(Received for publication, October 24, 1996, and in revised form, February 10, 1997)
From the Biology Department, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064
The vacuolar H+-ATPases (V-ATPases) of lemon fruits and epicotyls were detergent-solubilized, purified by column chromatography, and reconstituted into artificial proteoliposomes. During purification, a vanadate- and nitrate-sensitive ATPase activity, consisting of partially disassembled V-ATPase complexes, was resolved from the V-ATPase peak. ATPase and H+-transport activities of the purified, reconstituted V-ATPases of both fruit and epicotyl exhibited similar inhibitor profiles, except that the fruit V-ATPase retained partial vanadate sensitivity. Since the V-ATPase activity of native fruit tonoplast vesicles is insensitive to inhibitors (Müller, M. L., Irkens-Kiesecker, U., Rubinstein, B., and Taiz, L. (1996) J. Biol. Chem. 271, 1916-1924), membrane lipids or other factors may protect the fruit V-ATPase from inactivation in vivo. A kinetic analysis of H+-pumping and H+-leakage indicated that the reconstituted epicotyl V-ATPase exhibited twice as much intrinsic uncoupling or slip as the reconstituted fruit V-ATPase. Comparison of their subunit compositions by SDS-polyacrylamide gel electrophoresis indicated that the reconstituted fruit V-ATPase is enriched in two polypeptides of 33/34 and 16 kDa. Moreover, the stalks of negatively stained juice sac V-ATPases appeared thicker than those of epicotyl V-ATPases in electron micrographs.
The juice sacs of lemon fruits contain cells that can acidify
their vacuoles to as low as pH 2.2 (1). In contrast, the vacuoles of
the surrounding fruit tissues as well as those of vegetative tissues
are maintained in the typical vacuolar pH range, 5.0-6.0. The
occurrence in lemon of two types of vacuoles with vastly different
lumenal pH values provides a convenient experimental system to probe
the mechanisms underlying the control of steady state vacuolar pH. One
hypothesis to explain the extreme acidity of the juice sac vacuoles is
that their H+-ATPase
(V-ATPase)1 is a functionally specialized
isoform capable of generating a greater pH gradient than vegetative
V-ATPases. In an earlier report (2), we compared the ATP-driven
H+-pumping activities of tonoplast-enriched membrane
vesicles isolated from juice sacs and seedling epicotyls. In native
vesicles, the juice sac V-ATPase generated a steeper proton gradient
than the V-ATPase of epicotyls. However, since the epicotyl tonoplast
was more permeable to protons than the juice sac tonoplast, the steeper pH generated by the juice sac V-ATPase may have resulted from differences in membrane permeability rather than from intrinsic properties of the pumps. On the other hand, the two
H+-pumping activities differed with respect to several
kinetic parameters. The epicotyl activity showed a typical V-ATPase
profile with respect to ions and inhibitors (i.e.
stimulation by chloride, inhibition by nitrate, bafilomycin
A1, and N-ethylmaleimide (NEM), and
insensitivity to vanadate). In contrast, the proton pumping activity of
juice sac tonoplasts was insensitive to nitrate, bafilomycin, and NEM, and was partially inhibited by vanadate. Sensitivity of the juice sac
ATPase activity to nitrate and NEM increased following detergent treatment, consistent with the juice sac proton pump's identity as a
V-ATPase. However, evidence for the possible existence of a second
H+-ATPase on the juice sac tonoplast was also obtained. In
nitrate-induced V1-dissociation experiments, the epicotyl
vacuolar H+-pumping activity became inactivated with the
release of the catalytic subunit from the membrane. Despite the loss of
a major portion of the catalytic subunit, the juice sac membranes
retained 100% of their H+-pumping activity following
nitrate treatment, although vanadate sensitivity increased (2).
Solubilization of the fruit membranes with
n-dodecyl-
-D-maltoside and centrifugation on
a glycerol gradient resulted in the resolution of two peaks of ATPase
activity. The denser of the two peaks was strongly inhibited by
nitrate, partially inhibited by vanadate, and exhibited a typical
V-ATPase subunit composition on SDS-PAGE gels. The second peak was a
vanadate- and nitrate-sensitive ATPase of unknown identity (2).
It appears that the V-ATPases of lemon fruits and epicotyls may be strongly influenced by their native membranes. To compare the two proton pumps in the same membrane environment we have characterized the properties of purified and reconstituted V-ATPases from fruits and epicotyls. In addition, we have compared negatively stained juice sac and epicotyl tonoplast vesicles by electron microscopy. Our results suggest that native tonoplast lipids of the juice sacs play important roles not only in reducing proton permeability, but in protecting the V-ATPase from inactivation by inhibitors. However, the ability to generate a steeper pH gradient appears to be an intrinsic property of the juice sac V-ATPase. The nature of the vanadate-sensitive "second H+-ATPase" remains unresolved, but may represent partially disassembled V-ATPase complexes.
Lemon seeds (Citrus limon L. var.
Schaub Rough Lemon) were generously supplied by Willits & Newcomb,
Inc., Arvin, CA. Lemon fruits (var. Eureka) were harvested from trees
on the campus of the University of California, Santa Cruz. Bafilomycin
A1 was from Sigma, BCA protein assay reagents were from
Pierce, and n-dodecyl--D-maltoside was from
Calbiochem. Escherichia coli polar lipid extract was purchased from Avanti Polar Lipids; the NanoOrangeTM
protein quantitation kit and 9-amino-6-chloro-2-methoxy-acridine (ACMA)
were from Molecular Probes. All bulk chemicals were purchased from
Sigma and Fisher.
Tonoplast-enriched membranes from lemon fruit juice sacs and epicotyls were prepared as described previously (2). All steps were carried out at 4 °C, and the membranes were maintained on ice. Briefly, juice sacs of three lemons were released into 100 ml of cold fruit homogenization buffer (1.5 M MOPS-KOH, pH 8.5, 2.25% polyvinylpyrrolidone-40, 0.75% bovine serum albumin, 7.5 mM EDTA, 2 mM DTT, and 0.1 mM PMSF). They were ground using a mortar and pestle or, alternatively, homogenized with a Waring blender in a 250-ml flask filled to the top with juice sacs and homogenization buffer, and hermetically sealed to avoid oxidation. Epicotyls (40 g, fresh weight) were harvested with a razor blade and homogenized in 150 ml of cold epicotyl homogenization buffer (0.5 M MOPS-KOH, pH 8.5, 1.5% polyvinylpyrrolidone-40, 0.5% bovine serum albumin, 5 mM EDTA, 2 mM DTT, and 0.1 mM PMSF) using a mortar and pestle. Homogenates were filtered through a 0.28-mm nylon mesh and centrifuged at 12,000 × g for 15 min (Sorvall SS-34 rotor) to eliminate cellular debris, nuclei, and plastids. The supernatant was subjected to ultracentrifugation for 60 min at 132,000 × g in a Beckman SW-28 rotor. The microsomal pellet obtained was resuspended in 15 ml of resuspension buffer (RB; 10 mM BTP-Mes, pH 7.0, 20 mM KCl, 1 mM EDTA, 2 mM DTT, and 0.1 mM PMSF) and further purified on a 10%/35% sucrose step gradient made up in 10 mM BTP-Mes, pH 7.0, 10% glycerol, 20 mM KCl, 1 mM EDTA, 2 mM DTT, and 0.1 mM PMSF. After 60 min of centrifugation at 132,000 × g in a Beckman SW-28.1 rotor, the 10%/35% interface containing tonoplast-enriched membranes was recovered, diluted with RB, and pelleted for 20 min at 174,000 × g in a Beckman TLA-100.3 rotor. The tonoplast-enriched membranes were resuspended in RB at a final concentration of 10 µg of membrane protein/µl. In experiments involving inhibition by NEM, the membranes were resuspended in RB in the absence of DTT.
Membrane Solubilization and Partial Purification of ATPase Activities by Size Exclusion ChromatographyTonoplast-enriched
membranes were made up to 6 mg of protein/ml with RB and solubilized as
described previously (2) with an equal volume of 4% (w/w)
n-dodecyl--D-maltoside or 5% (w/w) octyl-
-glucoside in solubilization buffer (10 mM
BTP-Mes, pH 7.6, 10% glycerol, 1 mM EDTA, 8 mM
MgSO4, 50 mM DTT, 200 µg/ml L-
-phosphatidylcholine liposomes, and 0.012% butylated
hydroxytoluene). The solubilized membrane proteins were centrifuged for
15 min at 412,000 × g in a Beckman TLA-100.3 rotor,
and the supernatant was loaded on a 100 × 1-cm Sephacryl S-400 HR
(Pharmacia) chromatography column equilibrated in running buffer (10 mM Tris-Mes, pH 7.0, 0.3% (w/w) Triton X-100, 100 µg/ml
L-
-phosphatidylcholine, 10% glycerol, 1 mM EDTA, 4 mM MgCl2, 5 mM DTT, and 50 µM PMSF). The column was
eluted at 4.5 ml/h with running buffer. 1.5-ml fractions were collected
and assayed for ATPase activity in the presence or absence of vanadate
and nitrate.
After Sephacryl S-400 HR
chromatography, fractions making up the leading half of the V-ATPase
activity peak were pooled and further purified by Econo-Q anion
exchange (Bio-Rad). The proteins were loaded at 0.5 ml/min on a 5-ml
column equilibrated with Q buffer (QB; 5 mM Tris-HCl pH
6.0, 50 µg/ml [l--phosphatidylcholine, 10% glycerol, 1 mM EDTA, 4 mM MgCl2, 5 mM DTT, and 50 µM PMSF) and eluted with a
linear gradient of 0-0.3 M KCl. 1-ml fractions were
collected and assayed for ATPase activity in the presence or absence of
vanadate and nitrate. Both the lemon fruit and epicotyl preparations
showed an activity peak eluting at ~0.1 M KCl.
Additionally, the fruit preparation exhibited an equally nitrate- and
vanadate-sensitive activity peak eluting at ~0.065 M KCl.
The fractions making up the peak eluting at 0.1 M KCl were
diluted three times with QB, loaded again on an Econo-Q column, and
subjected to a second gradient elution. For reconstitution experiments,
ATPases bound to this second Econo-Q column were eluted with a 0.2 M KCl step to concentrate the activity in one fraction.
For chromatography elution buffers and
ATPase assays, L--phosphatidylcholine type IV-S (Sigma)
was dissolved to 10 mg/ml in a total volume of 10 ml of diethyl ether,
evaporated to dryness under a stream of nitrogen, lyophilized,
resuspended in 10 ml of water, and sonicated to clarity with a
Braun-Sonic U probe sonicator. For reconstitution experiments, 5 ml of
a 20 mg/ml E. coli polar lipid extract in chloroform (Avanti
Polar Lipids) was evaporated to dryness, resuspended together with 17.6 mg of cholesterol in 2 ml of diethyl ether, evaporated to dryness
again, lyophilized, resuspended in 2.35 ml of RB, and sonicated to
clarity. The liposomes (final concentration, 50 mg/ml) were aliquoted, frozen in liquid nitrogen, and stored at
20 °C. Immediately before being used, the liposomes were thawed and sonicated to clarity again.
The reconstitution procedure was based on the method of
Ward and Sze (3). 200 µl of 50 mg/ml E. coli/cholesterol
liposomes were added to the 0.2 M KCl step eluate of the
Econo-Q column containing the partially purified V-ATPase (1-ml
fraction, activity 0.08-0.35
µmol·ml1·min
1). The mixture was
incubated on ice for 30 min, frozen in liquid nitrogen, and thawed
again. 0.4 g of wet Bio-Beads SM-2 (Bio-Rad) prepared according to
Holloway (4) were added to the mixture, which was incubated for 30 min
at room temperature with gentle rocking. The beads were decanted, and
the supernatant was recovered and mixed with another 0.4 g of wet
Bio-Beads. The same incubation process was repeated, and the
supernatant was again recovered. 100 µl of additional E. coli/cholesterol liposomes were added to the reconstitution
mixture, which was again allowed to sit for 30 min on ice. The
reconstituted proteoliposomes were then diluted to a final volume of 10 ml with RB, incubated for 20 min at room temperature, and centrifuged
for 20 min at 174,000 × g (Beckman TLA-100.3). The
pellet containing the reconstituted vesicles was resuspended in 300 µl of RB containing 150 mM KCl. The sizes of the fruit
and epicotyl proteoliposomes were determined to be identical by
freeze-fracture electron microscopy (data not shown).
Proton pumping by tonoplast vesicles and reconstituted proteoliposomes was monitored by quinacrine or ACMA fluorescence quenching. The reaction mix contained 10 mM BTP-Mes, pH 7.0, 250 mM sorbitol, 100 mM KCl, 1 mM azide, 250 nM valinomycin, 2.5 mM ATP, and either quinacrine (10 µM) or ACMA (1.5 µM). For tonoplast-enriched vesicles, 50 µM vanadate was included in the mix. 100 µg of tonoplast-enriched membrane protein or 0.4-1.0 µg of reconstituted proteoliposomes were typically used, and the reaction was started with 4.5 mM MgSO4. Fluorescence quenching (quinacrine: 423 nm excitation, 502 nm emission wave lengths; ACMA: 430 nm excitation, 500 nm emission) was measured in a Perkin-Elmer LS-5 fluorescence spectrophotometer (Perkin-Elmer Corp.).
Calculation of Slip Rate ConstantsUncoupling or "slip" rates were estimated based on a kinetic model described by Tu et al. (7). According to this model, the proton pumping rate at any given time point during the formation of the gradient can be represented by the following equation,
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
![]() |
(Eq. 4) |
![]() |
(Eq. 5) |
ATP hydrolysis measurements were carried out
in a reaction mix containing 2.5 mM ATP, 4.5 mM
MgSO4, 100 mM KCl, 1 mM azide, 1 mM molybdate, 2 µM gramicidin, and 1 mg/ml
sonicated L--phosphatidylcholine liposomes in 25 mM BTP-Mes buffer, pH 7.0. The total reaction volume was
500 µl, and the reaction was started by adding the enzyme to the mix.
After 30 min at 37 °C, the reaction was stopped by adding 1.25 ml of
Fiske and Subbarow (5) reagent. After 30 min at room temperature,
absorbance of the samples at 660 nm was measured in a Spectronic
Genesis 5 spectrophotometer (Milton Roy, Rochester, NY). Boiled
membranes were used for background estimates. Where nitrate-sensitive
or vanadate-sensitive activity is reported, the results are expressed
as the difference in activity in the presence or absence of 400 mM KNO3 or 400 µM
Na3VO4, respectively.
Protein concentrations were measured routinely by a modified BCA protein assay (6) or with the NanoOrangeTM protein quantitation kit after precipitation of the proteins with cold acetone and delipidation with diethyl ether.
Gel ElectrophoresisSDS-PAGE was according to Laemmli (8) in 12 or 13.5% polyacrylamide gels. The samples were made up in sample buffer to a final concentration of 60 mM Tris-HCl, pH 6.8, 4% SDS, 5% DTT, 10% glycerol, and 0.0125% bromphenol blue. The gels were developed with silver.
Electron MicroscopyThe electron microscopy experiments were conducted at the laboratory of Prof. Ulrich Lüttge in Darmstadt, Germany. Tonoplast-enriched membranes were pelleted and resuspended at room temperature to a final concentration of ~1 mg/ml protein in 10 mM potassium phosphate buffer, pH 7.0, containing 5 mM ATP. Negative staining was performed with a solution of 2% methylamine tungstate according to the successive droplet method (9). A 5-µl droplet of membrane suspension was applied to a Formvar-coated 700-mesh/hexagonal grid. After 2 min, the droplet was wicked off with filter paper and replaced with a 5-µl droplet of 2% methylamine tungstate. After 15-20 s the stain was also wicked off, and the grid was allowed to dry. Specimens were examined and photographed with a Zeiss EM902 electron microscope (Carl Zeiss, Oberkochen, Germany) operated at 80 kV in the electron filter mode.
As a first step in the purification, detergent-solubilized
tonoplast-enriched membranes from epicotyls and fruits were layered onto a Sephacryl S-400 HR column, and the protein and ATPase activities were monitored. The protein distribution and ATPase activity profiles are shown in Fig. 1. An octyl--glucoside
solubilization resulted in a single peak of ATPase activity
corresponding to a molecular mass of about 4,500 kDa for both fruit and
epicotyl membranes (Fig. 1, A and B). Analysis of
the fractions by SDS-PAGE indicated that the peak fractions were
enriched in subunits for the V-ATPase (data not shown). The high
molecular mass of the complex indicated that the V-ATPase was migrating
as an aggregate. The epicotyl peak was inhibited by nitrate only,
whereas the fruit peak was sensitive to both nitrate and
vanadate.
If n-dodecyl--D-maltoside was used to
solubilize the membranes, two peaks of ATPase activity were obtained
for both fruit and epicotyl membranes, a nitrate-sensitive V-ATPase
activity peak, which migrated either as a 4,500-kDa aggregate as in the octy-
-glucoside experiment (Fig. 1D), or as a lower
molecular mass aggregate of about 1,500 kDa (Fig. 1C), and a
second peak with an apparent molecular mass of about 250 kDa (Fig. 1,
C and D). The second peak was both nitrate- and
vanadate-sensitive. Treatment of the octyl-
-glucoside peak fractions
with n-dodecyl-
-D-maltoside did not induce
the appearance of the second peak (data not shown). Hence the second
peak is not a degradation product of the first peak, but appears to be
specifically solubilized from the membrane by
n-dodecyl-
-D-maltoside.
The Sephacryl S-400 HR V-ATPase peak fractions from the
n-dodecyl--D-maltoside solubilization were
further purified on two successive Econo-Q anion exchange columns.
After a first passage over the column, both the fruit and the epicotyl
V-ATPases showed an activity peak eluting at 0.1 M KCl.
This peak was nitrate-sensitive and vanadate insensitive in
the epicotyl preparation, and nitrate-sensitive and partially
vanadate-sensitive in the case of the fruit. In addition, the fruit
preparation exhibited a second peak of activity at 0.065 M
KCl that was inhibited equally by nitrate and vanadate (data not
shown).
Both the single epicotyl ATPase activity peak and the fruit peak eluting at 0.1 M KCl contained typical V-ATPase subunits when analyzed by SDS-PAGE. The nitrate- and vanadate-sensitive fruit ATPase activity peak eluting at 0.065 M KCl appeared to co-purify with selected V-ATPase subunits rather than with any specific polypeptides. However, the presence of a low abundance contaminant with high ATP hydrolytic activity cannot be ruled out.
When the fractions making up the more nitrate-sensitive activity peak
were pooled and further purified on a second Econo-Q column, further
separation of the vanadate-sensitive from the nitrate-sensitive
activities was achieved (Fig. 2). The peak of maximum
nitrate-sensitive activity was further enriched in the complete set of
V-ATPase subunits and was depleted in contaminating bands, mainly a 100 kDa polypeptide (Fig. 2B, fraction 40). Bands at
97, 66, 55/56, 52, 42/43, 36, 33, 31, 17, 14, and 13 kDa co-migrated with the peak of activity. Most notably, the doublet at 33/34 kDa was
present only in the fruit preparation, and only the 33-kDa component of the doublet co-migrated with the more nitrate-sensitive activity peak. In most experiments, a 16-kDa band also co-migrated with
the nitrate-sensitive activity peak, although it appears to be shifted
to fraction 42 in the gel of Fig. 2B.
The nitrate- and vanadate-sensitive ATPase activity peak eluting at 0.065 M KCl was enriched in the 97- and 36-kDa bands, and in the 55/56-kDa doublet. The 33/34-kDa doublet was present, but only the 34-kDa component of the doublet co-migrated with the peak of nitrate- and vanadate-sensitive activity. The 66-kDa polypeptide (V-ATPase catalytic or A subunit) was also present, although in reduced amounts compared with the 55/56-kDa doublet (V-ATPase "regulatory" or B subunit). The strong doublet at 25/26 kDa, present in all fractions eluting from both Econo-Q columns, did not show a consistent pattern of co-migration with any of the two activities and is therefore thought to represent a contaminant.
The specific activities of the purified V-ATPases (average ± S.D.
of four purifications) were 9.5 ± 1.5 µmol of
Pi·mg1·min
1 and 6.9 ± 2.2 µmol Pi·mg
1·min
1 for
the epicotyl and fruit, respectively. The sensitivities of the two
purified V-ATPases to various inhibitors is shown in Fig. 3. Both V-ATPases were about equally inhibited by
nitrate (Fig. 3A), bafilomycin (Fig. 3B), and NEM
(Fig. 3C). In contrast, only the fruit V-ATPase showed
partial inhibition by vanadate (Fig. 3D). Fig. 3D
also shows that the fractions making up the V-ATPase peak after S-400
HR chromatography progressively lost their vanadate sensitivity with
subsequent Econo-Q column purifications. This indicates either that the
vanadate sensitivity is associated with a contaminating ATPase, or that
a subpopulation of V-ATPases, perhaps partial V1 complexes,
exhibit nitrate- and vanadate-sensitive ATPase activity. The latter
hypothesis is supported by the Econo-Q activity profiles and gels in
which the main vanadate-sensitive peak exhibited a subunit composition
compatible with that of a V1 complex partially depleted of
its catalytic subunit (Fig. 2).
Partially purified fruit and epicotyl V-ATPases from a step elution of the second Econo-Q column (see "Experimental Procedures") were reconstituted into artificial proteoliposomes, and their proton pumping activities were compared. When the proton gradients had stabilized, the reactions were stopped by adding EDTA, allowing the pH gradients to collapse due to proton leakage.
In Fig. 4, the upper panel shows six
different comparisons based on five different experiments (an epicotyl
trace is shown twice in B and E, and a fruit
trace is shown twice in A and B). Panels
A and C represent equal protein concentrations. The
proteoliposome concentrations in Fig. 4, B and C,
were chosen to give equal initial rates of proton pumping. In Fig. 4,
D and E, the protein concentrations were
normalized to generate equal fluorescence quenching at equilibrium. In
Fig. 4E the proteoliposomes also displayed equal proton
leakage rates. Fig. 4F represents aged proteoliposomes with
leaky membranes.
When equal protein concentrations of freshly prepared proteoliposomes were used (Fig. 4, A and C) or when the concentrations of reconstituted fruit and epicotyl proteoliposomes were adjusted to yield equal initial rates of proton pumping (Fig. 4, B and C), the reconstituted fruit V-ATPase consistently generated a steeper pH gradient than the reconstituted epicotyl enzyme. When the proteoliposome concentrations were adjusted so as to build up equal pH gradients at equilibrium, the initial rate of pumping by the fruit V-ATPase was lower than that by the epicotyl enzyme (Fig. 4D). This latter result was obtained even when the proteoliposomes exhibited equal leakage rates (Fig. 4E).
Leakage and intrinsic uncoupling or "slip" rates were estimated according to Tu et al. (7) as detailed under "Experimental Procedures." For each of the curves shown in Fig. 4, A-F, the rate constants ki, k3, and k2 are given in the table in the lower panel. If all five fruit and epicotyl traces are considered, the average leakage rate constants for the fruit and epicotyl proteoliposomes are approximately equal, 0.047 ± 0.029 and 0.045 ± 0.011, respectively. In contrast, the slip rate constants average 0.341 ± 0.121 for the fruit V-ATPase, and 0.687 ± 0.212 for the epicotyl enzyme. The average epicotyl/fruit slip ratio is 2.0 ± 0.3. These values were obtained by considering the initial third of the proton pumping curves and the second half of the leakage curves. If the entire curves were included in the calculations, the epicotyl/fruit slip ratio averaged 2.4 ± 0.4. Note that the slip rate of the reconstituted epicotyl V-ATPase was higher than that of the fruit enzyme under every condition tested.
The reconstituted proteoliposomes containing purified fruit and
epicotyl V-ATPases exhibited similar polypeptide profiles, as shown in
Fig. 5. Both had bands at 66, 55/56, 52, 42, 36, 31, 17, 16, and 13 kDa. In addition, the fruit V-ATPase contained bands at 100 and 78 kDa, as well as a doublet at 33/34 kDa. The 100- and 78-kDa
bands by themselves had no ATPase activity as shown by the second
Econo-Q profile (Fig. 2). Quantitative differences were also observed.
For example, the fruit enzyme was strongly enriched in a 16-kDa
polypeptide, it was slightly depleted in the catalytic subunit (66 kDa), compared with the epicotyl, and had a more pronounced doublet at
55/56 kDa.
Table I shows the sensitivities of the two reconstituted proton pumps to nitrate, bafilomycin, and NEM. Proton pumping by the reconstituted fruit V-ATPase was slightly less sensitive to nitrate and NEM than the epicotyl V-ATPase, especially at low concentrations, but it was as sensitive as the epicotyl V-ATPase to bafilomycin. The fruit V-ATPase also retained its partial sensitivity to vanadate (Fig. 6A). Because the fruit proteoliposomes were 100% sensitive to low concentrations of bafilomycin, the vanadate sensitivity of the pump cannot be due to a contaminating P-type ATPase (Fig. 6B). H+-pumping by the reconstituted epicotyl V-ATPase was completely insensitive to vanadate. Furthermore, the fruit V-ATPase, which was insensitive to oxidation in its native membrane (2), was now as prone to oxidation as the epicotyl V-ATPase (Fig. 7), and the inhibition could be partially reversed by 50 mM DTT.
|
Fig. 8 shows electron micrographs of tonoplast enriched
membrane fractions from lemon fruits and epicotyls, negatively stained with methylamine tungstate. Both membranes showed the typical ball-and-stalk structures of the vacuolar type H+-ATPases
previously described (9-11). Although the hydrophilic portion of both
complexes were roughly comparable in size, the stalk portions of the
epicotyl V-ATPases were barely visible, whereas those of the fruit
V-ATPases were quite prominent (see lower insets). The
thicker stalks of the fruit V-ATPases may reflect the presence of
additional subunits. Alternatively, the thinner stalk of most epicotyl
V-ATPases may represent artifactual loss of subunits during negative
staining.
The proton pumping activity of native tonoplast-enriched membrane
vesicles from lemon juice sacs exhibits an unusual insensitivity to
inhibitors of V-ATPases, including nitrate, bafilomycin, NEM, and
oxidation (2). Nevertheless, juice sac tonoplasts contain an authentic
V-ATPase that, when purified and reconstituted into artificial
liposomes, exhibit properties similar to those of other eukaryotic
V-ATPases. As in the case of the epicotyl V-ATPase, proton pumping and
ATPase activities of the reconstituted juice sac V-ATPase were
inhibited by nitrate, NEM, bafilomycin, and oxidation. These results
suggest that native membrane lipids play an important role in
protecting the fruit enzyme from inactivation in vivo. We
have previously shown that the juice sac tonoplast is less permeable to
protons than the tonoplast of epicotyls (2). Preliminary membrane
viscosity measurements indicate that the juice sac tonoplast is more
rigid than that of the epicotyl.2 Thus, the
specialized lipid composition of the juice sac tonoplast apparently
serves two important roles, reducing proton permeability and protecting
the V-ATPase against inactivation. Although the purified, reconstituted
fruit V-ATPase, unlike the V-ATPase in the native membrane, exhibited
an inhibitor profile similar to that of the epicotyl, it differed in
two respects: 1) it retained its sensitivity to vanadate, and 2) it
pumped protons with twice the efficiency, i.e. half the slip
rate, of the epicotyl V-ATPase. The latter observation suggests that
intrinsic structural features of the fruit V-ATPase are also important
determinants of the in vivo equilibrium pH.
During the course of purification, the chromatography fractions corresponding to the fruit V-ATPase progressively lost much of their vanadate-sensitive ATPase activity. The vanadate-sensitive activity that could be separated from the more nitrate-sensitive peak appeared to be composed of partial V-ATPase complexes, although we cannot rule out that an undetected minor contaminant, unrelated to the V-ATPase, may also be responsible for this activity. The most consistent feature of the vanadate-sensitive fractions was their depletion in 66-kDa (catalytic) subunit relative to the 55/56-kDa (regulatory) subunit. Interestingly, fractions depleted in the 66-kDa subunit were also enriched in a 34-kDa polypeptide, which may indicate that the 34-kDa band represents a breakdown product of the catalytic subunit. However, in contrast to the recent finding that a 35-kDa polypeptide cross-reacted with antibody to the catalytic subunit in salt stressed Citrus sinensis leaves (13), our 34 kDa polypeptide did not cross-react with antibody to the catalytic subunit (data not shown). The significance of the presence in juice sacs of a partial V-ATPase with vanadate-sensitive activity is unclear. Although it may represent a preparation artifact, it is interesting to note that native juice sac vesicles also showed a 30% inhibition of their proton pumping activity by vanadate (2). Moreover, we have recently determined that the octyl-glucoside-solubilized V-ATPase from acid lime juice sacs (pH 2.5) exhibits a single nitrate- and vanadate-sensitive activity peak after partial purification on Sephacryl S-400 HR, as the V-ATPase from lemon juice sacs. In contrast, the V-ATPase peak from sweet lime juice sacs (pH 6.0) was inhibited exclusively by nitrate, similar to the lemon epicotyl.3 Thus, the vanadate-sensitive ATPase activity is not merely a specific property of juice sacs, but is correlated with low vacuolar pH. It is proposed that a subpopulation of partially disassembled V-ATPases is normally present on the tonoplast of acidic juice sacs. These partially disassembled V-ATPases retain ATP hydrolytic activity which is more vanadate-sensitive than that of intact V-ATPases, and possibly carry out proton pumping as well. Further studies are needed to confirm this point.
It is unlikely that the partial vanadate sensitivity associated with the lemon fruit V-ATPase is related to a P-type inhibition mechanism for three reasons: 1) vanadate concentrations required for maximal inhibition of the juice sac V-ATPase were higher than those needed for inhibition of P-type ATPases; 2) the purified, reconstituted fruit V-ATPase was completely inhibited by bafilomycin; and 3) inhibition by vanadate has also been observed in other V-ATPases such as those from osteoclasts (14), yeast, chromaffin granules (15), and plants (16). So far, none of the nucleotide sequences obtained from these materials have shown a P-type motif in their catalytic site (17, 18). However, as shown by a recent report by David et al. (19), vanadate inhibition of the chicken kidney V-ATPase was dependent on the presence of ADP and was suggested to involve the formation of a vanadate-ADP complex at a nucleotide binding site. Although extremely high concentrations of vanadate were needed to inhibit the chicken kidney V-ATPase (IC50 = 1.58 mM), much higher than those used in this study, a similar mechanism could apply in the case of the lemon fruit and may be favored by the partial dissociation of the catalytic complex. This would be consistent with our previous observation that the proton pumping activity of tonoplast-enriched vesicles from lemon fruits became increasingly sensitive to vanadate after nitrate- and cold-induced release of the catalytic subunit. Although it is generally assumed that the whole catalytic complex is dissociated by treatment with chaotropic agents, the overall unchanged proton pumping activity measured with nitrate-treated lemon fruit vesicles implies that the enzyme remains functional despite the partial loss of subunits (2). Alternatively, it could be that a second nitrate-insensitive proton pump on the membrane becomes activated in response to a reduction in the membrane potential resulting from the inactivation of the V-ATPase. This second, vanadate-sensitive proton pump would thus compensate for the loss of the V-ATPase activity and maintain the total proton pumping activity unchanged.
Based on a measured H+/ATP stoichiometry of 2 (20-22), the
maximum pH that a V-ATPase can generate under typical physiological conditions is around 4 pH units (23). This is sufficient for a fully
coupled V-ATPase to reach a vacuolar pH of 2.5, provided it is
operating at thermodynamic equilibrium and the cytosol is slightly
acidified to pH 6.5. This latter condition seems to be fulfilled in the
mature lemon fruit as suggested by our preliminary 13C NMR
measurements.4 In contrast to the fruit,
the
pH built up across the epicotyl tonoplast is only ~2 units,
which indicates that the vegetative pump operates far from
thermodynamic equilibrium. Thus, the epicotyl V-ATPase, like other
typical V-ATPases of animals, plants, and fungi, is subject to some
type of kinetic inhibition. Moriyama and Nelson (24) have proposed that
V-ATPases are regulated by intrinsic uncoupling, or slip. We previously
provided evidence that H+-transport and ATP hydrolysis by
the epicotyl V-ATPase can be partially uncoupled in the absence of
chloride or in the presence of nitrate, whereas the juice sac V-ATPase
remains tightly coupled under these same conditions (2). Our
proton-pumping results with reconstituted proteoliposomes now confirm
that the juice sac V-ATPase is more tightly coupled than that of the
epicotyl, consistent with the slip model. According to our calculations based on the kinetic model described by Tu et al. (7), the slip ratio is 2.0-2.4 times higher for the epicotyl V-ATPase than for
the fruit enzyme.
What structural features of the juice sac V-ATPase might prevent slip, allowing it to generate a steeper pH gradient than the epicotyl V-ATPase? The subunit compositions of the two enzymes, as reflected in highly purified, delipidated preparations, are similar, except for a 33/34-kDa polypeptide doublet which was present in the fruit only, as well as some minor bands in the 20-30-kDa range (data not shown). The juice sac V-ATPase is also slightly depleted in the catalytic subunit, a feature that correlated with vanadate-sensitivity. Because the 33/34-kDa doublet was the predominant difference between subunit compositions of the two enzymes, it is possible that it represents an important structural feature of the juice sac V-ATPase which inhibits slip. In addition, bands at 100 and 86 kDa were present only in the reconstituted fruit preparation. The 100-kDa band represents a contaminant that is distinct from the 97-kDa integral membrane subunit identified in most V-ATPase preparations. Its presence is a consequence of the step elution used in reconstitution experiments to concentrate the V-ATPases attached to the second Econo-Q column. A linear KCl gradient elution of the column showed that this 100 kDa polypeptide did not co-migrate with the V-ATPase and that it did not exhibit any ATP hydrolytic activity (Fig. 2B). The 86-kDa polypeptide was not usually visible in gels of reconstituted fruit enzymes and may represent a complex of two or more subunits.
The dark 16-kDa band visible below the 17-kDa proteolipid in the fruit preparation was strongly enriched in six out of 10 fruit proteoliposome preparations and therefore appears to be characteristic of the reconstituted juice sac V-ATPase (the proteins of the other four preparations were run on SDS-polyacrylamide gels which did not resolve the low molecular weight bands). Although the function of this 16-kDa band is unknown, it is tempting to speculate that, together with the 33/34-kDa doublet, it is involved in tighter coupling of the fruit enzyme. A V-ATPase polypeptide, isolated from bovine chromaffin granules, and migrating on SDS-PAGE to an apparent molecular mass of 16 kDa, has recently been shown to be homologous to subunit b of the F0 complex (12). Since the b subunit in F-ATPases connects the catalytic subunit with the a subunit of the channel, it has been implicated in the coupling between the ATP-hydrolytic and proton transport sites (12). If the heavily stained 16-kDa band present in the reconstituted fruit enzyme were related to the b subunit of F-ATPases, its occurrence in multiple copies in the reconstituted fruit V-ATPase could explain the tighter coupling of the enzyme and its ability to build up a higher pH gradient across the membrane.
In electron micrographs of negatively stained tonoplast preparations, the "stalk" portion of the V1 complex of the juice sac V-ATPase appeared to be thicker than that of the epicotyl V-ATPase. This is consistent with the proposal that the catalytic complex of the fruit enzyme is more firmly anchored to the membrane than that of the epicotyl V-ATPase. Multiple copies of a "bridging" subunit might also account for the apparent presence in the fruit tonoplast of partially disassembled V-ATPases capable of both ATP hydrolysis and proton pumping. Finally, the increased stability afforded by a reinforced stalk might explain the failure of nitrate to inhibit proton pumping in native vesicles (2), despite the loss of a major portion of its catalytic subunits.
We thank Deborah Bailey and Kira Steinberg for technical assistance in the preparation of the membranes. We also gratefully acknowledge the invitation of Dr. Rafael Ratajczak and Prof. Ulrich Lüttge to carry out the electron microscopy experiments at Darmstadt.