From the Department of Physiology, Tufts University School of Medicine, Boston Massachusetts 02111
Received for publication, November 27, 2002, and in revised form, January 22, 2003
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ABSTRACT |
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Subunit A is the catalytic nucleotide binding
subunit of the vacuolar proton-translocating ATPase (or
V-ATPase) and is homologous to subunit The vacuolar proton-translocating ATPases (or
V-ATPases)1 are ATP-driven
proton pumps present in both intracellular compartments and the plasma
membrane of eukaryotic cells (1-8). They couple the energy released
upon ATP hydrolysis to the active transport of protons from the
cytoplasm to either the lumen of various intracellular compartments or
to the extracellular environment. Acidification of intracellular
compartments is important for such processes as receptor-mediated
endocytosis, intracellular trafficking of lysosomal enzymes,
degradation of macromolecules, uptake of neurotransmitters, and the
entry of various envelope viruses and toxins (1-8). Plasma membrane
V-ATPases have also been implicated in many normal and disease
processes, including bone resorption, renal acidification, pH
homeostasis, and tumor metastasis (9-13). Defects in specific V-ATPase
subunits have been shown to be responsible for a number of human
genetic diseases, including autosomal recessive osteopetrosis and renal
tubular acidosis (9, 14-16).
The V-ATPases are multi-subunit complexes composed of two functional
domains (1-8). The 640-kDa peripheral V1 domain is
responsible of ATP hydrolysis and consists of 8 different subunits
(subunits A-H) with molecular masses of 70-14 kDa. The V0
domain is a 260-kDa integral complex composed of five different
subunits (subunits a, d, c'', c', and c with molecular masses of
100-17 kDa) that function in proton translocation. The V-ATPases are
structurally related to the F-ATPases, which function as proton-driven
ATP synthases in mitochondria, chloroplasts, and bacteria (17-22). Both the A and B subunits of the V-ATPases participate in nucleotide binding, with the catalytic nucleotide binding sites located on the A
subunit and so called "non-catalytic" nucleotide binding sites
located on the B subunit (1-8). The A and B subunits are homologous to
the High resolution structural data of F1 reveal a hexameric
arrangement of Sequence alignment of the A subunit of V1 and the Materials and Strains--
Escherichia coli and yeast
culture media were purchased from Difco. Restriction endonucleases, T4
DNA ligase, and other molecular biology reagents were from Invitrogen.
Zymolyase 100T was purchased from Sekagaku America, Inc. Protease
inhibitors were purchased from Roche Molecular Biochemicals.
Concanamycin A was purchased from Fluka Chemical Corp.
9-Amino-6-chloro-2-methoxyacridine was purchased from Molecular Probes,
Inc. SDS, nitrocellulose membrane (0.45 µm pore size), Tween 20, horseradish peroxidase-conjugated goat anti-rabbit IgG, and horse
radish peroxidase-conjugated goat anti-mouse IgG were from Bio-Rad. The
chemiluminescence substrate for horseradish peroxidase was from
Kirkegaard & Perry Laboratories. Protein A-Sepharose, ATP,
polyoxyethylene 9 lauryl ether, and most other chemicals were obtained
from Sigma.
Mouse monoclonal antibodies 8B1-F3 against Vma1p, 13D11-B2 against
Vma2p, and 10D7 against Vph1p were purchased from Molecular Probes,
Inc. Rabbit polyclonal antibodies against Vma6p, Vma7p, Vma8p, Vma10p,
and Vma13p were generous gifts from Dr. Tom Stevens (University of
Oregon, Eugene, OR). The rabbit polyclonal antibody against Vma4p was
kindly provided by Dr. Daniel Klionsky (University of Michigan, Ann
Arbor, MI). The mouse monoclonal antibody 11E6 against Vma1p and a
mouse monoclonal antibody against Vma5p were generous gifts from Dr.
Patricia Kane (State University of New York Upstate Medical University, Syracuse).
Yeast strain SF838-5Aa vma1D-8 (MATa,
leu2-3, 112, ura3-52,
ade6, vma1D::LEU2) was used
for integration and subsequent biochemical characterization. Yeast
cells were grown in YPD media containing dextrose or YEP media without dextrose.
Construction of Mutants--
Mutations in the non-homologous
region of subunit A were made using the Altered Sites II in
vitro mutagenesis system (Promega) following the manufacturer's
protocol. The full-length VMA1 gene without the spacer
region (44) was cloned into pAlter-1 using BamH1 and
SalI sites. The mutagenesis oligonucleotides were as follows
with the substitution sites underlined: G150A,
5'-GGAAAGTTTCAAGTCGCCGATCATATTTCCGGT-3'; G156V,
5'-GATCATATTTCCGGTGTTGATATTTACGGTTCC-3'; D157E,
5'-CATATTTCCGGTGGTGAAATTTACGGTTCCGTT-3'; D157A,
5'-CATATTTCCGGTGGTGCTATTTACGGTTCCGTT-3'; V162L,
5'-GATATTTACGGTTCCCTTTTTGAGAATTCGCTA-3'; V162A,
5'-GATATTTACGGTTCCGCTTTTGAGAATTCGCTA-3'; E164D,
5'-ATTTACGGTTCCGTTTTTGACAATTCGCTAATTTCA-3'; E164A,
5'-ATTTACGGTTCCGTTTTTGCGAATTCGCTAATTTCA-3'; P177V,
5'-AGCCATAAGATTCTTTTGCCAGTAAGATCAAGAGGTACAATC-3'; G181A,
5'-CCACCAAGATCAAGAGCTACAATCACTTGGATT-3'; G205V,
5'-GAAGTTGAATTTGATGTCAAGAAGTCTGATTTC-3'; W216F,
5'-GATTTCACTCTTTACCATACTTTCCCTGTTCGTGTTCCAAGA-3'; W216A, 5'-GATTTCACTCTTTACCATACTGCGCCTGTTCGTGTTCCAAGA-3'; P217V,
5'-TTCACTCTTTACCATACTTGGGTTGTTCGTGTTCCAAGA-3'; R219K,
5'-CTTTACCATACTTGGCCTGTTAAAGTTCCAAGACCAGTTACT-3'; R219A, 5'-CTTTACCATACTTGGCCTGTTGCTGTTCCAAGACCAGTTACT-3'; P223V,
5'-GTTCGTGTTCCAAGAGTAGTTACTGAAAAGTTATCT-3'; P233V,
5'-AAGTTATCTGCTGACTATGTTTTGTTAACAGGTCAA-3'. All mutants were confirmed by DNA sequencing using an automated sequencer from
Applied Biosystems and subcloned into the YIp5 vector using BamHI and SalI sites along with wild type
VMA1. No other mutations were detected in the final product.
The wild type plasmid (YIp5-VMA1), vma1 mutant plasmids, or
YIp5 vector alone were linearized with ApaI to target
integration of the constructs to the URA3 locus. Transformation of
yeast cells (SF838-5A Isolation of Vacuolar Membrane Vesicles--
Vacuolar membrane
vesicles were isolated using a modified protocol described by Uchida
et al. (47). The yeast integrated with the wild type plasmid
(YIp5-VMA1), mutants, or the vector YIp5 alone were cultured
overnight in 1 liter of YPD (pH 5.5) to log phase. Cells were pelleted,
washed once with water, and resuspended in 100 ml of 10 mM
dithiothreitol and 100 mM Tris-HCl, pH 9.4. After
incubation at 30 °C for 15 min, cells were pelleted again, washed
once with 100 ml of YPD medium containing 0.7 M sorbitol, 2 mM dithiothreitol, 100 mM MES-Tris, pH 7.5, and
2 mg of zymolyase 100T, and incubated at 30 °C with gentle shaking for 60 min. The resulting spheroplasts were osmotically lysed, and the
vacuoles were isolated by flotation on two consecutive Ficoll
gradients. Protein concentrations were determined by Lowry assay
(48).
Immunoblot Analysis--
Whole cell lysates (prepared as
described previously (49)) and isolated vacuolar membrane vesicles were
treated with 50 mM Tris-HCl, pH 6.8, 8 M urea,
5% SDS, 1 mM EDTA, and 5% Biochemical Characterization--
ATPase activity was measured
using a coupled spectrophotometric method as described previously (51)
with some modification. Isolated vacuolar membrane vesicles were
incubated in ATPase assay buffer (50 mM NaCl, 30 mM KCl, 20 mM HEPES-NaOH, pH 7.0, 0.2 mM EGTA, 10% glycerol, 1 mM MgCl2,
1.5 mM phosphoenolpyruvate, 0.35 mM NADH, 20 units/ml pyruvate kinase, and 10 units/ml lactate dehydrogenase) with
0.1% Me2SO or 1 µM concanamycin A at room temperature for 10 min. The assay was initiated by the addition of 0.5 mM ATP, and the absorbance at 341 nm was measured
continuously using a Kontron UV-visible spectrophotometer.
ATP-dependent proton transport was measured as previously
described (52) using 10 µg of vacuolar protein in 50 mM
NaCl, 30 mM KCl, 20 mM HEPES-NaOH, pH 7.0, 0.2 mM EGTA, 10% glycerol, except that the initial rate of
fluorescence quenching (rather than the net fluorescence change) after
the addition of 0.5 mM ATP and 1.0 mM
MgCl2 was measured using the fluorescence probe
9-amino-6-chloro-2-methoxyacridine in the presence or absence of 1 µM concanamycin A using a PerkinElmer Life Sciences LS50B spectrofluorometer.
In Vivo Dissociation and Reassembly of the V-ATPase in Response
to Glucose Depletion and Readdition--
Dissociation of the V-ATPase
in response to glucose depletion and reassembly in response to glucose
readdition were measured as described previously (50) with some
modifications. The yeast integrated with the wild type plasmid
(YIp5-VMA1) or the mutants were grown in YPD media, pH 5.5, overnight to an absorbance at 600 nm of <1.0. The cells were converted
to spheroplasts by treatment with zymolyase 100T and incubated in YEP
media with or without 2% glucose for 40 min at 30 °C. To observe
reversal of dissociation of the V-ATPase upon glucose readdition,
spheroplasts were incubated in YEP media with or without 2% glucose
for 20 min. An aliquot of the spheroplasts incubated in the absence of
glucose was then incubated in YEP media with 2% glucose for an
additional 20 min. Spheroplasts were pelleted and lysed in
phosphate-buffered saline containing 1% polyoxyethylene 9 lauryl
ether, protease inhibitors (2 µg/ml aprotinin, 0.7 µg/ml pepstatin,
5 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl
fluoride), and 1 mM dithiobis(succinimidyl propionate). The
V-ATPase complexes were immunoprecipitated using monoclonal antibody
13D11 against the B subunit and protein A-Sepharose followed by
separation on 8% acrylamide gels and transfer to nitrocellulose. Western blotting was then performed using antibody 8B1-F3 against subunit A and antibody 13D11 against subunit B to detect the
V1 domain and antibody 10D7-A7 against Vph1p to detect the
V0 domain followed by incubation with horseradish
peroxidase-conjugated secondary antibody. Dissociation of the V-ATPase
complex is reflected as a reduction in the amount of the V0
subunit Vph1p, immunoprecipitated using the antibody directed against
subunit B of the V1 domain.
Effects of Mutations in the Non-homologous Region of Subunit A on
Growth Phenotype and Assembly of the V-ATPase Complex--
To
investigate the function of the non-homologous region of subunit A of
the V-ATPase, site-directed mutagenesis of the VMA1 gene
encoding subunit A in yeast was performed. Alignment of the sequence of
the non-homologous region of subunit A from 8 species is shown in Fig.
1. Of the 90 residues in this region, 13 are conserved in all eukaryotic A subunit sequences available. A total of 18 site-directed mutations were constructed at these 13 sites, with
in some cases both conservative and non-conservative substitutions tested. Each of the four conserved proline residues was replaced by
valine, whereas the four conserved glycine residues were replaced with
either alanine or valine. The three charged residues (Asp-157, Glu-164,
and Arg-219) were replaced with either alanine or a residue that
conserved the original charge. Finally, the two hydrophobic residues
(Val-162 and Trp-216) were replaced with either alanine or a similarly
bulky, hydrophobic amino acid to the original.
We first determined the effect of the introduced mutations on the
growth phenotype of the yeast strain expressing them. Deletion of genes
encoding subunits of the V-ATPase leads to a conditional lethal
phenotype (vma Effects of Mutations in the Non-homologous Region on Proton
Transport and ATP Hydrolysis by the V-ATPase--
We next tested the
effects of the mutations on the activity of the V-ATPase. Both
concanamycin-sensitive ATPase activity and ATP-dependent
proton transport (as assessed by fluorescence quenching using the probe
9-amino-6-chloro-2-methoxyacridine) were measured in isolated vacuolar
membrane vesicles at an ATP concentration of 0.5 mM. As can
be seen in Fig. 3, three of the mutations
(G156V, W216A, and R219K) resulted in loss of greater than 80% of both proton transport and ATPase activity. Of these, both G156V and W216A
showed severe defects in assembly that likely account for the observed
loss of activity. By contrast, the R219K mutant, which had less than
10% wild type activity, showed normal assembly on vacuolar membranes.
These results suggest that the R219K mutation directly affects V-ATPase
activity.
Four mutations (E164D, G181A, W216F, and R219A) reduce both proton
transport and ATPase activity in parallel by 50-80% relative to wild
type. Both G181A and W216F show partial or complete defects in
assembly, whereas E164D and R219A assemble normally. Interestingly, the
R219A mutant shows a severe growth phenotype (Fig. 2) despite possessing at least 40% of wild type levels of activity in
vitro.
Three mutations (P217V, P223V, and P233V) have significantly different
effects on ATPase activity and proton transport. Both P223V and P233V
show significant reductions in proton transport while retaining wild
type levels of ATPase activity, suggesting a decrease in coupling
between proton transport and ATP hydrolysis. By contrast, the P217V
mutant possesses only about 34% of wild type ATPase activity but 140%
of wild type proton pumping activity. These results suggest that
changes in the non-homologous region of the A subunit (and particularly
the region between residues 217 and 233) can result in significant
changes in coupling of proton transport and ATP hydrolysis by the
V-ATPase. It should be noted, however, that because the activities
reported in Fig. 3 correspond to measurements done at a single ATP
concentration (0.5 mM), which is below typical cytoplasmic
levels of ATP, the observed differences in coupling would not
necessarily occur under in vivo conditions. Also, because
this ATP concentration is below the Km measured for
the wild type enzyme (0.75 mM ATP), the observed
differences do not necessarily reflect differences in maximal velocity.
In Vivo Glucose-dependent Dissociation of the
V-ATPase--
In yeast, dissociation of the V1 and
V0 domains occurs in response to glucose depletion and
represents an important mechanism of regulating V-ATPase activity
in vivo (42). It has previously been demonstrated that
in vivo dissociation of the V-ATPase requires catalytic
activity (58, 59), although mutants of subunit D possessing as little
as 5-10% of wild type activity are still able to undergo dissociation
(60). To evaluate the effects of mutations in the non-homologous region
on in vivo dissociation of the V-ATPase, spheroplasts were
incubated in the presence or absence of glucose and then lysed with
detergent, and the V-ATPase was immunoprecipitated using the antibody
13D11 against subunit B of the V1 domain. After separation
of the immunoprecipitated polypeptides by SDS-PAGE, Western blot
analysis was performed using antibodies against subunits A and B of
V1 and subunit a of V0. Dissociation of the
V-ATPase appears as a reduction in the amount of subunit a (and hence,
V0) precipitated using an antibody that recognizes
V1, as previously described (50, 60). The four mutants
defective in normal assembly of the V-ATPase complex were not tested
for glucose-dependent dissociation. The remaining mutants
fell into one of five categories (Fig.
4). The first class is mutants possessing
greater than 30% wild type activity in vitro that show near
normal glucose-dependent dissociation and includes D157A,
V162L, V162A, E164A, and G205V. The second class (represented by R219K)
is inactive and, as predicted from previous results (50, 60), does not
undergo dissociation. The third class (that includes only P217V) shows
normal in vivo dissociation while displaying higher than
normal proton transport and somewhat reduced ATPase activity in
vitro. The fourth class, which includes E164D, R219A, and P233V,
possesses greater than 30% wild type activity and is partially blocked
in glucose-dependent dissociation but also show some defect
in assembly even in the presence of glucose. The fifth class, which
includes G150A, D157E, P177V, and P223V, possesses greater than 30%
wild type activity and shows normal assembly in the presence of glucose
but is completely blocked in glucose-dependent
dissociation. These results suggest that changes in the non-homologous
region can alter in vivo dissociation of the V-ATPase
independent of effects on catalytic activity.
Glucose-dependent Reassembly of the
V-ATPase--
Dissociation of the V-ATPase in response to glucose
depletion is reversed upon re-addition of glucose (42). To determine whether the glucose-dependent dissociation observed in
mutants of the non-homologous region was also reversible, spheroplasts were incubated in the presence or absence of glucose for 20 min or were
incubated in the absence of glucose for 20 min followed by incubation
in the presence of glucose for an additional 20 min. Solubilization of
the V-ATPase, immunoprecipitation, and Western blotting were then
performed as described above. As can be seen in Fig.
5, of the six mutants that exhibited
glucose-dependent dissociation, all showed reassembly upon
re-addition of glucose.
The structural similarity between the V- and F-ATPases has led to
the suggestion that both classes of proteins operate via a rotary
mechanism (1, 2). In this mechanism, ATP hydrolysis by nucleotide
binding subunits in the peripheral domain drives rotation of a central
stalk, which in turn causes rotation of a ring of proteolipid subunits
in the integral domain (31-38). It is the rotation of this proteolipid
ring relative to subunit a in the integral domain that is thought to
drive active proton translocation (31, 32). In order for ATP hydrolysis
and proton transport to be coupled in this mechanism, subunit a must be
held fixed relative to the nucleotide binding subunits or the energy released from ATP hydrolysis would be dissipated in non-productive rotation of the peripheral domain. This is accomplished by the presence
of a peripheral stalk, or "stator," that connects subunit a to the
nucleotide binding subunits. In the F-ATPases, this peripheral stalk is
composed of the Modeling of the nucleotide binding subunits A and B of the V-ATPases
based upon sequence homology with the The results presented in the current study suggest that mutations in
the non-homologous region of the A subunit do in fact lead to changes
in coupling of proton transport and ATP hydrolysis by the V-ATPases.
Regulation of coupling of proton transport and ATP hydrolysis has
previously been suggested to function in controlling acidification of
intracellular compartments (66, 67). For example, partial uncoupling of
the bovine V-ATPase has been reported to occur at ATP concentrations
above 0.5 mM, which is in the physiological range (66).
Mild proteolysis of the V-ATPase also leads to uncoupling (68), and in
the detergent-solubilized state, the bovine V-ATPase is not inhibited
by DCCD, suggesting a functional uncoupling of the V1 and
V0 domains (69). A 2-fold difference in coupling between
the V-ATPase of lemon epicotyl and fruit was reported (70), and
recently, a 4-5-fold difference in coupling efficiency of yeast
V-ATPase complexes containing two different isoforms of the a subunit
has been demonstrated (50). Moreover, mutations have been isolated in
several subunits, including subunits a (71) and D (60), which
lead to decreased coupling of proton transport and ATPase activity.
These mutations are similar to the P223V and P233V mutations described
in the current report that cause partial uncoupling of proton transport
and ATPase activity. Because, as noted above, higher concentrations of
ATP have been reported to lead to uncoupling of the bovine V-ATPase, it
is possible that some of the coupling effects observed may be the
result of changes in Km for ATP that cause a shift
in this optimal ATP concentration for coupling. The P217V mutant of the
non-homologous domain represents the first mutational change to lead to
a substantial increase in coupling efficiency. These results suggest
not only that changes in the non-homologous region can affect coupling efficiency but also that in the wild type complex, ATP hydrolysis and
proton transport are not optimally coupled.
Reversible dissociation of the V-ATPase has been demonstrated to occur
in both yeast and insect cells (42, 43) and represents an important
mechanism of controlling V-ATPase activity in vivo. Dissociation in yeast occurs rapidly and does not involve any of the
signal transduction pathways known to be activated by glucose deprivation (58), although an intact microtubular network appears to be
required for dissociation (72). In addition,
glucose-dependent reassembly (as well as normal assembly)
is promoted by a complex termed RAVE that includes as one of its
components the ubiquitin ligase subunit Skp1 (73, 74). Dissociation of
the V-ATPase has been shown to require catalytic activity (58, 59),
although the activity required for dissociation may be as low as
5-10% of the wild type level (60). This dependence may reflect the need to achieve a particular conformational state of the complex that
is only reached during turnover of the enzyme. Consistent with these
findings, the R219K mutant investigated in the present study was
inactive and failed to dissociate upon glucose withdrawal.
The most interesting mutants identified in the present study (and
exemplified by the G150A mutant) are those that show significant levels
of proton transport and ATPase activity (>30% relative to wild type)
but which are at least partially blocked in
glucose-dependent dissociation. Two mutations have
previously been reported in subunit G (Vma10p) that also show normal
ATPase activity and are partly inhibited in dissociation in response to
glucose withdrawal (75). Although proton transport was not measured in
these mutants, both showed a wild type growth phenotype, suggesting
substantially normal function. These mutations were proposed to
partially inhibit dissociation through stabilization of the complex, as
indicated by the presence of greater than wild type levels of
V1 subunits on the vacuolar membrane (72). It is possible
that a similar mechanism may be involved for the mutants in the
non-homologous region of the A subunit described in the present report,
and several of the mutants (including G150A, P177V, and P223V) seem to
show higher levels of assembly by immunoprecipitation (Fig. 4),
although no increase in V1 subunits associated with the
vacuolar membrane was detected (Fig. 2). These results suggest that
changes in the non-homologous region can directly affect in
vivo dissociation of the V-ATPase independent of effects on
activity. Whether this domain normally functions in control of this
process and what changes in this region may occur in vivo
remain to be determined.
of the
F1F0 ATP synthase (or F-ATPase). Amino
acid sequence alignment of these subunits reveals a 90-amino acid
insert in subunit A (termed the non-homologous region) that is absent from subunit
. To investigate the functional role of this region, site-directed mutagenesis has been performed on the VMA1
gene that encodes subunit A in yeast. Substitutions were performed on
13 amino acid residues within this region that are conserved in all
available A subunit sequences. Most of the 18 mutations introduced
showed normal assembly of the V-ATPase. Of these, one (R219K) greatly
reduced both proton transport and ATPase activity. By contrast, the
P217V mutant showed significantly reduced ATPase activity but higher
than normal levels of proton transport, suggesting an increase in
coupling efficiency. Two other mutations in the same region (P223V and
P233V) showed decreased coupling efficiency, suggesting that changes in
the non-homologous region can alter coupling of proton transport and
ATP hydrolysis. It was previously shown that the V-ATPase must possess
at least 5-10% activity relative to wild type to undergo in
vivo dissociation in response to glucose withdrawal. However,
four of the mutations studied (G150A, D157E, P177V, and P223V) were
partially or completely blocked in dissociation despite having greater
than 30% of wild type levels of activity. These results suggest that
changes in the non-homologous region can also alter in vivo
dissociation of the V-ATPase independent of effects on activity.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits of the F-ATPases, respectively (23, 24).
and
subunits surrounding a central cavity
containing the
subunit (25-27). The
and
subunits form a
central stalk that connects to a ring of proteolipid c subunits in the
F0 domain (27, 28). F1 and F0 are
also connected via a peripheral stalk (composed of the
and b
subunits) that links the
3
3 head to the a
subunit in F0 (29, 30). The F-ATPases are believed to operate via a rotary mechanism (31, 32) in which ATP hydrolysis in
F1 drives rotation of the central
subunit (33-35),
which in turn drives rotation of the ring of c subunits relative to
subunit a in the F0 domain (36-38). Subunit a is held
fixed relative to the hexameric head by the peripheral stalk (29, 30).
Rotation of the c subunit ring relative to subunit a leads to active
proton translocation across the membrane (31, 32).
subunit of F1 reveals the existence of a 90-amino acid
insert (termed the "non-homologous" region), which is conserved
among A subunit sequences but that is absent from the
subunit sequence (23, 39-41). To address the function of the
non-homologous region, site-directed mutations have been introduced at
conserved amino acid residues in the yeast V-ATPase A subunit (Vma1p).
The results obtained suggest that changes in the non-homologous region
can alter both coupling of proton transport and ATP hydrolysis and
dissociation of the V-ATPase complex in vivo. Reversible
dissociation has been shown to play an important role in regulation of
V-ATPase activity in cells (42, 43).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
vma1
-8) and selection of
transformants on Ura
plates were done as described
previously (45, 46). 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 YPD plates buffered with 50 mM KH2PO4 or 50 mM succinic acid to either pH 5.5 or pH 7.5.
-mercaptoethanol followed
by SDS-PAGE and transferred to nitrocellulose. Blots were then
incubated with antibodies against subunits of the yeast V-ATPase
followed by horseradish peroxidase-conjugated secondary antibodies as
previously described (50). Immunoblots were developed using a
chemiluminescent detection method from Kirkegaard & Perry Laboratories.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid alignment of the non-homologous
region of subunit A of the V-ATPases. Sequence alignment of eight
eukaryotic V-ATPase A subunit sequences are shown. Amino acid
residues identical in all known A subunit sequences are shown
shaded. The numbers shown above the sequence
correspond to the residue number in the yeast Vma1p sequence. S. cerevisiae, Saccharomyces cerevisiae; N. Crassa, Neurospora crassa; D. carota,
Daucus carota.
), in which yeast cells are not able to grow in media
buffered to pH 7.5 (40, 44, 53). Point mutations that cause a loss of
greater than 80% of wild type activity also result in a vma
phenotype (54, 55). As can be seen in Fig. 2, four of the mutations (G156V, W216A,
R219K, and R219A) led to a full vma
phenotype, three mutations
(G181A, W216F, and P223V) led to a very substantial reduction in growth
at pH 7.5, and four mutations (E164D, E164A, P177V, and G205V) resulted
in a mild growth defect at neutral pH. To determine whether the
observed growth defects were the result of defective assembly of the
V-ATPase, vacuolar membranes isolated from each of the mutant strains
were subjected to SDS-PAGE and analyzed by Western blot using
antibodies against 10 of the 13 V-ATPase subunits. Defects in V-ATPase
assembly are manifested as a reduction of one or more V-ATPase subunits from the vacuolar membrane (56, 57). As seen in Fig. 2, three mutations
(G156V, G181A, and W216A) led to severely defective assembly, whereas
the W216F mutation led to reductions in the levels of several V-ATPase
subunits on the vacuolar membrane. The remaining mutants appear to
assemble normally on the vacuolar membrane.
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Fig. 2.
Effect of mutations in the non-homologous
region on assembly of the V-ATPase complex. Vacuolar membrane
vesicles were isolated from the vma1 strain transformed
with YIp5 alone (Plasmid only), wild type VMA1 in
YIp5 (WT), or VMA1 containing the indicated
mutations in YIp5. The proteins were separated by SDS-PAGE and
transferred to nitrocellulose, and Western blotting was performed using
the indicated subunit-specific antibodies as described under
"Experimental Procedures." For subunits a, A, B, and H, 1 µg of
protein was used; for subunits C, d, and E, 10 µg of protein was
used; for subunits D, F, and G. 20 µg of protein was used. Also shown
is the growth phenotype of each strain on YPD agar plates buffered to
pH 7.5. +++ indicates wild type growth, ++ indicates partially
defective growth, + indicates substantially defective growth, and
indicates almost no growth. Growth was assessed by colony size.
Panel a, mutants G150A to P177V. Panel b, mutants
G181A to P233V.
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Fig. 3.
Effect of mutations in the non-homologous
region on concanamycin A-sensitive ATPase activity and
ATP-dependent proton transport in isolated vacuolar
membrane vesicles. Vacuolar membrane vesicles (10 µg of protein)
were isolated from cells expressing wild type or mutant forms of
subunit A and assayed for both concanamycin A-sensitive ATPase activity
(shaded bars) and concanamycin A-sensitive,
ATP-dependent proton transport (open bars), as
described under "Experimental Procedures." ATPase activity and
proton transport are measured at 0.5 mM ATP and are
expressed relative to values obtained for vacuoles isolated from the
strain expressing wild type subunit A (100%), with each value the
average of measurements on two or three independent vacuole
preparations (error bars correspond to the S.E.). The
specific activity of the concanamycin A-sensitive ATPase activity of
vacuoles containing the wild type subunit A is 0.13 µmol of
ATP/min/mg of protein at 0.5 mM ATP and 23 °C,
which is similar to values previously reported for this strain (55).
Panel a, mutants G150A to P177V. Panel b, mutants
G181A to P233V. Ywt, wild type.
View larger version (56K):
[in a new window]
Fig. 4.
Effect of mutations in the non-homologous
region on in vivo dissociation of the V-ATPase in
response to glucose withdrawal. The vma1 strain
expressing the wild type (WT) or mutant forms of the Vma1p
were grown overnight in YPD media and converted to spheroplasts by
treatment with zymolyase 100T. Spheroplasts were incubated for 40 min
in YEP media with or without 2% glucose at 30 °C (as indicated) and
then lysed in phosphate-buffered saline containing 1% polyoxyethylene
9 lauryl ether, protease inhibitors, and 1 mM
dithiobis(succinimidyl propionate). The V-ATPase complexes were
immunoprecipitated using the monoclonal antibody 13D11 against subunit
B and protein A-Sepharose. The proteins were separated by SDS-PAGE on
8% acrylamide gels followed by transfer to nitrocellulose. Western
blot analysis was performed using the monoclonal antibodies 10D7-A7
against subunit a (Vph1p), 13D11 against subunit B, or 8B1-F3 against
subunit A, as described under "Experimental Procedures."
Dissociation of the V1 and V0 domains is
reflected as a decrease in the amount of subunit a (part of the
V0 domain) immunoprecipitated using the antibody against
subunit B (part of the V1 domain).
View larger version (32K):
[in a new window]
Fig. 5.
Glucose-dependent reassembly of
the V-ATPase after in vivo dissociation. The
vma1 strain expressing the wild type (WT) or
mutant forms of the Vma1p were grown overnight in YPD media and
converted to spheroplasts by treatment with zymolyase 100T. The
spheroplasts were incubated for 20 min in YEP media with (+) or without
(
) 2% glucose. An aliquot of the spheroplasts incubated in the
absence of glucose was then incubated in YEP media with 2% glucose for
an additional 20 min (
+). The V-ATPase was then solubilized with
polyoxyethylene 9 lauryl ether and immunoprecipitated using the
antibody against subunit B followed by SDS-PAGE and Western blot
analysis as described in the legend to Fig 4.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit and the soluble domain of the b subunit
(29, 30). In the V-ATPases, the subunit composition of the peripheral
stalk is less certain, but cross-linking and immunoprecipitation
studies implicate subunits C, E, G, and H as well as the hydrophilic
domain of subunit a in this function (61-64).
and
subunits of
F1 and the available x-ray structural data (25-27) reveals
a very similar overall fold to the polypeptide chains (55, 62, 63, 65)
despite the existence of only about 25% amino acid sequence identity.
The non-homologous region of the V-ATPase A subunit represents a unique
protein domain that is present in all V-ATPase A subunits but which is
absent from the homologous
subunit of F1 (23, 39-41).
Because of the close packing of the
and
subunits in the
hexameric ring of F1 (25-27), the non-homologous region is
likely to form a unique domain located on the outer surface of the
V1 complex. Such a location suggests the possibility that
the non-homologous region may also form part of the peripheral stalk
connecting the V1 and V0 domains. Because of
the critical role of the peripheral stalk in coupling of ATP hydrolysis
to proton transport, it might be imagined that changes in this
structure could lead to changes in coupling efficiency.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Ting Xu, Yoichiro Arata, and Takao Inoue for many helpful discussions.
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FOOTNOTES |
---|
* This work was support in part by National Institutes of Health Grant GM 34478 (to M. F.). E. coli strains were provided through National Institutes of Health Grant DK34928.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.
Present address: Division of Biological Sciences, Institute of
Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan.
§ To whom correspondence should be addressed. Dept. of Physiology, Tufts University School of Medicine, 136 Harrison Ave., Boston MA 02111. Tel.: 617-636-6939; Fax: 617-636-0445; E-mail: michael.forgac@tufts.edu.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M212096200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: V-ATPase, vacuolar proton-translocating adenosine 5-triphosphatase; F-ATPase, F1F0 ATP synthase; MES, 4-morpholineethanesulfonic acid; YPD, yeast extract-peptone-dextrose; YEP, yeast extract-peptone.
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