Biochemical support for the V-ATPase rotary mechanism: antibody against HA-tagged Vma7p or Vma16p but not Vma10p inhibits activity
Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
Author for correspondence (e-mail:
nelson{at}post.tau.ac.il)
Accepted 16 June 2003
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Summary |
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Key words: V-ATPase, subunit, antibody, proton uptake, yeast, lemon, ATPase
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Introduction |
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The F-ATPase was directly shown to work by a rotary mechanism in which
conformational changes in the catalytic sector cause a rotation of the
subunit within it, leading to a counterclockwise turning of the membrane
sector c-ring against the membranous large a subunit. The
latter is held fixed relative to the headpiece by a peripheral stalk
(Noji et al., 1997
;
Omote et al., 1999
;
Sambongi et al., 1999
;
Panke et al., 2000
). A similar
method was recently used to show the rotation mechanism of V-ATPase
(Imamura et al., 2003
;
Yokoyama et al., 2003
;
Hirata et al., 2003
). In order
to support the rotary mechanism of the V-ATPase, and to elucidate the
participating rotating subunits, we used a biochemical approach involving
specific antibodies. In contrast to single molecule studies that frequently
rely on a small fraction of the total molecules, biochemical studies report on
the total population and are able to quantify the proton uptake and ATPase
activities simultaneously.
Antibodies have long been used as a powerful tool in the analysis of
structurefunction relationship in various enzymes and enzyme complexes.
The influence of antibody binding to enzyme or enzyme complex on their
activity can be positive, negative or neutral. The extent of inhibition or
enhancement of activity is a reflection of the nature and distribution of the
various antigenic determinants on the enzyme
(Arnon, 1975). Antibodies are
hydrophilic macromolecules and as such cannot penetrate biological membranes;
this makes them applicable to the study of the topology of membrane proteins
as was done for the F-ATPase c subunit
(Girvin et al., 1989
) and for
the V-ATPase c'' subunit (Vma16p), where it was shown that the C
terminus of Vma16p is cytoplasmic (Nishi
et al., 2001
). In studies of the F-ATPase, monoclonal or
polyclonal antibodies against the various subunits were used. Polyclonal
antibodies against the c subunit of E. coli, recognizing
epitopes on the cytoplasmic side (the loop region), prevented the binding of
F1 to F0 and blocked proton translocation through the
open F0 channel
(Deckers-Hebestreit and Altendorf,
1992
). It was shown that
subunit strongly binds the
subunit, and is located between the F1 and F0 portions
of the enzyme (Fillingame,
1999
; Tsunoda et al.,
2001
). Polyclonal anti-
sera caused near complete inhibition
of the F-ATPase activity (Smith and
Sternweis, 1982
). The
subunit was subsequently shown to be
non-rotary by the use of a monoclonal antibody against it. Binding of
gradually increasing moieties to the
subunit by using the
(Fab)2, complete antibody or a complete antibody with increasing
amounts of the secondary IgG did not change the extent of inhibition, which
was 50% at most; hence leading to the conclusion that rotational catalysis of
that subunit is most unlikely
(Moradi-Ameli and Godinot,
1988
).
When the effect of specific antibodies on V-ATPase was tested, only the
antibody raised against subunit F (Vma7p) inhibited activity. In tobacco
hornworm proton uptake as well as the ATPase activity could be inhibited to
the same extent (Gräf et al.,
1994). In the yeast S. cerevisiae, it was reported that
the ATP-dependent proton uptake was inhibited by anti-HA epitope antibody
added to vacuoles containing HA-tagged Vma7p V-ATPase
(Nelson et al., 1994
). Hence,
the interaction of antibody with the V-ATPase subunits can be used to test the
mobility of the various parts of the enzyme.
Up to now, homologues of Vma16p from plants have not been isolated
(Sze et al., 1999). Here we
report on the cloning of plant cDNAs encoding Vma16p and show that the subunit
lacks the first putative transmembrane span but nevertheless supplements the
yeast Vma16p-null mutant. Implications of these results on the yeast subunit
led to construction of the N-terminal HA-tagged Vma16p, which was used along
with tagged Vma10p and Vma7p V-ATPase subunits in our experiments. In order to
learn more about the structurefunction relationship of the specific
subunits, with respect to the proposed rotary mechanism, the effect of an
anti-HA antibody on V-ATPase containing a tagged subunit was tested. We report
that, in contrast to the HA-tagged Vma10p, the binding of anti-HA antibody to
epitope-tagged Vma7p or Vma16p-containing complexes inhibited both
ATP-dependent proton uptake and ATPase activity.
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Materials and methods |
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Plasmid constructs
The coding region of VMA7, VMA10 and VMA16 genes was
amplified from the yeast genomic DNA by polymerase chain reaction (PCR) with
specific primers. The DNA fragments were cloned into BFG-1 yeast shuttle
vector, which is a high copy number plasmid containing 2-micron (a yeast ori
of replications), LEU2 marker and a 3-phosphoglycerate kinase (PGK)
promoter, followed by three copies of hemaglutinin (HG) epitope, a multiple
cloning site and a PGK terminator. The yeast genes were cloned downstream to
the HA epitope, into the XbaI and EcoRI sites (except
VMA11 that was cloned into EcoRI and XhoI sites),
thus containing the tag in their N terminus. The cDNAs encoding Vma16p from
Arabidopsis thaliana and lemon fruit were cloned into pYES2 shuttle
vector (containing 2-micron and URA3 marker) under the inducible GAL
promoter.
Isolation of VMA16 cDNAs from Arabidopsis thaliana and
lemon fruit
A search in the Arabidopsis thaliana database revealed two clones
encoding Vma16p homologue. The longest one (At4g32530) was amplified by PCR
with specific primers from the Arabidopsis thaliana cDNA library
(ATCC). The degenerative primers used for the isolation of the lemon fruit
Vma16p homologue were prepared according to the deduced amino acid sequence of
the Arabidopsis thaliana clone: forward-AWGIYITG and reverse-NAFGVII.
These primers were used for PCR on the lemon fruit cDNA
(Aviezer-Hagai et al., 2000).
The DNA fragment that was obtained (240 bp) was used to construct specific
primers: forward-GAT GGC ATA TCC AGC TCT TAG and reverse-CAC CTC CAA GAA TCT
CAT CAG TGT. These primers were used by PCR with the lemon fruit cDNA N'
and C' set of primers (see construction of specific primers;
Aviezer-Hagai et al., 2000
) to
obtain overlapping fragments containing the 5' and 3' ends of the
lemon cDNA. For cloning into the pYES2 plasmid, the coding region of the lemon
fruit Vma16p homologue was obtained by PCR with primers containing the
initiator methionine or the stop codon.
Western analysis
Western blots were performed as described by Nelson et al.
(1994). The nitrocellulose
filters were subjected to the ECL amplification procedure (Perkin Elmer Life
Sciences, Boston, USA) and exposed to Kodak X-Omat LS film for 15 min.
The antibodies used in this study were: monoclonal antibodies against the
HA-tag (BabCO, 12CA5 mouse cell line) according to Nelson et al.
(1994
) and Vph1p (10D7-A7-B2;
Molecular Probes, Inc.), both at a dilution of 1:1000 (v/v) and polyclonal
antibodies against Vma5p and Vma1p from guinea pig at a dilution of 1:5000 and
1:1000 (v/v) (Supek et al.,
1994
), respectively. Secondary antibodies were horseradish
peroxidase (HRP)-conjugated sheep anti-mouse Ig (Amersham International,
Uppsala, Sweden), which also served as a primary antibody where indicated, and
HRP-conjugated rabbit anti-guinea pig antibody (Sigma, St Louis, USA) at a
dilution of 1:5000 (v/v).
Preparation of yeast vacuoles
Yeast vacuoles were prepared according to the method of Uchida et al.
(1985) with the required
modifications described by Perzov et al.
(2002
). For yeast strains
transformed with plasmid, the cells were grown overnight in 1 liter of minimal
medium lacking the appropriate amino acid to stationary phase; they were next
diluted to 0.5x absorbance at 600 nm in 5 liters YPD, pH 5.5, and grown
for 45 h. Then the cells were harvested and vacuolar membranes were
isolated as previously described (Supek et
al., 1994
). Specific activity of each preparation was established,
and to assess the effect of antibody on ATPase or proton uptake activity
assays, a comparable amount was taken to give similar basal activities in all
strains tested.
ATPase assay
Vacuolar membrane vesicles containing up to 1.5 mg protein were washed in 2
ml of 10 mmol l1 Tris-HCl, pH 7.5, 1 mmol
l1 EDTA, 2 mmol l1 dithiothreitol (DTT),
0.5 mmol l1 phenylmethylsulfonylfluoride (PMSF), and
recovered by centrifugation (Beckman Ti75, 200 000 g, 30 min).
The pellet was suspended in 500 µl of solubilization buffer containing 10
mmol l1 Tris-HCl, pH 7.5, 1 mmol l1 EDTA,
2 mmol l1 DTT, 0.5 mmol l1, 0.5 mmol
l1 PMSF and 4.5% glycerol. To this suspension the detergent
ZW3-14 was added at a final concentration of 0.5%, and after incubation at
4°C for 15 min (with gentle mixing every 5 min) it was centrifuged at 20
000 g for 30 min. 400 µl of the clear supernatant was
recovered and layered on top of a 20%50% (v/v) glycerol density
gradient containing 10 mmol l1 Tris-HCl, pH 7.5, 1 mmol
l1 EDTA, 2 mmol l1 DTT, 0.5 mmol
l1 PMSF and 0.005% (w/v) ZW3-14. The gradient was separated
by centrifugation in Beckman SW-60 435 500 g, for 5 h. 12
fractions of 0.35 ml each were collected from the bottom of the tube.
Bafilomycin A1-sensitive ATP hydrolysis was tested by measuring the
production of inorganic phosphate, using a modified McCusker et al.
(1987) assay. ATP hydrolysis
was assayed in 0.5 ml reaction mixture at a final concentration of 25 mmol
l1 MOPS-Tris, pH 7, 30 mmol l1 KCl, 50
mmol l1 NaCl, 5 mmol l1 MgCl2
and 5 mmol l1 Na2ATP. Briefly, 50 µl of
gradient fractions were preincubated in 200 µl reaction mixture without
Mg-ATP for 10 min at room temperature in the presence and absence of 4 µmol
l1 (final concentration) Bafilomycin A1. They
were then preincubated for another 1 h at room temperature in the presence and
absence of 50 µl of 1:1000 anti-HA monoclonal antibody (BabCO, 12CA5 mouse
cell line). Next, 10 µl of sonicated asolectin (5 mg ml1)
was added. The reaction was initiated by addition of 250 µl 5 mmol
l1 MgCl2 and 5 mmol l1
Na2ATP, allowed to proceed for 10 min at 30°C and stopped by
addition of 0.5 ml combined stopcolorimetric development reagent
consisting of 5% Fe2SO4, 1% ammonium molybdate and 1 mol
l1 H2SO4. Color development was
allowed to proceed for 15 min at room temperature and was monitored at 660 nm.
The same mixture but without the stop solution was used for the glycerol
gradient fractionation in order to separate the unbound anti-HA antibody and
demonstrate the anti-HA-V-ATPase complex.
ATP-dependent proton-uptake assay
Proton uptake activity was measured following the absorption changes at
490540 nm of Acridine Orange as previously described by Perzov et al.
(2002), with the appropriate
modifications. Various amounts of purified vacuoles were added to the reaction
mixtures (10 mmol l1 MOPS-Tris, pH 7, 15 mmol
l1 KCl, 135 mmol l1 NaCl) to achieve
similar proton pumping activity. From the wild type (WT) strain, 6 µg of
protein were used; from Vma10p-HA, 8 µg; from Vma7p-HA, 40 µg and from
Vma16p-HA, 15 µg were used for this assay. 30 min preincubation at room
temperature followed in the presence and absence of 15 µl of 1:1
diluted anti-HA monoclonal antibody (BabCO, 12CA5 mouse cell line). Next,
Acridine Orange to 30 µmol l1 final concentration were
added followed by 10 µl of 0.1 mol l1 MgATP. The reaction
was terminated by the addition of 1 µl of 1 mmol l1
carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). To
analyze the extent of antibody binding to the holoenzyme, the same vacuolar
preparation was used for glycerol gradient fractionation and the anti-HA
antibody assay was performed (see below).
Anti-HA antibody binding assay
The anti-HA binding to the intact vacuoles was tested in the following way:
200 µl of vacuoles (2 µg µl1) were diluted in
2 ml of the proton uptake assay buffer (see above) and incubated at room
temperature with 50 µl anti-HA monoclonal antibody (BabCO, 12CA5 mouse cell
line) for 30 min. The vacuoles were washed twice (Ti75, 165 000
g, 30 min) and suspended in 200 µl of the same buffer.
Next, the vacuolar vesicles were solubilized by incubation with 0.5% ZW3-14
(zwiterionic detergent), on ice for 15 min, and fractionated for 13 h (to
provide the same conditions as in the ATPase assay; see below), 435 500
g on a glycerol density gradient as detailed above. Anti-HA
binding to solubilized and fractionated vacuoles was tested following the
ATPase assay procedure, but without the addition of the stop solution. The
reaction mixture was then fractionated on a 20%50% glycerol density
gradient (as detailed above) for 13 h at 505 500 g.
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Results |
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Antibody interaction as a tool for structure-function
implications
The use of monoclonal antibodies against tagged V-ATPase subunits in order
to test their effect on the enzyme activity may advance our knowledge of the
proposed rotary mechanism of the enzyme and the structurefunction
relationship of its specific subunits. Binding of a large moiety such as an
antibody (approximately 150 kDa) to the rotating subunits should cause a
steric interference, which might decrease the rate of rotation and hence
inhibit activity. We tested the antibody effect on V-ATPase-containing tagged
Vma16p or Vma7p representing the proteolipid-ring and shaft subunits
(respectively), which are proposed to rotate against the stator subunits of
the enzyme, represented by the tagged Vma10p. For that purpose, we cloned the
yeast VMA16, VMA7 and VMA10 in-frame into BFG plasmid with
the three HA epitopes at their N termini. The resulting plasmids were used to
complement the corresponding yeast null mutants. Vacuoles containing the
tagged V-ATPase complexes were isolated, and the binding of anti-HA antibody
was tested. In general the activity of tagged strains was somewhat lower than
the WT; however, for the various strains variable amounts of the vacuoles were
used to give a similar basal activity in all the assays (see Materials and
methods).
Only if the epitopes are exposed and accessible will the antibodies bind to
them. Alternatively, when the HA-tag is on the lumenal side, detergents are
used, and a prerequisite to anti-HA binding is that the HA-tag will be intact
and not degraded by vacuolar proteases. This problem presented itself when the
Vma3p-HA and Vma11p-HA were tested. Although the total vacuolar preparation
displayed a positive signal with the anti-HA antibody, the glycerol gradient
fractionation showed that the HA containing fractions were not active, and the
active Bafilomycin A1-sensitive ATPase fractions were devoid of
HA-tag (results not shown). It was previously demonstrated
(Hirata et al., 1997) that the
tag on Vma11p-HA remained intact only in pep4
cells. However
the question remains as to why the other inactive fractions maintain their
HA-tag.
Binding of anti-HA antibody to tagged subunits in intact
vacuoles
In order to test the accessibility of the epitope tags to the antibody,
intact vacuoles containing the tagged subunits were preincubated for 30 min
with anti-HA antibody. Next, the unbound antibody was washed twice, and the
membranes were detergent-solubilized and immediately fractionated on a
glycerol density gradient. The distribution pattern of bound antibody and the
various subunit-containing fractions along the gradient were analyzed by
western blot. Anti-Vma5p and Vma1p antibodies were used as markers for the
catalytic sector of V-ATPase, anti-Vph1 antibody as a marker for the membrane
sector, anti-HA antibody as a marker for HA-tagged subunits, and
HRP-conjugated sheep anti-mouse Ig to trace the bound anti-HA antibody. The
heavy fractions positive in all antibodies indicated the presence of the
antibody-holoenzyme complex. As shown in
Fig. 2BDa, in the tagged
Vma7pHA, Vma16pHA and Vma10pHA, bound anti-HA antibody is detected by
anti-mouse Ig in heavier fractions (14). It coincides with the
distribution of all V-ATPase subunit signals
(Fig. 2BD), including
the HA. A small amount of non-specifically bound anti-HA-antibody is traced by
the anti-mouse Ig antibody in the WT (Fig.
2Aa, fractions 6,7); this may be due to insufficient washing of
the anti-HA prior to the application of the samples on the gradient.
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These results indicate that the HA-epitope on the tagged subunits in the
V-ATPase complex is exposed and accessible to the anti-HA antibody, that did
indeed bind to it. Specifically, these results support our previous assumption
regarding the number of TMs in Vma16p (four) and the topology of its N
terminus (cytoplasmic). While this manuscript was in preparation, Nishi et al.
(2003) reported that the
deletion of the first 41 amino acids from the Vma16p N terminus resulted in an
active V-ATPase. They also used cysteine labeling to conclude that the first
presumed helix is located on the cytoplasmic side of the vacuolar membrane
(Nishi et al., 2003
).
Effect of bound antibody on ATP-dependent proton uptake into
vacuoles
Once the binding of the anti-HA antibody was established, we investigated
its influence on ATP-dependent proton uptake activity into intact vacuoles
expressing the tagged subunits. To that end, vacuoles were preincubated with
anti-HA antibody, and proton uptake activity was then tested by following the
change in absorbency of Acridine Orange. As mentioned above, it was previously
shown that anti-HA antibody could inhibit the proton uptake activity of
V-ATPase containing Vma7p with a single HA-tag at its C terminus
(Nelson et al., 1994). As
shown in Fig. 3, the monoclonal
antibody strongly inhibited the proton uptake into vacuoles expressing
V-ATPase containing Vma7p-HA with three HA tags at its N terminus. Similarly,
the antibody inhibited V-ATPase containing Vma16p-HA. The inhibition observed
for both tagged complexes was dose-dependent, although the antibody had a more
detrimental effect on the Vma7p-tagged complex, where 2 µl of antibody
almost completely abolished the proton uptake by Vma7p-tagged complex, whereas
5 µl were needed for the Vma16p-tagged complex. In contrast, the anti-HA
antibody did not inhibit the V-ATPase containing Vma10p-HA, even though the
HA-epitope was retained on this subunit in the active enzyme and the anti-HA
binds to it, as shown in Fig.
2Cb. Actually, the proton uptake activity of WT cells (diluted
twice before the reaction) was slightly accelerated by the anti-HA antibody,
similar to the results obtained by others when non-specific antibody was used
(Gräf et al., 1994
).
These results show that the anti-HA antibody effect on the V-ATPase activity
is genuine and specific to the tagged subunits.
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Effect of bound antibody on ATPase activity of the solubilized
enzyme
In order to test the influence of the anti-HA antibody on the ATPase
activity of the V-ATPase containing tagged subunits, we first solubilized the
isolated vacuoles in order to allow maximal ATPase activity and to avoid
unnecessary background activity. The detergent-solubilized vacuoles were
fractionated on a glycerol density gradient for 5 h (see Materials and
methods). 12 fractions were collected from the bottom and assayed for their
Bafilomycin A1-sensitive ATPase activity. The most active fraction
was further tested for the influence of anti-HA antibody on the ATPase
activity. The samples were preincubated for 1 h at room temperature with an
excess of anti-HA monoclonal antibody in the ATPase reaction mixture without
ATP. Next, MgATP was added and the enzyme was allowed to work for 10 min at
30°C. The same treatment was performed for the control but without the
antibody. Finally, the reaction was stopped and monitored. The Bafilomycin
A1-sensitive ATPase activity of the solubilized vacuolar
preparation of each strain (without the antibody) served as a control (100%
activity) to the same strain's preparation treated with antibody. As shown in
Fig. 4, the anti-HA monoclonal
antibody had no effect on the Vma10p-HA containing V-ATPase complex and even
slightly increased the activity, similar to the WT yeast cells. However, there
was nearly 80% inhibition of the activity of Vma16p-HA containing V-ATPase,
and about 70% of the enzyme expressing the HA-tagged Vma7p, each in comparison
with their respective controls.
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Duplicate samples of the activity assays, without stop solution, were utilized to test the anti-HA binding to its specific tag in the solubilized V-ATPase. These samples were loaded directly on top of a glycerol density gradient and fractionated for 13 h, to allow a better separation of the unbound from the bound antibody as the excess of the unbound antibody was not washed out. 12 fractions were collected from the bottom and analyzed by western blot. The distribution of the HA-tagged subunits was detected by the use of the native anti-HA antibody as a primary antibody, while the pre-bound antibody was detected using an HRP-conjugated sheep anti-mouse Ig in the western analysis. Colocalization of the anti-HA and the anti-mouse Ig antibodies indicated successful antibody binding to the tag in the V-ATPase complex. In all tagged subunits, the anti-HA antibody and the anti-mouse Ig showed a common peak in heavier fractions, in addition to the peak of the non-specific or unbound anti-mouse Ig that was also present in the WT (results not shown).
In summary, the monoclonal antibody was able to bind to the HA-tagged subunits in intact vacuoles as well as in the solubilized enzyme. In the case of the HA-tagged Vma16p and Vma7p, binding of the antibody inhibited both the ATP-dependent proton uptake into vacuoles and the ATPase activity of the V-ATPase enzyme, yet it did not interfere with either activity in the Vma10p-HA containing V-ATPase.
The suggested model for the antibody inhibition
The fact that binding of the antibody to Vma10pHA did not affect the
activity of the enzyme, yet it inhibited the complexes with the other
HA-tagged subunits, supports the suggested rotation mechanism of action in
V-ATPase. By this assumption, Vma16p and Vma7p are part of the rotor and
shaft, respectively, whereas Vma10p is implicated in the peripheral complex,
serving as a stator that fixes the V1 sector to the a
subunit against which the c-ring is rotating.
Fig. 5 is a schematic representation of the subunits involved in the rotation within the ATPase enzyme and the alpha helix packing of the c-ring. In it, the putative TM1 and the C terminus of Vma16p are located on the cytoplasmic face of the membrane. The interference by the antibody bound to the HA epitopes is likely to be due to its collision with one of the other subunits that are attached to the membrane: for example, subunit a, which results in inhibition of rotation, hence the activity inhibition. We suggest that the binding of the antibody to Vma7p-HA inhibited activity by a similar mechanism. On the other hand, although accessible as are the above subunits, Vma10p is static during the catalysis; therefore the bound anti-HA has no effect on its V-ATPase activity.
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Discussion |
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Vma16p has two negatively charged glutamyl residues in the transmembrane
parts, but only the one in TM2 (previously referred to as TM3), the analog of
that in TM4 of Vma3p and Vma11p, is necessary for V-ATPase activity
(Hirata et al., 1997). These
features rendered the c-ring an asymmetric structure, in contrast to
the potentially symmetric structure of the corresponding part in F-ATPase
(Fillingame et al., 2002
).
From our results of antibody binding and those of others (Nishi et al.,
2001
,
2003
), the asymmetry of the
c-ring in V-ATPase is twofold: one is the different subunit
composition and the other is the opposite membrane orientation of subunit
Vma16p as compared to Vma3p and Vma11p. This puts the important catalytic
glutamyl residue of Vma16p in an nonparallel helix. It was proposed that the
special arrangement of the V-ATPase c-ring subunits is also the key
to understanding the inability of the enzyme to reach thermodynamic
equilibrium (Nelson et al.,
2002
; Moriyama and Nelson,
1988
). The lemon fruit is a rare example having very low pH
(pH
2) in the vacuole, where the V-ATPase might be operating close to its
thermodynamic equilibrium (Muller et al.,
1999
, 2002). In summary, even
though there are differences in subunit composition, in catalysis and in
coupling, the general structure of the two ATPases, the F- and V-ATPase, is so
similar that a rotary mechanism of action is the suggested mechanism.
Very recently, several reports have directly demonstrated the rotational
catalysis of single molecules of V-ATPase. In the thermophilic eubacterium
Thermus thermophilus an ATP-dependent, counterclockwise rotation of
beads attached to the D (Vma8p) or F (Vma7p) subunits was demonstrated when
the A subunit (Vma1p) of the V1 sector was immobilized onto a glass
surface (Imamura et al.,
2003). In the same manner of immobilization of the whole complex,
an ATP-dependent rotation of a bead attached to a proteolipid subunit of
Vo was obtained (Yokoyama et
al., 2003
). In the Saccharomyces cerevisiae V-ATPase,
counterclockwise rotation of an actin filament attached to the G (Vma10p)
subunit was observed when the enzyme was immobilized on a glass surface
through the c subunit (Hirata et
al., 2003
). In this report we use an in situ biochemical
approach to support the rotary mechanism. We report results of the effect of
monoclonal anti-HA antibody on the ATP-dependent proton uptake activity of the
tagged V-ATPase population embedded in the vacuolar membrane, and on the
Bafilomycin A1-sensitive ATPase activity of those membranes.
According to the suggested rotary mechanism, the most significant effect of
antibody inhibition on a V-ATPase subunit carrying an HA-tag will be exhibited
in the proteolipid subunits in the Vo sector and in the shaft
subunits of V1. The inhibition of the Vma7p-HA carrying enzyme has
previously been demonstrated (Gräf et
al., 1994; Nelson et al.,
1994
) and we used it as a control in our experiments. When we
tried to tag the membrane subunits Vma3p and Vma11p with the HA-tag, we
discovered that although the HA signal in total vacuolar preparations was
present, the ATPase active fractions did not contain this epitope on them. As
the N- and C-terminal segments of Vma3p and Vma11p are assumed to face the
lumen, the HA-tag was probably degraded by proteolytic activity in the vacuole
(Hirata et al., 1997
).
Therefore the only subunit remaining from the membrane sector for use in our
assay was Vma16p. The fact that the plant Vma16p lacking the first
-helix supplemented the phenotype of the yeast mutant, led us to assume
that the first
-helix of the yeast Vma16p might be outside the vacuole
(as was later demonstrated by Nishi et
al., 2003
) and would be suitable for HA-tagging. Indeed, the tag
on this subunit was retained in the active complex fractions and, as expected,
the enzyme was inhibited by the anti-HA antibody. These results show for the
first time that, by binding an antibody to tagged Vma16p-HA, we can inhibit
the activity of the holoenzyme, in the presence of ATP.
Subunit G (Vma10p) exhibits structural homology to subunit b of
F-ATPase (Supekova et al.,
1995), which forms a stator (peripheral stalk) together with
subunit a, and prevents the
3ß3
catalytic head from rotating when the c-ring and the shaft (subunits
and
) are rotating (Tsunoda
et al., 2001
). The main homology is in the N-terminal half of
these subunits, a major difference being the lack of a transmembrane domain in
subunit G (Supekova et al.,
1996
). Further analysis showed that the N terminus of the G
subunit might fold into an
helix in which one face has highly
conserved residues in both the V1 G and F0 b subunits
(Hunt and Bowman, 1997
).
Because of this structural resemblance, it was suggested that subunit G,
together with subunit E, act like a `hook' or a `stator' similar to b
in F-ATPase (Nelson and Harvey,
1999
; Arata et al.,
2002
). Mutational analysis of the conserved face of the G subunit
helix revealed that several mutations were tolerated and even
stabilized the complex, while others rendered the complex unstable
(Charsky et al., 2000
).
Therefore it was interesting to test the effect of antibody binding to
HA-tagged Vma10p. This binding does take place
(Fig. 2), which means that
subunit G is exposed in the V-ATPase complex, which supports previous results
obtained from studies on accessibility to trypsin cleavage
(Gruber et al., 2000
). The
bound antibody did not inhibit the activities of the Vma10p-HA-containing
V-ATPase (Figs 3,
4) which is in agreement with
Vma10p being a part of the stator, and not participating in the rotor
apparatus, as previously suggested (Nelson
and Harvey, 1999
; Charsky et
al., 2000
; Arata et al.,
2002
). It appears that the G subunit is present at 23
copies per complex (Supekova et al.,
1995
; Hunt and Bowman,
1997
). The large mass of the anti-HA antibody attached to the G
subunits does not interfere with the enzyme's activity because it binds to the
static part of the complex. On the other hand, the fact that the anti-HA
antibody, while bound to Vma7p-HA and Vma16pHA, inhibited both ATP-dependent
proton uptake and ATPase activities (Figs
3,
4), suggests that the two
subunits are located in the rotating segment of the V-ATPase. With this in
mind we suggest the model shown in Fig.
5 that depicts this steric interference of the antibody with the
rotary movement mechanism of the V-ATPase complex.
It would be interesting to test the other V-ATPase subunits by the same
method. Not all the HA-tagged or overexpressed V-ATPase subunits complement
their corresponding null mutants, either because of problems of overexpression
(Curtis and Kane, 2002), or
proteolytic degradation of the tag. Nevertheless we intend to exploit the same
method for analysis of other V-ATPase subunits.
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Acknowledgments |
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References |
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