(Received for publication, June 17, 1994; and in revised form, October 18, 1994)
From the
It has been claimed that the inorganic pyrophosphatase (PPase)
of the plant vacuolar membrane transports K in
addition to H
in intact vacuoles (Davies, J. M.,
Poole, R. J., Rea, P. A., and Sanders, D.(1992) Proc. Natl. Acad.
Sci. U. S. A. 89, 11701-11705). Since this was not confirmed
using the purified and reconstituted PPase consisting of a 75-kDa
polypeptide (Sato, M. H., Kasahara, M., Ishii, N., Homareda, H.,
Matsui, H., and Yoshida, M. (1994) J. Biol. Chem. 269,
6725-6728), these authors proposed that K
transport by the PPase is dependent on its association with other
membrane components lost during purification. We have examined the
hypothesis of K
translocation by the PPase using
native vacuolar membrane vesicles from Vitis vinifera suspension cells, multilabeled with fluorescent probes for
K
, H
, and membrane potential. This
material contained a high proportion of right-side-out, tightly sealed
vesicles, exhibiting high PPase activity which was strongly stimulated
by uncouplers and K
. Proton pumping occurred in
response to pyrophosphate addition in the absence of
K
. No K
incorporation into the
vesicles could be observed after PPase energization in the presence of
K
, although H
transport was highly
stimulated. The hydrolytic activity was stimulated by a protonophore
and by a H
/K
exchanger but not by the
K
ionophore valinomycin. No evidence could be obtained
supporting the operation of an endogenous
K
/H
exchanger capable to dissipate
the putative active K
flux generated by the PPase. We
conclude that PPase in native vacuolar membrane vesicles does not
transport K
.
Since the K concentration in plant vacuoles can
reach 40-200 mM (Lüttge and
Higinbotham, 1979; MacRobbie, 1970), the thermodynamic equilibrium
value predicted from cytosolic K
concentration and
vacuolar membrane potential may be exceeded. Thus, the possibility of
an active K
translocate in the vacuole has to be
considered (Rea et al., 1992b and references therein). The
plant vacuolar inorganic pyrophosphatase (EC 3.6.1.1, PPase) (
)is known to function as an electrogenic H
pump, translocating H
from the cytosol to the
vacuole lumen (Hedrich and Shroeder, 1989; Rea and Sanders, 1987; Sze,
1985). Considerable interest has resulted from recent
electrophysiological data suggesting that the PPase of beet root
vacuolar membrane transports K
together with
H
. Patch clamping intact vacuoles (Davies et
al., 1992) revealed an outwardly directed current dependent on
vacuolar K
after PPase activation in the reverse mode (i.e. PP
synthesis). Since the reversal potential
of this current was determined by both H
and
K
Nernst potentials, it was ascribed to simultaneous
H
and K
transport out of the vacuole.
The PPase would thus be the first primary active K
transport system identified in plants.
However, recent work
with highly purified pumpkin PPase reconstituted into proteoliposomes
showed that K
transport was independent
on the activation of H
translocation by the PPase and
could be ascribed to passive diffusion. These results led to the
conclusion that this enzyme does not translocate K
(Sato et al., 1994). A possible explanation for the
discrepancy between these results and those of Davies et al. (1992) is that another membrane protein is necessary to confer
K
translocation ability on the PPase. Alternatively,
isolation and purification of the PPase may have altered its function.
We took advantage of several characteristics of the vacuolar
membrane vesicles from Vitis vinifera cells to examine this
question, using three independent criteria, viz. (i)
direct, simultaneous determination of intravesicular
K, pH, and membrane potential, (ii) ionophore
effects on hydrolysis activity, and (iii) K
effects on transport and hydrolytic properties of the PPase. The
results show that H
transport by the PPase is not
directly linked to K
transport in native vacuolar
membranes from V. vinifera.
Unless otherwise stated, aliquots of PBFI and pyranine (1
mM and 0.5 mM final, respectively) were added to a
vacuolar membrane vesicle suspension (150 µg of proteins)
containing 9% (v/v) glycerol, 2 mM Mg-MES, 10 mM potassium iminodiacetate (KIDA), and 50 mM BTP-MES (pH
7.5). This mixture was frozen in liquid nitrogen, thawed at room
temperature, and sonicated twice for 1 min. Non-encapsulated probes
were removed by gel filtration through a Sephadex G-50 column. The
volume recovered was within 90-100% of the applied volume. A
10-µl aliquot (12 µg of proteins) of this volume was added to
the assay cuvette (2 ml final) containing 9% glycerol, 2 mM Mg-MES, 10 mM KNO or KIDA, 100 µM EGTA, 50 nM of the permeant dye oxonol VI, and 50 mM BTP-MES (pH 7.5). The cuvette was stirred and maintained at 30
°C.
Fluorescence intensities of the probes pyranine, PBFI, and
oxonol VI were simultaneously measured with a multichannel photon
counting spectrofluorometer SLM-Aminco 8000 C interfaced with an IBM
computer as described previously (Venema et al., 1993). The
T-shaped configuration of this apparatus allows two simultaneous
fluorescence measurements using two emission channels A and B (fitted
with a monochromator and a 495-nm long-pass cut on filter,
respectively). Furthermore, different excitation/emission wavelengths
can be selected during brief macroprogrammed acquisition cycles.
Briefly, channel B was used to acquire pyranine signals after
excitation at 460 and 410 nm, corresponding to the non-protonated and
protonated species, respectively. Channel B was also use to acquire
alternatively the PBFI signal after excitation at 336 nm. Channel A was
used to acquire the oxonol signal at 646 nm after excitation at 614 nm.
Each individual fluorescence signal was first ratioed with the signal
of a rhodamine reference cell (channel C) and corrected for the
fluorescence of the buffer solution. The oxonol response was calibrated
by imposing a H diffusion gradient through tonoplast
membranes suspended in the same medium as that described for
measurements plus 3 µM FCCP. A calibration curve was
obtained: E
= -20.6 (F/F
)
+ 154.4 F/F
+ 5.88, where E
is the Nernst membrane potential for H
and F is the maximum fluorescence minus the final fluorescence (F
) obtained after clamping the potential to zero
with 30 mM KSCN. In experiments where the PPase-generated
potential was measured, F was corrected by the percentage of
active vesicles (right-side-out vesicles). The ratio of the
unprotonated to protonated forms of pyranine corrected by the
percentage of active vesicles (sealed right-side-out) was used to
monitor the internal pH changes in the vesicles. The signal was
calibrated using the following equation: pH = 7.6 + 2.9 log (F
). The calibration curve was obtained as
for oxonol calibration by imposing pH gradients through tonoplast
membranes in the presence of 100 nM valinomycin plus 3
µM FCCP. Potassium concentration in the vesicles was
monitored using the PBFI signal ratioed by the pyranine signal at its
420-nm isosbest.
The quenching of the fluorescence of the permeant amines quinacrine (5 µM) and ACMA (1 µM) was assayed in the same standard medium described above. The excitation and emission wavelengths were 420 and 495 nm for quinacrine and 415 and 485 nm for ACMA, respectively.
The hydrolytic
activities of the two vacuolar membrane markers in the absence and in
the presence of the permeabilizing agent Triton X-100 (0.02%) gave
similar latencies (about 30%) (Table 1), suggesting that 70%
of vacuolar membrane vesicles were oriented right-side-out. The basal
activity of the PPase (i.e. the one of right-side-out vesicles
in the absence of permeabilizing agent) assayed in the presence of KIDA
was 2-fold stimulated by the protonophore FCCP (Table 2), proving
that native vesicles were tightly sealed to H
and
small ions. These data together indicate that cell suspensions of V. vinifera are a suitable source of native vacuolar
membrane vesicles for studying the transport properties of the PPase in
the direct mode (PP
hydrolysis and H
translocation into the vacuole).
Figure 1:
Effect of NO and IDA
on the pH gradient and membrane
potential generated by the PPase. Vacuolar membrane vesicles (12 µg
of protein) in 9% glycerol, 2 mM Mg-MES, 10 mM KIDA,
50 mM BTP-MES (pH 7.5), containing 0.5 mM pyranine
were added to 2 ml of reaction medium containing 9% glycerol, 2 mM Mg-MES, 100 µM EGTA, 50 nM oxonol VI, 50
mM BTP-MES (pH 7.5), and 10 mM KIDA (A) or
10 mM KNO
(B). At the indicated times (dashed lines) aliquots of PP
(0.15 mM),
valinomycin (20 nM), or FCCP (5 µM) were added to
the reaction medium. Internal pH and membrane potential were determined
from the variations of the pyranine and oxonol signals, respectively,
as described under ``Experimental
Procedures.''
The anionic
selectivity disappeared and a maximum basal activity was reached in the
presence of the protonophore FCCP (Table 2), which totally
dissipated both the pH gradient and the membrane potential gradient
generated by the PPase (Fig. 6A). This confirms that
the stimulation of PPase hydrolytic activity by anions depends on their
capacity to dissipate the membrane potential and not on some specific,
direct interaction with the PPase. We conclude that the PPase from V. vinifera is a highly electrogenic H proton pump, and its activity is sensitive to both electrical and
osmotic components of the H
electrochemical gradient.
Figure 6:
Simultaneous determinations of membrane
potential, H, and K
concentrations in
vacuolar membrane vesicles after PPase activation in the presence of
FCCP and 10 mM K
. Measurements were made in a
medium containing 9% glycerol, 2 mM Mg-MES, 100 µM EGTA, 10 mM KIDA, 5 µM FCCP, and 50 mM BTP-MES (pH 7.5) in the absence (A) or the presence (B) of 0.08% (w/v) DOC. At the indicated times (dashed
lines), aliquots of PP
(0.15 mM), valinomycin
(20 nM), and KIDA were added to the reaction medium. Internal
pH and membrane potential were, respectively, determined from the
variations of the pyranine and oxonol signals as described under
``Experimental Procedures.'' Internal K
was
monitored using the PBFI fluorescence ratio after excitation at 336 and
420 nm.
Figure 2:
Response of the pH probes ACMA (A) and pyranine (B) to PPase energization in the
absence or the presence of K. Vacuolar membrane
vesicles (12 µg of protein) containing 9% glycerol, 2 mM Mg-MES, 5 mM BTP-MES (pH 7.5), and 0.5 mM pyranine encapsulated as described under ``Experimental
Procedures'' were added to 2 ml of reaction medium containing 9%
glycerol, 2 mM Mg-MES, 100 µM EGTA, 1 µM ACMA, 10 mM BTP-NO
, and 5
mM BTP-MES (pH 7.5). At the indicated times (arrows),
aliquots of PP
(0.15 mM), FCCP (5
µM), or (NH
)
(10
µM) were added to the reaction medium. Fluorescence
signals are given in arbitrary units.
Figure 3:
Stimulation of vacuolar membrane PPase
hydrolytic and proton pumping activities by K.
Vacuolar membrane vesicles suspended in 0.25 M sucrose, 2
mM Mg-MES, and 10 mM BTP-MES (pH 7.5) were diluted in
a reaction medium of the same composition plus 100 µM EGTA
and 0-140 mM KNO
. PP
hydrolysis (A) was measured on the same samples as H
transport (B). The later was monitored by the quenching
of quinacrine. PanelC represents the relationship
between the data of panelsA and B.
Therefore, the use of the
non-permeant dye pyranine demonstrated that H translocation by the PPase does not actually depend on the
presence of K
, as expected from an obligatory
H
/K
cotranslocation mechanism.
It
has been proposed that each of the transport sites of the PPase might
be occupied by K or H
, resulting in a
variable H
/K
stoichiometry (Davies et al., 1992). In such a system, a pure H
transport (0 K
/n H
)
would be possible and would explain the results of Fig. 2. We
further examined the K
stimulation of the PPase by
measuring hydrolytic and H
pumping activities in the
same assay sample. A permeant amine dye (quinacrine) was used in this
experiment despite a lower sensitivity than pyranine because such a dye
was not saturated upon establishment of high pH gradients. Both
H
pumping and hydrolytic activities were strongly
stimulated by K
(K
= 10
mM) but remained linearly correlated (Fig. 3), which
suggests that K
did not compete with H
ions for a common translocation site, as previously suggested
(Davies et al., 1992).
The hypothesis of a
K/H
cotranslocation by the PPase was
also examined by studying the effect of K
and
H
ionophores on the PPase hydrolytic activity. A
stimulation of the hydrolytic activity should be expected from the
dissipation of such electrochemical gradients. As showed in Table 2, FCCP strongly stimulated the hydrolytic activity of the
enzyme in agreement with the total dissipation of the large
H
electrochemical gradient generated by the pump ( Fig. 1and 6A). Addition of the K
ionophore valinomycin to dissipate the putative K
electrochemical gradient did not stimulate the activity,
irrespective of the K
concentration used in the assay
medium ( Table 2and Fig. 4). Moreover, no additional
stimulation was observed when valinomycin was used together with FCCP.
On the other hand, high stimulations were observed in the presence of
the H
/K
exchanger nigericin. These
experiments indicate that the PPase responded to the dissipation of the
electrochemical gradient of H
but not to that of
K
.
Figure 4:
Effect of H and
K
ionophores on the PPase hydrolytic activity at
different K
concentrations. Vacuolar membranes
(1-2 µg) suspended in 0.25 M sucrose, 2 mM Mg-MES, and 10 mM BTP-MES (pH 7.5) were diluted in a
reaction medium containing 0.25 M sucrose, 2 mM Mg-MES, 100 µM EGTA, 10 mM BTP-MES (pH 7.5),
and 0-150 mM KNO
. Where appropriate,
valinomycin, FCCP or nigericin (5 µM) were added. The
ionic strength was kept constant over all the range of K
concentrations by adding BTP-NO
(BTP
+ K
= 150
mM).
In
the first experiment (Fig. 5A), the PPase was activated
in the presence of the non-permeant anion IDA. Energization of the
PPase generated a membrane potential up to 270 mV, and a small active
H influx associated with a passive K
efflux. This could mean either that the PPase does not transport
K
in these conditions or that K
is
recirculated by, for example, a K
channel. Addition of
valinomycin caused a rapid additional K
efflux
associated with a transient depolarization of the vacuolar membrane.
This suggests that the vacuolar membrane did not contain an efficient
K
uniport system, capable of short-circuiting the
PPase at low internal K
concentration (10
mM). When the PPase was energized after addition of
valinomycin (Fig. 5B), the initial observed membrane
potential was lower (200 mV). This was associated with a higher
K
efflux, as shown by the 4-fold increase in the rate
of PBFI fluorescence variation. However, after a few minutes, the
membrane potential was similar to that observed in the absence of the
K
ionophore. Thus, valinomycin failed to permanently
short-circuit the PPase, owing to the small intravesicular volume which
was rapidly depleted in K
. This result is in agreement
with the absence of a stimulation of the hydrolytic activity by
valinomycin ( Table 2and Fig. 4), since hydrolysis was
measured over a 30-min period.
Figure 5:
Simultaneous determinations of membrane
potential, H, and K
concentrations in
vacuolar membrane vesicles after PPase activation in the absence or the
presence of valinomycin. Vacuolar membranes were incubated during 10
min in the absence (A) or the presence (B) of 20
nM valinomycin. The reaction medium contained 9% glycerol, 2
mM Mg-MES, 100 µM EGTA, 10 mM KIDA, and
50 mM BTP-MES (pH 7.5) with or without valinomycin. At the
indicated times (dashed lines) PP
(0.15
mM), valinomycin (20 nM), KIDA (12 mM), or
FCCP (3 µM) were added to the reaction medium. Internal pH
and membrane potential were, respectively, determined from the
variations of the pyranine and oxonol signals as described under
``Experimental Procedures.'' Internal K
was
monitored using the PBFI fluorescence ratio after excitation at 336 and
420 nm.
In a second experiment (Fig. 6A), the H electrochemical
gradient was clamped to zero using FCCP, which led to a 2-fold increase
in the hydrolytic activity (Table 2). According to the
H
/K
cotranslocation model, a
K
influx should be observed after energization. Data
in Fig. 6A indicate that the internal K
concentration did not vary after energization of the pump.
However, a K
influx could have been detected in such
an experiment. After valinomycin addition, K
supply to
the outside induced a significant increase in the PBFI signal,
associated with an alkalinization of the interior of the vesicles, as
expected from the catalysis of H
and K
fluxes by the two ionophores. When the vesicles were
permeabilized with 0.08% (w/v) deoxycholate (DOC), only the PBFI
response was observed, confirming that the
H
/K
exchange previously observed was
due to a true coupled process (Fig. 6B).
A
H/K
antiporter has been shown at the
vacuolar membrane of Brassica napus (Cooper et al.,
1991). It could therefore be hypothesized that the putative
K
electrochemical gradient generated by the PPase
would be partly dissipated by such a system and transformed into an
additional H
influx. However, imposition of inward
K
gradient to non-energized membranes did not create a
detectable H
efflux (Fig. 7), in spite of the
high sensitivity of the pyranine probe. Addition of FCCP and
valinomycin was necessary to observe a H
/K
exchange. Finally, energization of the PPase in a pH clamping
experiment using FCCP was repeated in the presence of 50 mM K
(Fig. 8), a condition thought to inhibit
the oilseed rape H
/K
antiporter
(Cooper et al., 1991). In the absence of a permeant anion,
active K
translocation by the PPase should produce an
alkalinization of the interior of the vesicle associated with the
establishment of a H
diffusion potential (positive
inside). Neither pyranine nor oxonol responded to pump energization
under these conditions (PBFI fluorescence was not measured since it is
saturated at 50 mM K
). Thus, the hypothesis
of a H
/K
antiport was ruled out.
Figure 7:
Alkalinization of non-energized vacuolar
membrane vesicles in response to an imposition of a K gradient. The reaction medium contained 9% glycerol, 2 mM Mg-MES, 100 µM EGTA, 10 mM KIDA, 50 mM BTP-MES (pH 7.5) (A) plus 5 µM FCCP and 50
nM valinomycin (B), or 5 µM FCCP, 50
nM valinomycin, and 0.08% (w/v) DOC (C). At the
indicated times (dashed lines) aliquots of KIDA (10
mM) were added to the reaction medium. Internal pH was
determined from the variations of the pyranine signals as described
under ``Experimental
Procedures.''
Figure 8:
Simultaneous determinations of membrane
potential and H concentration in vacuolar membrane
vesicles after PPase activation in the presence of FCCP and 50 mM K
. Vacuolar membranes vesicles were suspended in
a medium containing 9% glycerol, 2 mM Mg-MES, 50 mM KIDA, and 5 mM BTP-MES (pH 7.5). Measurements were made
in a medium containing 9% glycerol, 2 mM Mg-MES, 100
µM EGTA, 50 mM KIDA, 6 µM FCCP, and
5 mM BTP-MES (pH 7.5). At the indicated times (dashed
lines) aliquots of PP
(0.15 mM), valinomycin
(20 nM), and KIDA (200 mM) were added to the reaction
medium. Internal pH and membrane potential were respectively determined
from the variations of the pyranine and oxonol signals as described
under ``Experimental
Procedures.''
PP hydrolysis by the vacuolar membrane
PPase from V. vinifera suspension cells was strongly
stimulated by K
(Fig. 3). However, we showed
that a significant H
translocation occurred in the
absence of K
(Fig. 2). This indicates that the
putative K
translocation site of the PPase does not
display a strict requirement for K
. The fractional
K
/H
stoichiometry of transport (1.7
K
/1.3 H
) deduced from the
electrophysiological measurements in vacuoles from red beet
roots suggests that K
and H
could
compete for three transport sites (Davies et al., 1992).
However, the relationship between H
translocation and
PP
hydrolysis remained linear when using increasing
concentrations of K
(Fig. 3), indicating that
K
and H
probably did not compete for
the same translocation sites. Despite the tight energetic coupling
between PP
hydrolysis and the H
electrochemical gradient, demonstrated by the large stimulation
of hydrolysis by FCCP, the K
ionophore valinomycin had
no effect on PPase hydrolytic activity at any K
concentration used ( Table 2and Fig. 4). The strong
stimulation of the PPase by K
does not seem to be
associated with the creation of a K
electrochemical
gradient, as would be expected from an electrogenic cotranslocation of
K
and H
.
Finally, the hypothesis
of a K translocation by the PPase has been examined
directly using the PBFI probe under conditions (10 mM K
) where PBFI could detect K
net
influx as well as net efflux ( Fig. 5and Fig. 6).
Clamping the membrane potential at zero using FCCP, we have shown that
the energization of the PPase does not affect the K
equilibrium (Fig. 6).
We also examined the existence of
a K/H
antiporter with a high affinity
for K
, as demonstrated in vacuolar membrane from
oilseed rape hypocotyls (Cooper et al., 1991). Indeed, the
cooperation between a H
/K
active
symporter (i.e. the PPase) and such an antiporter would be
expected to partly dissipate the K
electrochemical
gradient. Nevertheless, K
/H
exchange
across non-energized vacuolar membrane from V. viniferacould not be observed in the absence of H
and K
ionophores (Fig. 7). Since the
antiporter described in the vacuolar membrane of rape hypocotyls was
inhibited at a K
concentration greater than 50
mM, the response of the pH probe pyranine was studied after
energization of the PPase at a high K
concentration
and in the presence of FCCP (Fig. 8). No net H
efflux was detected as expected from the activation of a
H
/K
cotransporting PPase in the
presence of a protonophore.
In conclusion, the present study of the in vitro energization of the PPase in native vacuolar membrane
vesicles from V. vinifera does not confirm the
H/K
cotranslocation indicated by the
electrophysiological analysis performed on isolated vacuoles from B. vulgaris (Davies et al., 1992). Using
K and a highly purified and reconstituted PPase, Sato and
colleagues(1994) also could not find evidence of such an active
K
translocation. The use of native vacuolar membrane
vesicles makes unlikely the possibility of a regulation of PPase
transport properties by other polypeptides present in the native
vacuolar membrane such as the 20-24-kDa polypeptides previously
described in the literature (Britten et al., 1992). In some
experiments, we used a vesicle sonication and freeze-thawing procedure
to load the non-permeant fluorescent indicators. Only 2% of the
membrane proteins were lost as a result of this procedure, but neither
a change in the PPase specific activity nor in its stimulation by
K
and H
ionophores was observed (data
not shown). Furthermore, it has been found (Kim et al., 1994)
that the heterologous expression of the gene encoding for the 66-kDa
PPase unit in yeast is sufficient to fully restore all the known
properties of this enzyme, including its K
stimulation. It is important to emphasize that our study and that
of Sato and colleagues(1994) were performed in the direct mode
(PP
hydrolysis), whereas the electrophysiological one was
performed in the reverse mode (PP
synthesis) and that these
studies were made on different species. We conclude that K
transport is not a general property of the vacuolar membrane
PPase. Like this enzyme, the plant plasma membrane ATPase was thought
to transport K
in addition to H
, as a
result of the stimulatory effect of K
on its activity.
This hypothesis has been made unlikely because (i) there is a
significant H
transport in the absence of
K
, provided that permeant ions ensure electrical
charge compensation for the H
translocation, and
(ii) the K
site is on the wrong side of the
ATPase (Gibrat et al., 1990). The stimulation of ATPase and
PPase activities by K
may reflect some structural
requirement for this cation, which is the major ionic species in the
natural environment of these enzymes.