effect on V-ATPase activity, replacement with nonaromatic residues (such as alanine or serine) caused either a complete loss of activity (F452A) or a decrease in the affinity for ATP (Y532S and F538A). The
F452A mutation also appeared to reduce stability of the V-ATPase complex. These results suggest that aromatic or hydrophobic residues at
these positions are essential to maintain activity and/or high affinity
binding to the catalytic sites of the V-ATPase.
Site-directed mutations were also made at residues (Phe479 and Arg483) that are postulated to be contributed by the A subunit to the noncatalytic nucleotide binding sites. Generally, substitutions at these positions led to decreases in activity ranging from 30 to 70% relative to wild type as well as modest decreases in Km for ATP. Interestingly, the R483E and R483Q mutants showed a time-dependent increase in ATPase activity following addition of ATP, suggesting that events at the noncatalytic sites may modulate the catalytic activity of the enzyme.
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INTRODUCTION |
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The vacuolar proton-translocating ATPases (or V-ATPases)1 are a class of enzymes that couple the hydrolysis of ATP to the transmembrane movement of protons. This proton movement results in the generation of a pH gradient and the acidification of intracellular compartments (1-9) or, for certain specialized cells, the extracellular space (10-12). The V-ATPases therefore play an essential role in many basic cellular functions which require acidification, including protein processing and degradation, receptor-mediated endocytosis, intracellular membrane trafficking, and coupled transport (for review, see Refs. 1-9).
The V-ATPases are composed of two domains, a 500-kDa peripheral
V1 domain with the structure
A3B3C1D1E1F1G1H1,
which is responsible for ATP hydrolysis, and a 250-kDa V0
domain with the structure a1d1c"1(c,c)6, that is
responsible for proton translocation (1, 13). In Saccharomyces
cerevisiae, the V-ATPase subunits are encoded by at least 14 genes, including VMA1, which encodes the 69-kDa A subunit
(14, 15), and VMA2, encoding the 60-kDa B subunit (16).
Sequence homology indicates that the A and B subunits of the V-ATPases
(14-22) are related to the
and
subunits, respectively, of the
F-type ATPases (F-ATPases), which function in mitochondria, chloroplasts, and bacteria to synthesize ATP (23-28). The level of
sequence identity between these proteins is only 20-25%, however, and
the A subunit contains a large (100-amino acid) insertion not present
in
, suggesting that significant structural and functional differences may exist between these proteins.
From the x-ray crystal structure of the F1 domain
from bovine heart mitochondria it was confirmed that the nucleotide
binding sites are at the interface between the and
subunits,
with the catalytic site primarily on
and the noncatalytic site
primarily on
(29). Previous biochemical experiments on the V-ATPase suggest that the catalytic nucleotide binding site is located on the A
subunit while the noncatalytic site resides on the B subunit. For the
catalytic site, these data include ATP-protectable labeling of the A
subunit by reagents such as N-ethylmaleimide and
7-chloro-4-nitrobenz-2-oxa-1,3-diazole, which correlates with inhibition of activity (for references, see Forgac (30)). In addition,
modification of a single A subunit cysteine residue (Cys254
of the bovine A subunit) by N-ethylmaleimide, cystine, or by disulfide bond formation with Cys532, leads to inactivation
of the enzyme (31-33). Also, labeling of the A subunit by the
photoaffinity ATP analog 2-[azido-32P]ATP
correlates well with inhibition by this reagent (34). Finally, the A
subunit, unlike the B subunit, contains several consensus sequences
that appear to be critical for ATP hydrolysis in the homologous
subunit (35-37), including the glycine-rich loop.
There is limited information concerning the structure of the nucleotide
binding sites on the V-ATPase, although mutagenesis studies in yeast
have begun to identify residues important for V-ATPase activity
(38-40). To investigate residues involved in formation of the
nucleotide binding sites, site-directed mutagenesis of the
VMA1 gene in yeast was performed. Based on the crystal structure of the F1 subunit and sequence alignment of
the
and A subunits, mutations were made in several residues
proposed to form part of the adenine binding pocket at the catalytic
site, including Phe452, Tyr532, and
Phe538 (corresponding to Tyr345,
Phe418, and Phe424, respectively, of the bovine
mitochondrial
subunit) (29). In addition, mutations were
constructed at two residues, Phe479 and Arg483
(corresponding to Tyr368 and Arg372 of the
bovine F1
) (29), which are postulated to be contributed by the A subunit to the noncatalytic nucleotide binding sites. The
effects of these mutations on activity, kinetics and assembly of the
V-ATPase complex were assessed.
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EXPERIMENTAL PROCEDURES |
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Materials and Strains-- Zymolyase 100T was obtained from Sekagaku America, Inc. Concanamycin A was obtained from Fluka Chemical Corp. Tran35S-label was purchased from ICN Biomedicals. Leupeptin, aprotinin, and pepstatin were all purchased from Boehringer Mannheim. Yeast extract, dextrose, peptone, and yeast nitrogen base were purchased from Difco. Zwittergent 3-14 was purchased from Calbiochem-Novabiochem Corp. Molecular biology reagents were from Promega and New England Biolabs. ATP and most other chemicals were purchased from Sigma.
Yeast strain SF838-5AMutagenesis-- Mutagenesis was performed on the wild type VMA1 cDNA using the Altered Sites II in vitro mutagenesis system (Promega) following the manufacturer's protocol. The full-length VMA1 cDNA lacking the endonuclease spacer region (15) was cloned into pAlter-1 using BamHI and SalI sites. The mutagenesis oligonucleotides were as follows with the substitution sites underlined: F452Y, GAAAGCATTACCCATCTATC; F452W, CAAAGAAAGCATTGGCCATCTATCAAC; F452A, CAAAGAAAGCATGCCCCATCTATCAAC; F479Y, CAATTACCCTGAATATCCTGTTTTAAG; F479A, CAATTACCCTGAAGCTCCTGTTTTAAGAG; F479W, CAATTACCCTGAATGGCCTGTTTTAAGAG; R483K, GAATTTCCTGTTTTAAAAGATCGTATG; R483E, GAATTTCCTGTTTTAGAAGATCGTATGAAG; R483Q, GAATTTCCTGTTTTACAAGATCGTATGAAG; Y532F, CAACAAAATGGTTTCTCCACTTATG; Y532S, CAACAAAATGGTTCCTCCACTTATGATG; F538Y, CACTTATGATGCTTACTGTCCAATTTGG; F538W, CACTTATGATGCTTGGTGTCCAATTTGG; and F538A, CACTTATGATGCTGCCTGTCCAATTTGG.
All mutations were confirmed by DNA sequencing using the dideoxy method. Cassettes containing each mutation were subcloned using HpaI and SalI into a wild type pAlterI/VMA1 plasmid that had not been subjected to mutagenesis. The full-length mutant vma1 cDNAs were then subcloned, along with wild type VMA1 for a positive control, into the yeast integration vector YIp5 (New England Biolabs) using BamHI and SalI sites.Transformation--
Wild type VMA1 in YIp5
(WT-VMA1) as a positive control, YIp5 vector alone
(vma1/YIp5) as a negative control, or vma1
mutant YIp5 plasmids were linearized with ApaI to target the
integration of the constructs to the URA3 locus. Yeast cells
SF838-5A
vma1
-8 were transformed with the linearized
constructs using the lithium acetate method (41). The transformants
were then selected on Ura
plates as described previously
(42). Chromosomal DNA was isolated from the transformed yeast cells and
the VMA1 gene was amplified by a polymerase chain reaction.
The presence of the site-directed mutations was confirmed by sequencing
the polymerase chain reaction products. Growth phenotypes of the
mutants were assessed on YEPD plates buffered with 50 mM
KH2PO4 and 50 mM succinic acid to
either pH 5.5 or 7.5. Plates containing 50 mM
CaCl2 were buffered to pH 7.5 using 50 mM
MES/MOPS (43).
Purification of the V-ATPase Enzyme--
Yeast integrated with
either wild type plasmid (WT-VMA1), vma1 mutant
plasmids, or the vector YIp5 alone (vma1/YIp5) as a negative control were cultured overnight in 1 liter of YEPD pH 5.5 to
log phase. Vacuoles were isolated as described previously (44). For
purification of the V-ATPase, vacuolar membranes were washed three
times with 10 mM Tris-HCl, pH 7.5, and 1 mM
EDTA, and solubilized in buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 0.5 mM
phenylmethylsulfonyl fluoride, and 2 mM dithiothreitol) with Zwittergent 3-14, and the V-ATPase was isolated by glycerol density gradient sedimentation on 20-50% glycerol gradients as described previously (45).
Immunoblot Analysis--
Whole cell lysates and solubilized
vacuoles were prepared using 50 mM Tris-HCl, pH 6.8, 8 M urea, 5% SDS, 1 mM EDTA, and 5% -mercaptoethanol, as described previously (46). Samples were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with
the monoclonal antibody 8B1-F3 against the yeast V-ATPase A subunit
(Molecular Probes, Inc.), followed by horseradish peroxidase-conjugated secondary antibody (Bio-Rad). Immunoblots were developed using a
chemiluminescent detection method following the manufacturer's protocol from Kirkegaard & Perry Laboratories.
Metabolic Labeling and Immunoprecipitation of the
V-ATPase--
Yeast strains WT-VMA1,
vma1/YIp5, and vma1 mutants were grown in
synthetic dextrose-methionine-free medium overnight, converted to
spheroplasts, and metabolically labeled with Tran35S-label
(50 µCi/5 × 106 spheroplasts) for 60 min at
30 °C. Spheroplasts were pelleted, washed, and lysed in
solubilization buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 10% glycerol) with
C12E9, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin). The V-ATPase was cross-linked
using dithiobis(succinimidyl propionate) and immunoprecipitated using the monoclonal antibody 8B1-F3 against the yeast A subunit and protein
A-Sepharose (Pharmacia Biotech Inc.). Samples were subjected to
SDS-PAGE on a 12% acrylamide gel, fixed in 30% methanol and 7.5%
acetic acid for 1 h, incubated in Enlightning solution (NEN Life
Science Products) for 30 min, dried, and analyzed using
autoradiography.
Modeling of the Catalytic Nucleotide Binding Site on the V-ATPase
A Subunit--
A model for the V-ATPase was created from the 2.8 Å resolution structure of bovine heart mitochondrial
F1-ATPase (29). The amino acid sequences were first aligned
using the Genetics Computer Group (GCG) sequence analysis software
package. Approximately 23% sequence identity was obtained after
sequence alignment, with good agreement in predicted secondary
structure (47). The positions of insertions and deletions created by
the alignment procedure were examined on the model of
F1-ATPase using InsightII (Biosym Technologies, San Diego,
CA), and were found to be mostly in loops at the surface of the
molecule. A few were located within secondary structure elements and
the sequence alignment was adjusted to move these by a few residues to
the end of the structural elements. The large insertion of about 100 amino acids present in the A subunit (but not ) was not modeled. The
resultant structure now containing the amino acid sequence of V-ATPase
was subjected to 200 steps of energy minimization using X-PLOR
(48).
Other Procedures-- ATPase activity was measured using a coupled spectrophotometric assay (44) in the absence or presence of 1 µM concanamycin A, a specific V-ATPase inhibitor (49). ATPase assays performed on vacuoles or on purified enzyme contained 1 mM ATP, 2 mM MgCl2 and were done at 37 °C. SDS-PAGE was carried out as described by Laemmli (50). Protein concentrations were determined by Lowry et al. (51) assay, and determinations on purified enzyme included initial protein precipitation with 10% trichloroacetic acid.
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RESULTS |
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Growth Phenotypes for vma1 Mutants--
Site-directed mutations of
the VMA1 cDNA encoding the yeast V-ATPase A subunit were
constructed as described under "Experimental Procedures." Three of
the residues mutated, Phe452, Tyr532, and
Phe538, are located in the C-terminal domain of the A
subunit and are candidates for contributing to the adenine binding
pocket of the catalytic nucleotide binding site. Two additional A
subunit residues, Phe479 and Arg483, are
postulated on the basis of sequence alignment and the crystal structure
of F1 to form part of the noncatalytic nucleotide binding site located on the B subunit. The mutant vma1 cDNAs
were subcloned into the yeast integration vector YIp5 and expressed in
a vma1 strain in which the VMA1 gene was
deleted. The deletion of genes encoding subunits of the V-ATPase
results in a conditional lethal phenotype (14, 15, 52), which is also
used to screen for mutants defective in vacuolar acidification. These
strains are able to grow on medium buffered to acidic pH (5.0-5.5) but
are unable to grow at neutral pH (7.5) or in neutral medium containing 50 mM CaCl2. Table
I summarizes the growth phenotypes for
these vma1 mutants. The wild type strain WT-VMA1
grew under each condition and the negative control
vma1
/YIp5 strain exhibited the expected conditional
lethal phenotype. F452A was unable to grow at pH 7.5 in the absence or
presence of elevated extracellular calcium, exhibiting a growth
phenotype similar to the vma1
strain. F538W was unable to
grow at pH 7.5 in the presence of high calcium. The remaining mutants,
including those at the noncatalytic site, showed relatively normal
growth at pH 7.5. It has previously been determined that 20% of
V-ATPase activity is sufficient to rescue the growth phenotype (39).
These results thus suggest that the F452A and F538W mutants are
deficient in vacuolar acidification, but that the remainder of the
mutant strains have a V-ATPase activity that is at least 20% of
wild-type. It was next necessary to determine whether these mutations
directly caused a loss of V-ATPase activity.
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ATPase Activities in Purified Vacuoles from vma1 Mutants--
Fig.
1 shows the V-ATPase activities of the
wild type WT-VMA1, vma1/YIp5, and
vma1 mutant strains. Fig. 1A shows the relative ATPase activities for the strains containing mutations at the catalytic
site, and Fig. 1B the activities for the strains with the
proposed noncatalytic site mutations. V-ATPase activities were measured
in isolated vacuoles using a coupled spectrophotometric assay at 1 mM ATP and 37 °C as described under "Experimental
Procedures." Activities are expressed relative to the
WT-VMA1 strain, defined as 100%, which had a specific
activity of 0.19 (± 0.04) µmol of ATP/min/mg
protein.2 The negative
control, vma1
/YIp5 strain, had no measurable activity. F452Y, Y532F, F538Y, and F538A had nearly wild type levels of activity,
whereas F452W and Y532S had approximately 50% and F538W had 20% of
wild type levels. F452A had less than 5% of wild type activity and was
comparable to the deletion strain. Mutations at the noncatalytic site
all appear to reduce activity by 30-70% as shown in Fig.
1B. These data are consistent with the growth phenotypes.
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A Subunit Expression and V-ATPase Assembly in vma1 Mutants--
It
was next necessary to determine whether the observed decrease in
activity was due to decreased protein levels or to decreased V-ATPase
assembly. Fig. 2 shows the effects of the
mutations on A subunit stability and the presence of the A subunit on
the vacuolar membrane, which provides an initial measure of V-ATPase
assembly. Fig. 2 is an immunoblot using the monoclonal antibody 8B1-F3
against the yeast A subunit to determine levels of the A subunit in
either whole cell lysates (CELL) or isolated vacuoles
(VAC). The top panel shows the immunoblots for
the wild type WT-VMA1 and for vma1/YIp5. The
middle and bottom panels show the immunoblots for
the strains containing mutations at the catalytic site, and the
proposed noncatalytic site, respectively. Of the mutations tested, only
R483E showed somewhat lower levels of A subunit in the whole cell
lysate compared with the wild type. However, all of the mutated
proteins, including R483E, showed normal levels of A subunit on the
vacuolar membrane.
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Kinetic Analysis of vma1 Mutants-- To determine if any of the mutations resulted in changes in KmATP or Vmax for ATP hydrolysis, kinetic analysis on the purified enzymes was performed (Table II). Vacuoles were isolated from wild type WT-VMA1 and vma1 mutant strains and the V-ATPases were solubilized and purified by glycerol density gradient sedimentation as described under "Experimental Procedures." ATPase activities of the purified enzymes were measured over a range of ATP concentrations from 100 µM to 2.5 mM ATP, while the MgCl2 was maintained at 1 mM above the ATP concentration. Km and Vmax values were calculated from double reciprocal plots of ATP concentration versus ATPase activity. WT-VMA1 had a specific activity of 1.8 (±0.1) µmol/min/mg of protein at 1 mM ATP, a Km of 0.72 (±0.19) mM ATP and a Vmax of 2.7 (±0.1) µmol/min/mg of protein. As shown in Table II, the Km and Vmax for F452Y and F538Y were not significantly different from wild type, whereas F452W, F538W, and Y532F had a decreased Km, suggesting an increase in substrate affinity. kcat/Km values for the former two mutations were reduced by about 2-fold, whereas for the Y532F mutation, a small increase in kcat/Km was observed. F538A had a Km value approximately 2 times that of WT-VMA1, suggesting a small decrease in ATP affinity. Because the reaction rate for the Y532S mutant showed no sign of saturating, even at the highest ATP concentrations tested (2.5 mM), precise values for Km and Vmax could not be determined (data not shown). However, we estimate that the Km for ATP is greater than 5 mM, thus reflecting a significant decrease in affinity relative to the wild type. Some of the mutations proposed to be at the noncatalytic site also resulted in kinetic changes. F479W, F479A, and R483K all showed significant decreases in both Km and Vmax, although values of kcat/Km were either unchanged (F479A) or reduced by at most 2-fold. Interestingly, both R483Q and R483E exhibited a time-dependent increase in ATPase activity, as depicted for the R483Q mutant in Fig. 4.
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DISCUSSION |
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Although mutagenesis has been employed in several studies to
identify A subunit residues important for activity of the V-ATPases (39, 40, 53), there is currently no information concerning the residues
that may participate in formation of the adenine binding pocket on the
A subunit. In the F-ATPase subunit, the adenine ring appears to be
in close contact with a hydrophobic surface formed by aromatic rings,
in particular Tyr345 of the bovine mitochondrial enzyme
(29). To determine whether aromatic rings are important in this region
of the A subunit, site-directed mutagenesis was performed on
Phe452, Tyr532, and Phe538, located
in the C-terminal domain of the A subunit. Conservative substitutions
(such as Phe to Tyr and Tyr to Phe) as well as nonconservative substitutions (such as Phe to Ala or Tyr to Ser) were carried out.
Substitution with a bulkier amino acid (Trp) was also performed.
From the data presented, it appears that an aromatic ring or a
hydrophobic residue is important at each of these positions, particularly Phe452. Of the catalytic site mutations
tested, none significantly reduced stability of the A subunit and only
one (F452A) reduced stability of the V-ATPase complex as evidenced by
the inability to immunoprecipitate the intact complex from
metabolically labeled cells. With respect to their effects on activity
of the V-ATPase complex, the conservative mutations F452Y, Y532F, and
F538Y had little effect on enzyme activity whereas the F452W mutation
reduced activity by 50-70%, although this mutation also resulted in a
lower value for Km, suggesting an increased affinity
for ATP. Interestingly, the F452A mutant, which could be isolated as an
intact complex from vacuoles, was completely inactive, suggesting that
an aromatic or sufficiently large hydrophobic residue at this position
is crucial. Phe452 corresponds to Tyr345 of the
mitochondrial subunit, which appears from the crystal structure of
F1 to be in direct contact with the adenine ring (29).
Tyr345 of the mitochondrial
subunit is labeled by the
photoaffinity analog 2-[azido-32P]ATP (54) and
substitution of tryptophan for Tyr331 (which corresponds to
Tyr345 in the Escherichia coli
subunit)
results in a reduction in both Km and
Vmax by about 2-fold (55), whereas substitution with alanine reduces Vmax by 5-fold and
increases Km by 9-fold (56). It should be noted that
substitution of leucine for Tyr331 in the E. coli F-ATPase results in a 7.5-fold increase in
Km for ATP with little effect on
Vmax (56), suggesting that a hydrophobic residue
can partially substitute for the aromatic function at this position.
Our data suggest that Phe452 plays a comparably important
role in nucleotide binding to the catalytic site on the V-ATPase A
subunit. The inability to immunoprecipitate the intact V-ATPase from
cells bearing the F452A mutation suggests that this substitution may
also lead to a reduced stability of the V-ATPase complex.
Mutations at Tyr532 and Phe538 had rather
different effects on activity. In particular, Y532S appeared to show a
significantly reduced affinity for ATP, again consistent with a role of
this residue in ATP binding. This residue corresponds to
Phe418 of the bovine mitochondrial subunit which is
situated near the end of the adenine binding pocket (29). Replacement
of Phe538 with alanine also significantly reduced the
affinity for ATP (approximately 2-fold), consistent with its
interaction with the adenine ring. The corresponding residue in the
F1
subunit, Phe424, appears to interact
with the adenine ring from the opposite side of Tyr345
(29). It should be noted that the effects of mutations in these
subunit residues (Phe418 and Phe424) on
nucleotide binding to the F-ATPases have not been reported. These
results support a model in which aromatic or hydrophobic residues in
the C-terminal domain of the A subunit contribute to nucleotide binding
to the catalytic site of the V-ATPase, with their absence resulting in
substantial changes in either nucleotide binding or activity. These
results also point up the likely similarity in structure of the
catalytic nucleotide binding sites of the V and F-ATPases.
Using the recently released coordinates of the bovine heart
mitochondrial F1 ATPase (29) and sequence alignment of the
F-ATPase subunit and V-ATPase A subunit, we have carried out energy
minimization to arrive at a tentative model of the catalytic nucleotide
binding site on the V-ATPase (Fig. 5).
Shown are the aromatic residues investigated in the current study
(Phe452, Tyr532, and Phe538) as
well as A subunit residues previously characterized by site-directed mutagenesis (40), including Glu286, Lys263,
Cys261, and Cys539. As for the
subunit,
both Glu286 and Lys263 are in close proximity
to the triphosphates of bound ATP, consistent with their proposed role
in catalysis (29). Cys261 and Cys539 are
predicted to be approximately 13 Å apart, suggesting that some
distortion of the nucleotide binding site may need to occur in order
for these two residues to undergo disulfide bond formation (33). All
three aromatic residues are predicted to be in relatively close
proximity to the adenine binding pocket, with Phe452 nearly
stacked with the adenine ring. This is consistent with the mutagenesis
data, where the largest effects on activity were observed with the
F452A mutation.
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We have previously observed that mutations in B subunit residues
postulated to be contributed to the catalytic site have dramatic effects on activity (38), suggesting that, as with the F-ATPases, the
catalytic sites of the V-ATPases are located at the interface between
the two nucleotide binding subunits. We also wished to determine the
effects of mutations in A subunit residues postulated to be contributed
to the noncatalytic sites on the B subunit. These include
Phe479 (corresponding to Tyr368 of the bovine
F1 subunit) and Arg483 (corresponding to
Arg372 of F1
). This is particularly
important as the function of the noncatalytic nucleotide binding sites
remains uncertain. Interestingly, all the mutations tested (even the
conservative F479Y and R483K mutations) decreased enzyme activity in
purified vacuoles by 30-70% and reduced
kcat/Km by at most 2-fold.
These data are consistent with our previous findings that B subunit
mutations at the noncatalytic site also resulted in modest (30-60%)
decreases in activity (38). These results suggest that changes in the noncatalytic site produce an enzyme that is not substantially altered
in its catalytic efficiency.
Although several of the mutations (including F479W and R483E) showed slightly reduced assembly as assessed by immunoprecipitation, none of them completely abolished assembly. On the other hand, both of these mutations showed somewhat reduced labeling of the 50-kDa region of the immunoprecipitates, which might reflect partial loss of the VMA13 gene product. Vma13p (a 54-kDa V1 subunit) is required for activity but not assembly of the V-ATPase complex (57). Among the most interesting mutations identified were R483E and R483Q, both of which resulted in a time-dependent increase in enzyme activity. One possible explanation for this effect is the loss during purification of the mutant enzymes of an endogenous nucleotide (possibly at the noncatalytic site) required for activity. This endogenous nucleotide would then be restored on addition to the assay. Alternatively, these mutations may result in slowed release of an inhibitory nucleotide which, in the wild type, is released rapidly. Sulfite has been suggested to activate the yeast V-ATPase by promoting the release of inhibitory ADP (58). Nevertheless, the effects of these mutations suggest that changes in the noncatalytic nucleotide binding sites can modulate the catalytic activity of the V-ATPase.
We have provided preliminary evidence for the role of A subunit residues Phe452, Tyr532, and Phe538 in formation of the catalytic nucleotide binding site of the V-ATPase, possibly through contribution to the adenine binding pocket. We have also demonstrated that mutations in residues on the A subunit, Phe479 and Arg483, that are postulated to form part of the noncatalytic site on the B subunit, can have effects on V-ATPase activity. Further work will be required to more completely define the function of these sites in the control of activity.
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ACKNOWLEDGEMENT |
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We thank Dr. Patricia Kane, Department of
Biochemistry and Molecular Biology, SUNY, Syracuse, for the
generous gift of the yeast strain SF838-5A vma1
-8, the
plasmid pPK17-7, and for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 34478 (to M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Tufts University, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6939; Fax: 617-636-0445.
1
The abbreviations used are: V-ATPase, vacuolar
proton-translocating adenosine triphosphatase; YEPD, yeast
extract-peptone-dextrose; PAGE, polyacrylamide gel electrophoresis;
MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic
acid; AMP-PNP, 5-adenylyl
,
-imidodiphosphate; PAGE,
polyacrylamide gel electrophoresis; WT, wild type.
2 The specific activity reported is that for unwashed vacuoles at 1 mM ATP. Higher specific activities are obtained for washed vacuoles at saturating ATP concentrations.
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REFERENCES |
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