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(Received for publication, June 28,
1995; and in revised form, November 14, 1995) From the
Lemon fruit vacuoles acidify their lumens to pH 2.5, 3 pH units
lower than typical plant vacuoles. To study the mechanism of
hyperacidification, the kinetics of ATP-driven proton pumping by
tonoplast vesicles from lemon fruits and epicotyls were compared. Fruit
vacuolar membranes were less permeable to protons than epicotyl
membranes. H
Eukaryotic cells contain a variety of acidic intracellular
compartments, including coated vesicles, secretory vesicles, Golgi
bodies, endosomes, lysosomes, and vacuoles, which have in common that
their membrane transport processes are energized by the vacuolar
H In animal cells, different
compartments of the endocytotic pathway have characteristic lumenal
pHs, ranging from pH 6.5 in the coated vesicles to pH 5.0 in the
lysosomes, suggesting that the lumenal pH of each organelle is tightly
regulated(6) . A number of observations suggest that the pH of
plant vacuoles is also regulated. In plants with crassulacean acid
metabolism for example, the vacuolar pH of the leaves varies diurnally,
from pH 3 at night to pH 6 in the day(7) . In stomatal guard
cells, the vacuolar pH is 4.5 in the dark when the stomata are closed,
and 6 in the light when the stomata are open(8) . During fruit
development the vacuolar pH often changes, becoming either more or less
acidic as ripening progresses. Such fluctuations indicate that the
vacuolar pH is under metabolic and developmental control. However, even
in the case of vacuoles with a constant pH the V-ATPase may be
continually regulated, inasmuch as the typical steady state
Bennett and Spanswick (9) determined an
H In lemon fruit juice sacs, the vacuolar pH declines from
6.5 to as low as 2.2 during maturation(20) . Assuming a
cytosolic pH of
Figure 1:
ATP-dependent proton pumping and
membrane permeability of tonoplast enriched vesicles isolated from
lemon epicotyls and juice sacs. Membrane protein concentrations were
adjusted to give the same initial rates of H
Figure 2:
Effect of chloride on H
When ATPase activity was measured under
similar conditions, the epicotyl membranes exhibited only a 15%
chloride stimulation (Fig. 2B). If only the
nitrate-sensitive activity is considered, the percent chloride
stimulation increases to 30%. There remains a marked discrepancy
between the chloride dependence of proton pumping and ATPase activities
in the epicotyl. In contrast, both the proton pumping and ATP
hydrolytic activities of juice sacs were equally insensitive to
chloride. Thus, the apparent uncoupling of H
Figure 3:
Determination of the pH optima for
H
Figure 4:
Immunoblot of membrane proteins from lemon
epicotyls and juice sacs. Tonoplast-enriched membranes were separated
by SDS-PAGE on a 15% polyacrylamide gel, transferred onto
nitrocellulose, probed with a polyclonal antibody raised against the
70-kDa subunit of the corn V-ATPase, and stained using horseradish
peroxidase. 10 and 20 µg of membrane proteins of juice sacs and
epicotyls were loaded on the gel. Standard proteins and their molecular
mass in kDa are given on the right of the sample
lanes.
Whereas the epicotyl tonoplast proton pumping activity
showed a typical inhibition by nitrate and bafilomycin A
Figure 5:
Nitrate inhibition of the V-ATPases of
lemon epicotyls and juice sacs. A, the effect of increasing
concentrations of tetramethylammonium nitrate on the initial rate of
proton pumping by tonoplast-enriched membranes (100 µg of protein)
as measured by quinacrine quenching. B, the effect of
KNO
Figure 6:
Inhibition of ATP-dependent H
The
proteolipid-binding inhibitor, N,N`-dicyclohexylcarbodiimide
(DCCD), inhibited both the juice sac and the epicotyl H
Figure 7:
Inhibition of ATP-dependent H
Figure 8:
Inhibition of ATP-dependent H
Azide (1 mM) and vanadate (50
µM) were routinely included in the reaction mix to
suppress residual plasma membrane ATPase and mitochondrial ATPase
activities, respectively. As shown by the dose-response curves in Fig. 9, these two contaminants would represent no more than
Figure 9:
Effects of vanadate and azide on
ATP-dependent H
Figure 10:
Inhibition of H
Figure 11:
Oxidative inactivation of the V-ATPases
of epicotyls and juice sacs. Tonoplast-enriched membranes of lemon
epicotyls and juice sacs were incubated either at 4 °C or at 20
°C, and the initial rates of ATP-dependent proton pumping were
assayed at different time intervals. A, the H
To
further characterize the oxidative inactivation of the epicotyl
V-ATPase, the ability of agents other than ATP to protect the enzyme
was examined. As shown in Table 2, a variety of chelators failed
to protect against oxidation, arguing against a role for metals as
cofactors in an enzymatic oxidation process. Catalysis is not required
for protection, since ADP was shown to be as effective as ATP in
protecting the enzyme. Surprisingly, 100 mM sulfate not only
protected the enzyme against inactivation, it also caused a doubling of
the original activity. The effects of sulfate and nucleotides were not
additive (Table 2). Other anions also protected against oxidation
to varying degrees, including chloride and nitrate. The ability of
nitrate to protect against oxidation is surprising inasmuch as nitrate
is a potent inhibitor of catalysis. The combination of nitrate plus ATP
or MgATP was less effective than nitrate alone in protecting against
oxidation. Nevertheless, it clearly protected relative to the control.
Figure 12:
Dissociation of the V
Figure 13:
Solubilization of fruit juice sac
membranes with dodecylmaltoside. Tonoplast-enriched membranes of lemon
juice sacs were solubilized with dodecylmaltoside added to the
concentrations indicated. After centrifugation, equal volume fractions
of the pellets and the supernatants were assayed for ATPase activity in
the presence or the absence of 200 mM KNO
Partial purification of the
solubilized enzymes by density gradient centrifugation on a linear
15-30% glycerol gradient resulted in the separation of the
nitrate-sensitive from the vanadate-sensitive ATPase activities.
However, the nitrate-sensitive peak also appeared to be partially
vanadate-sensitive, and the vanadate-sensitive peak was partly
inhibited by nitrate (Fig. 14). The vanadate-sensitive peak was
missing in glycerol gradients of solubilized epicotyl membranes and is
thus specific for the fruit membranes (data not shown).
Figure 14:
Glycerol density gradient centrifugation
of solubilized fruit juice sac membrane proteins. Tonoplast-enriched
membranes were solubilized with 2% dodecylmaltoside and layered on a
15-30% glycerol gradient. After centrifugation for 16 h at
177,000
During maturation, the vacuolar pH of the juice sac cells of
lemon fruits declines gradually from pH 6.0 to pH 2.2, a process that
may take over a year. This prolonged acidification is accompanied by
citrate accumulation in the form of the free acid to a final
concentration of about 300 mM(20) . Since citrate
typically enters vacuoles as citrate
Mature juice sacs are hypoxic, which raises the
question of whether or not there is sufficient ATP energy to maintain
the observed pH gradients. Based on the published values of 16
µM ATP, 21 µM ADP(34) , and 3 mM PO
Oxidation of the lemon
epicotyl V-ATPase was temperature-dependent and was blocked by ATP and
ADP. Feng and Forgac (19) showed that the oxidation of the
coated vesicle V-ATPase was also blocked by nucleotides and was partly
prevented by 100 mM KCl, which they attributed to an effect of
ionic strength. Our results indicate that the protective effect of
salts is anion-specific. Sulfate, which has been shown to inhibit the
V-ATPase of chromaffin granules (38) not only protects the
lemon epicotyl V-ATPase against oxidation, it strongly activates it.
However, neither sulfate nor other anions protected against inhibition
by NEM. We also
tested the ability of sulfate to substitute for chloride in the proton
pumping assay. As previously reported for chromaffin granules (38) , chloride was required by the epicotyl V-ATPase even in
the presence of sulfate, suggesting that there are two distinct anion
binding sites. In contrast to the epicotyl, the juice sac V-ATPase
was insensitive to NEM, and exhibited no DTT-reversible oxidation.
However, NPM was more inhibitory than NEM, suggesting that the critical
cysteine is in a hydrophobic pocket. In accord with this view,
detergent solubilization of the juice sac V-ATPase increased the
sensitivity of the juice sac V-ATPase to NEM. The insensitivity of the
juice sac V-ATPase to oxidation may be another factor which contributes
to the unusually low pH of juice sac vacuoles. Inasmuch as maturation
in lemon fruits occurs over a year, and during much of that time the
tissue is hypoxic, a highly stable V-ATPase is needed to maintain the
steep
Volume 271,
Number 4,
Issue of January 26, 1996 pp. 1916-1924
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
COMPARISON OF THE VACUOLAR H-ATPase ACTIVITIES OF
FRUITS AND EPICOTYLS (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
pumping by epicotyl membranes was
chloride-dependent, stimulated by sulfate, and inhibited by the
classical vacuolar ATPase (V-ATPase) inhibitors nitrate, bafilomycin, N-ethylmaleimide, and N,N`-dicyclohexylcarbodiimide.
In addition, the epicotyl H
pumping activity was
inactivated by oxidation at room temperature, and oxidation was
reversed by dithiothreitol. Cold inactivation of the epicotyl V-ATPase
by nitrate (
100 mM) was correlated with the release of
V
complexes from the membrane. In contrast, H pumping by the fruit tonoplast-enriched membranes was
chloride-independent, largely insensitive to the V-ATPase inhibitors,
and resistant to oxidation. Unlike the epicotyl
H
-ATPase, the fruit H
-ATPase activity
was partially inhibited by 200 µM vanadate. Cold
inactivation treatment failed to inhibit H
pumping
activity of the fruit membranes, even though immunoblots showed that
V
complexes were released from the membrane. However, cold
inactivation doubled the percent inhibition by 200 µM vanadate from 30% to 60%. These results suggest the presence of
two H-ATPases in the fruit preparation: a V-ATPase and
an unidentified vanadate-sensitive H
-ATPase. Attempts
to separate the two activities in their native membranes on linear
sucrose density gradients were unsuccessful. However, following
detergent-solubilization and centrifugation on a glycerol density
gradient, the two ATPase activities were resolved: a nitrate-sensitive
V-type ATPase that is also partially inhibited by 200 µM vanadate, and an apparently novel vanadate-sensitive ATPase that
is also partially inhibited by nitrate.
-ATPase
(V-ATPase)(
)(1, 2, 3) . In
addition to the V-ATPase, plant vacuolar membranes contain an
H-pyrophosphatase (H
-PPase), although
in most tissues the V-ATPase is the dominant
pump(4, 5) .
pH
across the tonoplast appears to be considerably less than the
theoretical maximum. From the H/ATP stoichiometry of
the pump (n), the membrane potential (
), the
Faraday constant (F), and the
G,
the maximum
pH at equilibrium can be calculated according to the
equation:
/ATP stoichiometry of 2 for the plant V-ATPase, which
was confirmed by Guern et al.(10) . Schmidt and
Briskin (11) extended these studies to the
H
-PPase and included estimates of internal buffering
capacity. They confirmed an n value of 2 for the V-ATPase and
calculated a maximum possible
pH across the tonoplast of
5.0-5.4 units when the membrane potential is +20 mV. The
authors concluded that the V-ATPase normally functions far from
equilibrium and is regulated by factors other than energy supply.
Mechanisms that have been proposed to regulate the V-ATPase include
``slip''(12) , cytosolic activators or
inhibitors(13, 14, 15) ,
chloride(12, 16) , cytosolic pH(17) , and
oxidation/reduction(18, 19) . As yet, none of these
mechanisms has been shown to correlate with in vivo proton
gradients.7.2, the final pH corresponds to a
pH of
5 units. Thus, the fruit V-ATPase either operates near
thermodynamic equilibrium, or, alternatively, the pump
H
/ATP stoichiometry is <2. If the V-ATPase operates
near thermodynamic equilibrium, it follows that the regulatory
mechanisms that normally prevent the V-ATPase from reaching equilibrium
are absent or deficient in the fruit juice sac cells. On the other
hand, if the proton pump responsible for hyperacidification has an n < 2, it could be either a V-ATPase with variable
H
/ATP stoichiometry (17) or a novel type of
tonoplast H
-ATPase with an n = 1. Here
we report that the H
pumping activity of juice sac
tonoplast-enriched membrane vesicles is relatively insensitive to a
variety of V-ATPase inhibitors and is inhibited
30% by 200
µM vanadate. Centrifugation of the detergent-solubilized
fruit membranes on linear glycerol gradients resulted in the separation
of two peaks of ATPase activities: a nitrate-sensitive V-ATPase that is
partially inhibited by 200 µM vanadate, and an apparently
novel vanadate-sensitive ATPase that is partially inhibited by high
nitrate concentrations. The possibility that these two proton pumps may
interact with each other during vacuolar hyperacidification is
discussed.
Materials
Lemon seeds (Citrus limon 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 A was from Sigma, BCA protein assay reagent was
obtained from Pierce and n-dodecyl-
-D-maltoside
from Calbiochem. All bulk chemicals were purchased from Sigma and
Fisher.Juice Sac Membrane Preparation
Tonoplast-enriched
membrane vesicles were prepared from lemon fruit juice sacs as follows.
Three lemons were peeled, and their segments were carefully separated.
The surrounding endocarp tissue was slit longitudinally along the
narrow edge of each segment using a single-edged razor blade, and the
juice sacs were released into 100 ml of cold homogenization buffer (HB;
1.5 M MOPS-KOH, pH 8.5, 1.5% polyvinylpyrrolidone-40, 0.75%
bovine serum albumin, 7.5 mM EDTA, 2 mM DTT, and 0.1
mM PMSF). All subsequent steps were carried out at 4 °C
and the membranes were maintained on ice. The juice sacs were ground
using a mortar and pestle and filtered through a 0.28-mm nylon mesh.
Cellular debris, nuclei and plastids were removed by centrifugation at
12,000 
g for 15 min in a Sorvall RC2-B refrigerated
centrifuge (SS-34 rotor). The supernatant was subjected to
ultracentrifugation in a Beckman SW-28 rotor at 132,000 g for 60 min, and the pellet was resuspended in 15 ml of
resuspension buffer (RB; 10 mM BTP-Mes, pH 7.6, 20 mM KCl, 1 mM EDTA, 2 mM DTT, and 0.1 mM PMSF). The microsomal fraction was further purified on a 10%/35%
sucrose step gradient made up in gradient buffer (10 mM BTP-Mes, pH 7.6, 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 two volumes of RB, and pelleted at 174,000
g for 20 min 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. Unless otherwise
stated, when membranes were prepared for experiments involving
oxidation or inhibition by N-ethylmaleimide (NEM) or N-phenylmaleimide (NPM), they were resuspended in RB in the
absence of DTT.Epicotyl Membrane Preparation
Lemon seeds were
germinated and grown for 4 weeks at 29 °C, in the dark, in flats
containing moist vermiculite. Epicotyls (40 g fresh weight) were
harvested with a razor blade and homogenized in 150 ml of cold HB using
a mortar and pestle. All other steps were as described in the fruit
membrane preparation.Liposome
Preparation
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.Membrane Solubilization
Membranes were made up to
6 mg of protein/ml with RB. One volume of 4% (w/w) n-dodecyl--D-maltoside in solubilization buffer
(10 mM BTP/Mes, pH 7.6, 10% glycerol, 1 mM EDTA, 8
mM MgSO
, 50 mM DTT, 200 µg/ml
sonicated liposomes, and 0.012% butylated hydroxytoluene) was added
drop by drop to the membranes while stirring on ice. The mixture was
further incubated 30 min on ice with gentle stirring before being used
in ATP hydrolysis experiments or further purified by glycerol gradient
centrifugation.Glycerol Density Gradient
Centrifugation
Solubilized membrane proteins (3.5 mg) were
centrifuged for 15 min at 412,000 g (4 °C) in a
Beckman TLA-100.3 rotor. The supernatant was filtered through a
0.45-µm cellulose nitrate filter and loaded on a linear
15-30% glycerol gradient in 10 mM BTP-Mes, pH 7.0, 0.1%
Triton X-100, 1 mM EDTA, 4 mM MgSO, 10
mM KCl, 50 µg/ml sonicated liposomes, and 20 mM DTT. After centrifugation for 16 h at 177,000 g (4 °C) in a Beckman SW-41 rotor, 0.5-ml fractions were
collected and analyzed for ATPase activity in the presence or absence
of nitrate and vanadate.Dissociation of V
Membranes
were made up to 0.3 mg of protein/ml in 2.5 ml of RB containing 5
mM ATP, 7 mM MgSO Complex, and 0, 20, 100, 200,
or 500 mM KNO
or KI. They were incubated on ice
for 1 h and centrifuged 15 min at 412,000 g (Beckman
TLA-100.3). The membrane pellet was resuspended in 750 µl of RB and
used for proton pumping and immunoblotting. The proteins in 50 µl
of the resuspended pellet, and 167 µl of the supernatant were used
for immunoblotting after being precipitated with 10% trichloroacetic
acid, washed with cold acetone, lyophilized, and separated by SDS-PAGE. Proton Pumping Assays
Proton pumping by tonoplast
vesicles was monitored by the quenching of quinacrine fluorescence as
described previously(21) . The reaction mix contained 10 mM BTP-Mes, pH 7.0, 250 mM sorbitol, 100 mM KCl, 50
µM vanadate, 1 mM azide, 250 nM valinomycin, 2.5 mM ATP, and 10 µM quinacrine unless otherwise stated. 100 µg of membrane
proteins were typically used and the reaction was usually started with
3.5 mM MgSO. In kinetic experiments, ionic
interactions were taken into account by means of the SOLCON computer
program (generously provided by D. C. S. White, University of York,
York, United Kingdom, and Y. E. Goldman, University of Pennsylvania,
Philadelphia, PA) using logarithmic association constants of all
metal-ligand complexes present in the assay mix(22) . Proton
pumping was initiated with MgSO at the concentration
indicated to provide the required concentration of MgATP. Typically,
the concentration of MgSO had to be present at
approximately 2 mM in excess of the sum of the concentrations
of ATP and ADP present in the assay mix. Fluorescence quenching (423 nm
excitation, 502 nm emission) was measured in a Perkin-Elmer LS-5
fluorescence spectrophotometer. Proton pumping activity was expressed
as % quench
mgmin.
ATPase Assays
Native or solubilized membranes were
added to a reaction mix containing 2.5 mM ATP, 2.5 mM MgSO, 100 mM KCl, 1 mM azide, 1
mM molybdate, 2 µM gramicidin D, and 1 mg/ml
sonicated liposomes in 25 mM BTP/Mes buffer, pH 7.0. The total
reaction volume was 300 µl. After 30 min at 37 °C, the reaction
was stopped by adding one volume of 10% trichloroacetic acid, 4%
perchloric acid (ice-cold) to the reaction mix. The mixture was
maintained on ice for 5 min, centrifuged 4 min at 14,000 g (4 °C) in a Tomy MTX-150 microcentrifuge, and the supernatant
assayed for released inorganic phosphate by the Fiske and Subbarow
reaction(23) . Boiled membranes were used for background
estimates. Where nitrate-sensitive activity is reported, 200 µM vanadate were present in the reaction mix and the results are
expressed as the difference in activity in the presence or absence of
200 mM KNO. Where vanadate sensitive activity is
reported, 200 mM KNO were present in the mix and
the results show the difference in activity in the presence or absence
of 200 µM vanadate.Immunoblotting
One-dimensional SDS-PAGE of
tonoplast enriched membranes was performed as described by Laemmli (24) on 12% polyacrylamide gels. The proteins from SDS gels
were electrotransferred to 0.45-µm nitrocellulose filters (35 V,
200-300 mA, overnight; or 100 V, 250-350 mA for 1 h) in a
Transphor TE 50 transfer cell (Hoefer Scientific Instruments, San
Francisco, CA) or in a Mini Trans-Blot® electrophoretic transfer
cell (Bio-Rad). The blots were blocked with 3% nonfat instant milk in
TPBS (10 mM Na
HPO, 0.05% Tween®
20, and 0.9% NaCl, pH 7.5, with NaHPO) for 1 h
before being incubated for 7 h in TPBS containing 0.5% bovine serum
albumin and the primary antibody to the 70-kDa subunit of the corn
V-ATPase(25) . The blots were then washed in TPBS and
visualized by a peroxidase-coupled secondary antibody reaction
(Vectastain ABC kit, Vector Laboratories, Burlingame, CA).Antiserum Purification
Preimmune serum and
antiserum raised against the 70-kDa subunit of the corn V-ATPase were
purified on a Protein A-Sepharose column according to Sambrook et
al.(26) . The sera were made up to 2.25 µg of
protein/µl in 100 mM TrisHCl, pH 8.0, before use in
proton pumping and ATP hydrolysis experiments.Protein Concentration
Estimates of protein
concentrations were done routinely by a modified BCA protein assay
after trichloroacetic acid precipitation(27) .Statistical Treatment of the Results
All
experiments were performed at least three times in duplicates. Proton
pumping results are given as the means (± range) of the
duplicates of one representative experiment. For ATP hydrolysis
experiments, the mean (± S.D.) of two independent experiments
are presented. K
and V
values were calculated from Hanes-Woolf plots.
The proton
pumping activities of the tonoplast-enriched membrane fraction from
epicotyls and mature fruits were compared by quinacrine fluorescence
quenching. When the fruit membranes were diluted to give the same
initial rate of HpH and Membrane Permeability pumping as the epicotyl vesicles,
they developed a greater pH gradient (total quench) than the epicotyl
membranes (Fig. 1). When the gradient had stabilized, 10 mM EDTA was added to stop the reaction by chelating
Mg
. In the absence of MgATP, the fruit membranes
maintained 80% of the pH gradient for up to 2 h after an initial
leakage, whereas the pH gradient across the epicotyl tonoplast
collapsed within 20 min. Addition of gramicidin at the end of the
reaction confirmed that a pH gradient was still present across the
fruit vesicle membranes. We conclude that the juice sac tonoplast is
less permeable to protons than the epicotyl tonoplast.
pumping
(70 µg of fruit protein and 100 µg of epicotyl protein were
used in this representative experiment). The assay mix was as described
under ``Experimental Procedures,'' and the reaction was
started with MgSO
. After equilibrium was reached, 10 mM EDTABTP (pH 7.0) was added to chelate the
Mg. After a new equilibrium was reached, the residual
pH gradient was collapsed with 4 µM gramicidin.
Chloride Dependence of H
As shown in Fig. 2A, the
H Pumping Versus
ATPase Activity
pumping activity of the epicotyl V-ATPase was almost
completely dependent on chloride. Because the reaction was started by
adding membranes loaded with 20 mM KCl, and valinomycin was
present in the assay mix, chloride stimulation of H
pumping is probably due to direct activation of the enzyme rather
than to the ability of chloride to collapse the membrane potential.
Similar results were obtained in the presence of valinomycin and 50
mM potassium iminodiacetate in the assay mix (data not shown).
In this latter case, the membrane were allowed to equilibrate for
20-30 min in the reaction mix containing 50 mM potassium
iminodiacetate and the proton-pumping reaction was started with
Mg
. Because K
and valinomycin were
present in the mix, the buildup of a positive
should again
have been prevented. In contrast to epicotyl membranes, juice sac
vesicles did not require chloride for proton transport (Fig. 2A).
pumping and ATP hydrolysis by tonoplast-enriched membranes from
lemon epicotyls and juice sacs. For proton pumping (A), the
reaction mix contained 2.5 mM ATP, 3.5 mM MgSO
, 0.25 µM valinomycin, 50 µM vanadate, 1 mM azide, sorbitol, 10 µM quinacrine, and cholineCl at the concentrations indicated.
Constant osmolarity was achieved by balancing cholineCl and
sorbitol in the mix. Each proton pumping reaction was started by adding
10 µl of membranes (100 µg of protein), preloaded with 20
mM KCl by freeze-thawing to collapse the membrane potential
(, epicotyl; 
, juice sac). Experimental conditions for ATP
hydrolysis (B) were similar to those for H pumping, except that 1 mM molybdate was present in the
mix, and quinacrine was omitted.
-transport
from ATP hydrolysis observed in the epicotyl V-ATPase in the absence of
chloride, defined as ``slip,'' does not occur in the juice
sac V-ATPase.
pH Optima, K
As
shown in Fig. 3, the pH optima for the fruit and the epicotyl
V-ATPases were both , and V7.0. Table 1gives the K
and V values for the fruit and epicotyl
membranes based on proton pumping activity. Whereas the K values for MgATP of the fruit and epicotyl
membranes were similar, the average V of the
fruit enzyme was about double that of the epicotyl.
pumping by the epicotyl and juice sac V-ATPases. 10
µl of tonoplast enriched membranes (100 µg of protein) were
assayed for ATP-dependent proton pumping in a reaction mix containing
2.5 mM ATP, 100 mM KCl, 0.25 µM valinomycin, 50 µM vanadate, 1 mM azide, and
10 µM quinacrine. The mix was buffered with 10 mM BTP-Mes to the indicated pH. The reaction was started with 3.5
mM MgSO
, and the initial rate of quinacrine
quenching is reported (, epicotyl; , juice
sac).
Immunoblotting
The higher V of the fruit V-ATPase could reflect a higher density of V-ATPases
on the membrane. Immunoblots probed with antibodies to the 70-kDa
subunit of the corn V-ATPase (25) suggest that the fruit
tonoplast vesicles actually contain less V-ATPase per milligram of
protein than the epicotyl membranes (Fig. 4). This implies
either a higher proportion of active enzyme on the juice sac
tonoplast or a higher K. Alternatively, the
antibody may have a lower affinity for the fruit V-ATPase than for the
epicotyl enzyme, which would imply that the fruit V-ATPase is a
different isozyme.
Inhibitors
Both the fruit and the epicotyl
V-ATPases were inhibited by ADP. Inhibition was largely competitive,
although the sigmoidal nature of the curves at low
[ATP]/[ADP] ratios suggested allosteric inhibition
as well (data not shown). The average K
values for
ADP calculated from replots of the slopes of double-reciprocal plots
were about 30 µM for both the fruit and epicotyl
membranes.,
the juice sac tonoplast vesicles were remarkably insensitive to these
inhibitors (Fig. 5A and Fig. 6). Measurements of
ATP hydrolytic activity also indicated that the fruit V-ATPase was less
sensitive to nitrate than the epicotyl enzyme (Fig. 5B, dashed lines). Detergent solubilization increased the nitrate
sensitivity of the fruit, and decreased that of the epicotyl, so that
both activities were equally sensitive to nitrate at 200 mM (Fig. 5B, solid lines).
on ATP hydrolysis in native and solubilized tonoplast
vesicles (60 µg of membrane protein), as monitored by P release. H pumping was initiated with 3.5 mM MgSO
. ATP hydrolysis was initiated by adding the
membranes to the mix (- -- -, epicotyl membranes; - -- -,
juice sac membranes; --, solubilized epicotyl
membranes; --, solubilized juice sac
membranes).
pumping by bafilomycin A
in tonoplast-enriched
vesicles from epicotyls and juice sacs. Bafilomycin A at
the concentrations indicated and 10 µl of tonoplast-enriched
membranes (100 µg of protein) were added to the reaction mix
(composition as under ``Experimental Procedures''). Each
reaction was started with 3.5 mM MgSO and the
initial rates of quinacrine fluorescence quenching are reported
(, epicotyl; , juice sac).
pumping, although the inhibition was less pronounced in juice sac
membranes than in epicotyl membranes (Fig. 7). The sensitivity
of both enzymes to low concentrations of the sulfhydryl reagent, NEM,
is shown in Fig. 8. The epicotyl V-ATPase was highly sensitive
to NEM, with 94% of its activity being inhibited by 50 µM NEM, whereas the fruit enzyme was almost unaffected at this
concentration. The more hydrophobic sulfhydryl reagent, NPM, was more
effective than NEM in inhibiting proton pumping by the fruit V-ATPase (Fig. 8).
pumping by DCCD in tonoplast-enriched vesicles from epicotyls and
juice sacs. The reaction mix was as described under ``Experimental
Procedures''. 100 µg membrane proteins and DCCD at the
concentrations indicated were added. Each reaction was started with 3.5
mM MgSO
and the initial rates of quinacrine
fluorescence quenching were determined (--, epicotyl;
--, juice sac).
pumping by NEM and NPM in tonoplast-enriched vesicles from
epicotyls and juice sacs. 100 µg membrane proteins and NEM or NPM
at the concentrations indicated were added to a reaction mix (see
``Experimental Procedures''). Each reaction was started with
3.5 mM MgSO
, and the initial rates of fluorescence
quenching were measured (--, epicotyl, NEM;
--, juice sac, NEM; - -- -, juice sac,
NPM).
10% of the total activity in the fruit membranes under standard
assay conditions. Moreover, the membrane-bound vanadate-sensitive
activity could not be separated from the nitrate-sensitive activity by
linear sucrose or dextran gradients (data not shown), suggesting that
the nitrate- and vanadate-sensitive activities are on the same
membrane.
pumping by tonoplast-enriched vesicles
from epicotyls and juice sacs. A, the effect of vanadate on
the initial rates of H
pumping as measured by
quinacrine quenching. B, the effect of azide on the initial
rates of H
pumping. The reaction mix contained 2.5
mM ATP, 100 mM KCl, 0.25 µM valinomycin,
10 µM quinacrine, and either sodium vanadate or sodium
azide at the concentrations indicated. In the vanadate experiment,1
mM azide was included into the mix and in the azide experiment
50 µM vanadate was present. Each reaction was started with
3.5 mM MgSO
(, epicotyl; , juice
sac).
Inhibition by 70-kDa Antibody
When fruit and
epicotyl membranes were treated with polyclonal antibody to the
catalytic subunit of the corn V-ATPase, the proton pumping activity of
the epicotyl was inhibited by up to 80%, whereas the fruit membranes
were inhibited by only 25% (Fig. 10A). However,
following solubilization, the nitrate-sensitive ATPase activities of
the fruit and epicotyl were equally inhibited by the 70-kDa antibody (Fig. 10B).
pumping
and ATPase activity by antibody to the 70-kDa subunit of corn. A, the effect of polyclonal antibody to the corn 70-kDa
subunit on the initial rate of proton pumping by lemon
tonoplast-enriched membranes measured by quinacrine fluorescence
quenching. B, the effect of the same antibody on ATP
hydrolysis by the solubilized enzymes, as monitored by P
release. Preimmune serum was used in both cases as a control. For
H pumping and ATP hydrolysis assay conditions, see
``Experimental Procedures.'' 100 µg of membrane proteins
were added to the mix in proton pumping experiments and each reaction
was started with 3.5 mM MgSO
. In ATP hydrolysis
experiments, each reaction was started by adding 20 µg of
solubilized membrane proteins to the mix and the nitrate-sensitive
activity was determined (--, inhibition of the
epicotyl V-ATPase by the 70-kDa antibody; --,
inhibition of the juice sac V-ATPase by the 70-kDa antibody; - --
-, effect of preimmune serum on the epicotyl V-ATPase; - -- -,
effect of preimmune serum on the juice sac
V-ATPase).
Oxidation/Reduction
The sensitivities of the
fruit and epicotyl V-ATPases to DTT-reversible oxidation was examined
by measuring the decay of proton pumping activity over time at 20
°C, and the restoration of activity by 50 mM DTT. The
membranes were resuspended either in RB containing 1 mM DTT (Fig. 11A), or RB without DTT (Fig. 11, B and C). As shown in Fig. 11(A-C),
the epicotyl V-ATPase was almost completely inhibited after a 4-h
incubation in the presence or absence of 1 mM DTT. In
contrast, the activity of the fruit V-ATPase was stable over the same
time period in the presence of 1 mM DTT and was only slowly
inactivated in the absence of DTT. Incubation of the epicotyl membranes
in the presence of 50 mM DTT or 5 mM ATP over the
same time period completely protected the epicotyl V-ATPase from
inactivation (Fig. 11, A and C). Addition of
50 mM DTT to the oxidized epicotyl preparation restored proton
transport activity to >60% of the activity at time 0. When epicotyl
membranes were incubated on ice, inactivation was greatly reduced, and
was completely reversible with DTT, demonstrating that the oxidation
was temperature-dependent. In contrast, the slow loss of activity of
the fruit V-ATPase that occurred in the absence of DTT was
temperature-independent and could not be reversed by DTT (Fig. 11B). In addition, ATP did not protect against
inactivation of the juice sac V-ATPase (Fig. 11C). To
determine whether the greater oxygen sensitivity of the epicotyl
membranes was due to the release of a soluble oxidant, a 1:1 mixture
(µg protein) of fruit and epicotyl membranes was incubated at 20
°C and the inactivation kinetics measured. The oxidation curve of
the mixture corresponded to the average curve of the two membrane
fractions measured separately (data not shown). If a soluble oxidant
were involved, the loss of activity would have been additive.
pumping activities of membranes incubated in the presence of 1
mM DTT (-
-, epicotyl, 20 °C;
--, juice sac, 20 °C; --,
epicotyl, 4 °C) or in the presence of 50 mM DTT (-
-
- -, epicotyl, 20 °C). Where indicated, 50 mM DTT
was added to the membranes. B, H pumping
activities of membranes incubated in absence of DTT
(-
-, epicotyl, 20 °C; --, juice
sac, 20 °C; - -- -, epicotyl, 4 °C; - -- -, juice
sac, 4 °C; --, 1:1 mixture (µg of protein)
of epicotyl and juice sac membranes). At 240 min, 50 mM DTT
was added to all membranes. C, H
pumping
activities of membranes incubated at 20 °C in the presence or
absence of 5 mM ATP (-
-, epicotyl,
-ATP; - -- -, epicotyl, +ATP; --,
juice sac, -ATP; - -- -, juice sac, +ATP). At time 240
min, 50 mM DTT was added to all membranes. The composition of
the reaction mix was as described under ``Experimental
Procedures,'' 100 µg of membrane proteins were assayed in each
case, and the reactions were started with 3.5 mM MgSO.
Dissociation of the V
We hypothesized that the nitrate insensitivity of the
fruit membranes might be due to a tighter attachment of the peripheral
V Complex with Chaotropic
Agents complex to the membrane-bound portion, V
. To
test this, dissociation of V from V in fruit
and epicotyl membranes was induced by KNO, on ice, in the
presence of 5 mM MgATP. As shown in Fig. 12(A and B), the proton pumping activity of the epicotyl
membranes was strongly inhibited at nitrate concentrations > 20
mM, and the inhibition was correlated with the release of
V complexes from the membrane, as shown by immunoblotting.
Surprisingly, the total proton pumping by the fruit membranes was
insensitive to as high as 500 mM nitrate, even though the
pattern of V dissociation paralleled that of the epicotyl.
The only detectable effect of nitrate on the fruit proton pumping
activity was a progressive increase in the proportion of
vanadate-sensitive activity and a concomitant decrease in the
vanadate-insensitive activity (Fig. 12B). Similar
results were obtained with KI (data not shown). The data suggest the
presence of a second, vanadate-sensitive proton pump on the fruit
tonoplast.
complex
with nitrate. A, the effect of increasing concentrations of
nitrate on the cold release of V complex, as probed on
immunoblots with antibody to the catalytic subunit of the corn
V-ATPase. Tonoplast-enriched vesicles were incubated for 1 h, on ice,
in the presence of 5 mM MgATP and KNO as
indicated. After centrifugation, equal volume fractions of the pellets
and the supernatants were analyzed by SDS-PAGE and blotted onto
nitrocellulose. B, the effect of V release on the
initial rate of proton pumping by the pelleted and resuspended
membranes (--, epicotyl membranes in the presence of
50 µM vanadate; - -, fruit juice sac
membranes in the absence of vanadate; --, fruit juice
sac membranes in the presence of 500 µM vanadate; -
-- -, resultant of the difference of the two precedent curves
(vanadate-sensitive activity).
Solubilization and Glycerol Gradient
Centrifugation
Following solubilization of the fruit
tonoplast-enriched membranes with 2% dodecylmaltoside and
centrifugation, the nitrate-sensitive ATPase activity released into the
supernatant was double that of the initial membrane-bound activity (Fig. 13A). The total nitrate-sensitive activity after
solubilization (pellet plus supernatant) was 2.5 times that of the
native membrane (Fig. 13B). In contrast, the
nitrate-insensitive activity remained constant over the range of the
detergent concentrations used.
. A, the nitrate-sensitive ATPase activities remaining in the
pellets (- -- -) and the nitrate-sensitive activities released
into the supernatants (--). B, the total
ATPase activities after solubilization (pellet plus supernatant).
- -, total activity; --,
nitrate-sensitive activity; - -- -, nitrate-insensitive
activity.
g, 0.5-ml fractions were collected and
analyzed for vanadate-sensitive (- -- -) and nitrate-sensitive
(--) ATPase activities (activities in the presence or
absence of 400 mM nitrate and 200 µM vanadate). Fraction 1 represents the top of the gradient, fraction
21, the bottom. The amount of activity recovered from the
gradients was 50% of the initial
activities.
(28) ,
it would tend to bind protons and raise the vacuolar pH rather than
lower it. Thus, the dramatic decline in pH is brought about by active
proton pumping, rather than by citrate uptake. The
H
-PPase can be ruled out as the primary proton pump
because: 1) H
-PPase activity is negligible in juice
sacs, (
)and 2) calculations indicate that under typical
cellular conditions the H-PPase is incapable of
generating such a large pH gradient(11, 17) .
Membrane Permeability
The equilibrium pH across the
tonoplast is governed by two main factors: proton pumping and proton
leakage across the membrane(29) . We have shown that juice sac
tonoplast vesicles are less permeable to protons than epicotyl
vesicles. The slower leak rate of the juice sac tonoplast allows the
buildup of a steeper pH gradient in the in vitro assays and
undoubtedly plays an important role in vivo. However, it is
unlikely that the vacuolar pH in epicotyls is regulated by proton
leakage alone since futile cycling of protons would be costly
energetically. Thus, V-ATPases must be kinetically regulated in
vivo.The Juice Sac Tonoplast Contains a Vanadate-sensitive
V-ATPase
The H-ATPase activity of the native
tonoplast-enriched membrane vesicles from juice sacs was insensitive to
a wide array of V-ATPase inhibitors, including nitrate, bafilomycin
A
, NEM, and a polyclonal antibody to the 70-kDa subunit of
corn. Nevertheless, a V-type H-ATPase appears to be
present based on the following criteria. First, both the fruit and
epicotyl H
pumping activities have similar pH optima
and K
values for MgATP. Second, although the
fruit-derived vesicles were insensitive to NEM, the hydrophobic
sulfhydryl inhibitor, NPM, was a more effective inhibitor. Third,
detergent solubilization increased the sensitivity of the fruit ATPase
to inhibition by nitrate by 2.5-fold, indicating that the lack of
sensitivity to nitrate is caused by molecular interactions in the
native membrane. Fourth, the solubilized ATPase activity was inhibited
by antibody to the 70-kDa antibody and by NEM. ()Fifth, cold
treatment in the presence of MgATP and nitrate caused the release of
immunodetectable V complexes from the fruit membrane.
Interestingly, the solubilized, partially purified nitrate-sensitive
V-ATPase from the fruit is also partly inhibited by 200 µM vanadate. Since the epicotyl V-ATPase, when treated in similar
fashion, is not inhibited, ()the fruit V-ATPase appears to
be a different isozyme. Vanadate-sensitive V-ATPases have previously
been shown to be present on the plasma membranes of animal osteoclast
cells, although they are more sensitive to vanadate than the fruit
enzyme(30) . More recently, a vanadate- and nitrate-sensitive
V-ATPase has been demonstrated on the tonoplast of vacuoles isolated
from Acer pseudoplatanus cell cultures(31) . Whether
or not vanadate-sensitive V-ATPases are specifically adapted for
hyperacidification remains to be determined.Regulation by ADP
ADP has been suggested to be
involved in the kinetic regulation of the V-ATPase(32) . In
lemon, ADP inhibition of the V-ATPases appears to have both a
competitive and an allosteric component. The K values determined for the competitive ADP inhibition of proton
pumping for both the fruit and the epicotyl V-ATPases were surprisingly
low (20-40 µM) compared to previously published
values for plant V-ATPases (140-180 µM(32) ,
120 µM(33) ). Since the K for
MgATP is about 0.8-0.9 mM, the low K value for ADP suggests that the V-ATPase in epicotyls is
partially inhibited by ADP in vivo, assuming that epicotyl
cells contain typical millimolar concentrations of ATP and
ADP(9) .(20) , and using -8.8 kcal/mol for
G`(35) , the calculated
G in mature juice sacs is around -12
kcal/mol. Since this value is in the same range as in normal vegetative
tissues(11) , there is sufficient free energy to drive proton
transport in mature juice sacs, although the rate will be slower than
in epicotyls because of the low substrate concentration.
Regulation by Slip
Moriyama and Nelson (12) proposed that the vacuolar H-ATPase of
chromaffin granules is controlled by slip, defined as ATPase catalytic
activity uncoupled from proton transport. In epicotyl tonoplast
vesicles, the proton pumping activity was strongly stimulated by
chloride in the presence of K
and valinomycin to
collapse
. Under the same conditions, ATPase activity was
only marginally stimulated by chloride. Since the H
pumping in the epicotyl was much more dependent on chloride than
the ATPase activity, the epicotyl V-ATPase appears to undergo slip when
chloride is limiting. In contrast, the H
pumping and
ATPase activities of the juice sac V-ATPase were both equally
insensitive to chloride. One interpretation of the results is that the
juice sac V-ATPase remains coupled under low chloride conditions and
does not undergo slip. Alternatively, the chloride-independent activity
could be due to the presence of a second proton pump on the membrane
that does not require chloride.
Regulation by Oxidation/Reduction
The epicotyl
V-ATPase was strongly inhibited by the sulfhydryl reagent, NEM. It is
now well established that the highly conserved cysteine residue in the
catalytic site of V-ATPases (Cys in the bovine coated
vesicle H
-ATPase and Cys
in yeast) is
the site of NEM binding and inhibition(18, 36) .
Moreover, Krauss et al.(37) and Feng and Forgac (18, 19) have provided evidence that the V-ATPase can
undergo DTT-reversible oxidative inactivation. Our results show that
DTT-reversible inactivation of the lemon epicotyl V-ATPase occurred in
native tonoplast vesicles upon incubation at 20 °C. Moreover, the
reduction in the V
of the epicotyl V-ATPase
compared to the fruit enzyme appeared to be proportional to the amount
of oxidized enzyme. When 20 mM DTT was included in the vesicle
isolation buffers, instead of the usual 2 mM, the V of the epicotyl enzyme doubled, making it
equal to that of the fruit. This is similar to the results
of Feng and Forgac (19) for bovine coated vesicles and it
suggests that as much as 50% of the epicotyl V-ATPase may be in the
oxidized, inactive state in vivo. This suggests that the site of protection against
oxidation is distinct from the NEM-binding site. The ability of anions
to block oxidation could be due to a conformational change.pH across the tonoplast over the entire life of the fruit.A Second Vacuolar H
Under
conditions known to cause the physical dissociation of the catalytic
V-ATPase?
complex from the membrane sector, V-ATPases from juice
sacs and epicotyls released comparable amounts of V complexes into the incubation medium. Whereas V release from epicotyl membranes was correlated with a decrease in
the total H pumping activity, the total H
pumping activity of the fruit membranes was unaffected. The net
increase in vanadate-sensitive H
pumping activity
accompanying the dissociation of the catalytic complex of the juice sac
V-ATPase suggests that a second, vanadate-sensitive
H
-ATPase is activated under the conditions promoting
V
dissociation. This interpretation is supported by the
identification of a vanadate-sensitive ATPase peak that separates from
the nitrate-sensitive V-ATPase peak after centrifugation of the
detergent-solubilized fruit enzymes on linear glycerol gradients. This
activity is not likely to be due to a contaminating plasma membrane
H-ATPase because: (a) the concentration of
vanadate needed to inhibit it is 10
greater than that needed to
inhibit a normal P-type ATPase, and (b) the activity is also
nitrate-sensitive. Moreover, if the two proton pumps were to reside on
different membranes, one would expect the two activities to be
additive. The cold inactivation experiment shows, however, that this is
not the case. Rather, the putative second proton pump appears to take
over the function of the V-ATPase as the V complex becomes
dissociated, so that the initial rate of proton pumping by the vesicles
remains the same. A corollary of the two proton pump model is therefore
that the two H-ATPases interact in some way on the
membrane. Reconstitution studies are currently under way to determine
whether the two pumps behave differently when reconstituted into
artificial liposomes either individually or as a pair.
)-ATPase;
BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; DCCD, N,N`-dicyclohexylcarbodiimide; DTT, dithiothreitol; HB,
homogenization buffer; H
-PPase,
H
-pyrophosphatase; Mes,
2-(N-morpholino)ethanesulfonic acid; MOPS;
3-(N-morpholino)propanesulfonic acid; n,
H
/ATP stoichiometry; NEM, N-ethylmaleimide;
NPM, N-phenylmaleimide; PMSF, phenylmethylsulfonyl fluoride;
RB, resuspension buffer;
, membrane potential; PAGE,
polyacrylamide gel electrophoresis.
)))
We thank Megan Jensen for technical assistance in the
preparation of membranes. We also thank Ronald Poole, Roger Spanswick,
Richard Cross, Don Briskin, Dale Sanders, and Roberto Bogomolni for
valuable discussions.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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