(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 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.
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-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) .
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 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:
Bennett and Spanswick (9) determined an
H/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.
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 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.
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 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 EDTA
BTP (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.
Figure 2:
Effect of chloride on H 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 choline
Cl at the concentrations indicated.
Constant osmolarity was achieved by balancing choline
Cl 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.
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-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.
Figure 3:
Determination of the pH optima for
H 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).
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,
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).
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 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).
Figure 6:
Inhibition of ATP-dependent H 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).
The
proteolipid-binding inhibitor, N,N`-dicyclohexylcarbodiimide
(DCCD), inhibited both the juice sac and the epicotyl H 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).
Figure 7:
Inhibition of ATP-dependent H 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).
Figure 8:
Inhibition of ATP-dependent H 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).
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
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.
Figure 9:
Effects of vanadate and azide on
ATP-dependent H 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).
Figure 10:
Inhibition of H 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).
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 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
.
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 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).
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. 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.
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 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.
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(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) .
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(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.
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. 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.
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 pH across the tonoplast over the entire life of the fruit.