Plant Physiol. (1985) 77, 329-334
0032-0889/85/77/0329/06/$01.00/0
Electrogenic Proton Translocation by the ATPase of Sugarcane
Vacuoles'
Received for publication August 1, 1984 and in revised form October 19, 1984
MARGARET THOM* AND EWALD KOMOR
Hawaiian Sugar Planters'Association, Aiea, Hawaii 96701 (M.T.); and Botanisches Institut der
Universitit Bayreuth, D8580, Bayreuth, West Germany (E.K.)
ABSTRACT
Existence of a proton-translocating ATPase on the tonoplast of higher
plants has been further confirmed by use of two experimental systems:
(a) intact isolated vacuoles from sugarcane cells and (b) vesicles prepared
from the same source. Addition of MgATP to vacuoles polarized the
tonoplast by 40 millivolts to a value of +20 millivolts, but a large
preexisting pH gradient across the membrane restricted the pH change
to 0.2 unit. In vesicle preparations, the tonoplast was polarized to +66
millivolts by the addition of MgATP and the intravesicular space was
acidified by 1 pH unit to pH 5.5. Proton translocation equilibrium is
controlled by the protonmotive potential difference, maximal at 125
millivolts for sugarcane cells. Energization of the tonoplast occurred at
physiological concentrations of MgATP. Specificity of MgATP for proton translocation was indicated by a much smaller effect of MgADP and
MgGDP on the electrochemical gradient, although these substrates were
also hydrolyzed by tonoplast preparation.
In recent years, availability of better methods of isolating intact
functional vacuoles from higher plants has permitted better
insight into the physiology and biochemistry of this organelle (5,
20,25). A tonoplast-bound ATPase which functions in energizing
the membrane exists in higher plants (4, 24, 26). The evidence
for this phenomenon comes predominantly from small fungal
vacuoles (10), lysosome-like membrane vesicles (13), and microsomal membrane fractions (3,6, 19). Evidence from intact, largesized, higher plant vacuoles is less abundant. Wagner and Lin
(26) reported that ATP added to tulip vacuoles caused an acidification of the intravacuolar space and a slight polarization of
the membrane potential. Miller et al. (15) noted a small depolarization of the membrane potential of red beet vacuoles in the
presence of ATP. These findings are in agreement with widespread observations that plant vacuoles, in situ, have an acidic
interior (9) and possess a positive membrane potential oriented
toward the cytoplasm (2).
Intact vacuoles can be isolated from sugarcane cells grown in
suspension cultures. These isolated vacuoles hydrolyze ATP (23,
24) and transport sugars against a concentration gradient (22).
Addition of MgATP to isolated sugarcane vacuoles polarized the
membrane potential towards a more positive value, but the
voltage when measured as SCN- accumulation was significantly
less (25 mv) than had been measured for vacuoles in situ (60
mv) (11, 24). A concentration of 0.5 M sucrose has been measured
in vacuoles of sugarcane. Hence, these cells provide a suitable
experimental system to study the mechanism of sugar accumulation.
In this study, optical probes which give continuous responses
were used to measure the membrane potential and pH gradient
of isolated vacuoles and of tonoplast vesicles prepared from
similar vacuoles. The effect of MgATP on the electrochemical
gradient was also determined. The results indicate that the tonoplast-bound ATPase of sugarcane functions as a proton translocator.
MATERIALS AND METHODS
Plant Material and Vacuole Isolation. Sugarcane cell suspensions (a subclone of Saccharum sp. hybrid H50-7209) were grown
in White's inorganic salt mixture supplemented with yeast extract, arginine, sucrose, vitamins, and 2,4-D (17). The cells,
harvested 9 d after subculture, constituted 3 to 4 g fresh weight/
100 ml medium. Vacuoles were isolated from protoplasts by
centrifugation on a Ficoll cushion (21). Tonoplast vesicles were
prepared by breaking vacuoles in a glass homogenizer (about 40
strokes) in a large volume of medium containing 25 mm TricineMes (pH 6.5), 10 mm MgSO4, and 250 mM mannitol. The
membrane suspension was layered over 10% dextran T70 in
homogenization buffer and centrifuged at 100,000g for 60 min.
The membrane fraction used for experiments sedimented in a
layer above the dextran. The volumes of vacuoles and tonoplast
vesicles were calculated from the water-permeable but dextranimpermeable space.
Membrane Potential Measurements. Absorption changes of
oxanol VI' and di O-C5(3) were used to measure membrane
potential (1). Vacuoles or tonoplast suspension was added to a
solution containing 25 mM Tricine-Mes (pH 6.5), 10 mm MgSO4,
4 AM of the optical probe, and 500 mM (vacuoles) or 250 mM
mannitol (tonoplasts). The change in absorbance at 603 and 580
nm for oxanol VI and 500 and 480 nm for di O-C5(3) was
followed on a dual wavelength spectrophotometer. Artificially
imposed K+ gradients in the presence of 10 fg ml-' valinomycin
were used to calibrate the membrane potential. Membrane potential was calculated using the change in absorbance from the
time immediately after the initial shift caused by addition of
MgATP until the steady state value is reached. Potassium diffusion potential was calculated according to the Nernst equation.
Potassium concentration was measured using a specific ion electrode.
pH Measurements. The internal pH of vacuoles and tonoplast
'Supported by a grant from the Deutsches Forschungsgemeinschaft.
2 Abbreviations: oxanol VI, bis(3-propyl-5-oxoisoxazol-4-yl)pentoPublished with the approval of the Director as Paper No. 582 in the
Journal Series of the Experiment Station, Hawaiian Sugar Planters' methine oxanol; di O-C5(3), 3,3'-dipentyloxacarbocyanine iodide; FCCP,
Association.
p-trifluoromethoxycarbonyl cyanide.
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THOM AND KOMOR
330
Plant Physiol. Vol. 77, 1985
calibration of the response showed the same MgATP-catalyzed
polarization of the membrane potential (Table I).
When the 'resting potential' of vacuoles was decreased to -10
mv by addition of 100 mm KCl, MgATP-dependent polarization
was reduced to 34 mv (Fig. 2). The plateau level of the membrane
,M acridine orange was added and changes in fluorescent emis- potential was, therefore, the same as in the control, i.e. without
sion were measured at 425 505 nm (excitation emission) additional K+. The addition of Ki after MgATP-induced polarfor quinacrine or 493 530 nm for acridine orange. The pH ization had no further effect on the membrane potential (Fig. 3).
gradient was calculated from the fluorescence quench and the The sensitivity of the membrane potential to FCCP and to
volume of vacuoles or tonoplast vesicles.
valinomycin plus nigericin suggests that the positive polarization
Chemicals. Oxanol VI and di O-C5(3) were gifts from G. catalyzed by MgATP is caused by proton translocation.
Hauska (Regensburg, West Germany). Fluorescent amines were
Membrane Potential of Tonoplast Vesicles. In the presence of
from Sigma Chemical and nigericin and valinomycin were from MgATP, the membrane potential of intact vacuoles was positive,
Calbiochem.
but the value was much lower than values reported for microsomal fractions (3). Tonoplast vesicles derived from sugarcane
vacuoles exhibited a greater response to MgATP than did intact
RESULTS
vacuoles. The membrane potential change was more than 60 mv
Membrane Potential of Isolated Vacuoles. Isolated vacuoles of (Table I) and was completely reversed by valinomycin plus
sugarcane have a negative inside membrane potential when nigericin (Fig. 4). This quantitative difference between vacuoles
measured as the accumulation of tetraphenylphosphonium ion and
tonoplast vesicles may be due either to the efflux of easily
(11), but the value is close to zero when measured as the permeable
intravacuolar cations acting to counterbalance the
accumulation of thiocyanate (24). Both values must be regarded MgATP-catalyzed
influx and thereby reducing the degree
with caution since it is difficult to correct for a significant amount of polarization, or cation
to
the
pH gradient in intact vacuoles,
of probe adsorption. Furthermore, each of these probes is appli- which, for thermodynamiclarger
reasons, stalls the membrane potencable only if the membrane potential is significantly greater than tial
at a lower value.
zero in either direction. Optical probes were substituted in this
Gradient of Isolated Vacuoles. Isolated vacuoles retained
study to assess the reliability of the previous measurements and anpH
acid
interior (1 1). Addition of vacuoles to a solution of the
to follow the effects of nucleotides and uncouplers. Oxanol VI
amine quinacrine caused a fluorescence quench which
permeant
to
vacuoles
Addition
of
gave the largest response.
valinomycin
indicated
a
pH gradient of 1.4 units (Table II). The addition of
in the 'resting state,' i.e. without addition of MgATP, changed
caused
a very slight, barely detectable acidification of
MgATP
the oxanol VI absorption only slightly. Therefore, the membrane
potential in the resting state is very close to the potassium 0.2 pH unit (Table II). Addition of valinomycin plus nigericin
diffusion potential which was -26 mv (Table I). The internal collapsed the pH gradient and reversed the fluorescence quench.
concentration of potassium in these vacuoles was 10.5 mm. The It is difficult to reconcile this small acidification of the intraaddition of the uncoupler FCCP caused additional polarization vacuolar space with the concept of proton translocation by the
of the tonoplast to -79 mv (Table I)-an expected consequence tonoplast-bound ATPase. However, it is possible that either the
preexisting pH gradient does not allow further acidification, or
if the gradient of 1.4 pH units was abolished.
The addition of MgATP caused a rapid decrease of oxanol VI the buffering capacity of the vacuolar solute makes it difficult to
absorption followed by a new steady state after 2 min (Fig. 1, detect a pH change. These possibilities were tested with tonoplast
plot A). This change corresponded to about a 40 mv positive vesicles from which intravacuolar solutes were removed during
polarization ofthe membrane potential. In the presence of FCCP, their preparation.
pH Gradient of Tonoplast Vesicles. A much more vivid effect
the MgATP-dependent polarization of the membrane potential
was much slower and smaller (Fig. 1, plot B). Valinomycin and of MgATP-dependent quenching of both acridine orange (Fig.
nigericin completely inhibited the response (Fig. 1, plot C). 5) and quinacrine (Fig. 6) fluorescence was obtained when toMgADP addition caused only a small change of the membrane noplast vesicles were used. The pH gradient catalyzed by MgATP
was 0.9 to 1.2 units (Table II). The addition of valinomycin plus
potential (Fig. 1, plot D; Table I).
When 4 AsM of the carbocyanine dye, di O-CA(3), was used to nigericin collapsed the pH gradient and reversed the fluorescence
measure membrane potential, results paralleled those obtained quenching (Figs. 5 and 6). When valinomycin plus nigericin was
with oxanol VI. The changes in absorbance were smaller but the added before the addition of MgATP, the acidification was
vesicles
was
measured
as
the fluorescence quenching of the
permeant amines quinacrine and acridine orange ( 12). Vacuoles
or tonoplast vesicles were suspended in 25 mm Tricine-Mes (pH
6.5), 10 mm MgSO4, 5 mM KC1, and either 500 mM (vacuoles)
or 250 mM mannitol (tonoplasts). Either 10 jAM quinacrine or 5
--
--
--
Table I. Membrane Potential of Sugarcane Vacuoles and Tonoplast Vesicles
Membrane potential was determined as described in "Materials and Methods." MgATP and MgADP were
added to give a final concentration of 0.9 mm and FCCP final concentration was 50 gM. Values are the mean
of at least three determinations ± SD.
Change of Membrane
Plant
Probe
Addition Final Membrane
Material
Potential by Addition
Potential
mv
Vacuoles
K+
-26 ± 11
oxanol VI
-23 ± 6
oxanol VI
FCCP
-79 ± 20
-54 ± 28
oxanol VI
+19 ± 12
+42 ± 19
MgATP
+14 ± 7
di O-C5 (3) MgATP
+39 ± 19
-14 ± 6
+7 ± 5
oxanol VI
MgADP
Tonoplast vesicles
oxanol VI
oxanol VI
MgATP
0
+66 ± 5
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Copyright © 1985 American Society of Plant Biologists. All rights reserved.
+66 ± 5
PROTON TRANSLOCATION IN SUGARCANE VACUOLES
FIG. 1. Change in absorbance of oxanol VI by vacuoles. Vacuoles (20
Ml internal volume, 200 mg protein) were suspended in 1 ml 25 mM
Tricine-Mes (pH 6.5), 10 mM MgSO4, and 500 mM mannitol and 4 ;uM
of oxanol VI. MgATP or MgADP was added to give a final concentration
of 0.9 mM MgATP2-. Final FCCP concentration was 50 ,M; valinomycin
and nigericin, 5 gM each. A, Change in absorbance on addition of
MgATP; B, effect of FCCP on ATP-dependent change in absorbance; C,
effect of valinomycin plus nigericin on ATP-dependent change in absorbance; D, change in absorbance on addition of MgADP.
inhibited. Valinomycin alone did not prevent the MgATP-dependent acidification (Fig. 6), but the subsequent addition of
nigericin not only prevented acidification but collapsed the pH
gradient. MgADP and MgGDP had no effect on the internal pH
of tonoplast vesicles (Fig. 6; Table II). Results from these experiments confirm previous findings (I 1, 24) that the tonoplastbound ATPase of sugarcane catalyzes electrogenic proton translocation.
DISCUSSION
The membrane potential of isolated sugarcane vacuoles was
in the negative range (-20 to -30 mv), similar to the range in
lutoids (7) and sugar beet vacuoles (8, 15). The addition of
MgATP shifted the membrane potential toward the more positive direction by 40 to 50 mv so that it was in the range of 20
mv. In lutoids and sugar beet, the membrane potential remained
negative with the addition of ATP (7, 8, 15), and in tulip vacuoles
the ATP-catalyzed polarization was only 2 to 5 mv (26). This
difference may arise either from (a) different techniques of measurement, (b) different physiological conditions of isolated vacu-
331
FIG. 2. MgATP-dependent shift in absorbance of oxanol VI after K+
addition. Experimental conditions were the same as described in Figure
1. K+ was added as KCL.
oles, (c) different plant material, or (d) different permeable
intravacuolar content. Reports on chloride stimulation (3) and
nigericin stimulation (20) of ATPase suggest that the vacuolar
ATPase is controlled by the proton potential so that the emphasis
of the protonic force shifts between the membrane potential and
the pH gradient. This phenomenon has been known since the
protonmotive force was first found in chloroplasts and mitochondria (16). A preexisting pH gradient in the deenergized
vacuoles might prevent a strong polarization by the ATPase.
Release of internal solutes and consequent dissipation of the pH
gradient during the preparation of tonoplast vesicles from sugarcane vacuoles led to a larger MgATP-catalyzed positive polarization of the membrane potential. This change of the membrane
potential (60-70 mv) is comparable to the published values for
other tonoplast vesicles (3, 13).
ATP-dependent acidification of the intravacuolar and intravesicular space has also been reported in red beet (4), oat root
(6), corn coleoptile (14), and tulip petals (26). In sugarcane,
however, very little MgATP effect was noted in isolated vacuoles,
while in tonoplast vesicles MgATP elicited greater acidification.
This difference was due not only to the greater surface/volume
ratio of vesicles, but also to the thermodynamic force of the
proton potential of intact vacuoles. In the presence of MgATP,
isolated vacuoles have an electrochemical gradient of 115 mv
and tonoplast vesicles of 125 mv. While this is a small difference,
the pH gradient is large in vacuoles; it was 1.5 pH units even in
the nonenergized state. The control of the energization by the
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332
Plant Physiol. Vol. 77, 1985
THOM AND KOMOR
FIG. 3. Effect of K+ addition of the MgATP-polarized membrane potential in vacuoles. Experimental conditions were the same as described in
Figure 1. K+ was added as KCL.
FIG. 4. MgATP-dependent shift in absorbance of
oxanol VI by tonoplast vesicles. Tonoplast vesicles
(10 ul internal volume, 115 Mg protein) were suspended in 3 ml 25 mm Tricine-Mes (pH 6.5), 10
mM MgSO4, and 250 mM mannitol and 4 Mm oxanol
VI at pH 6.5. Final concentration of MgATP was 3
mM; valinomycin, 5 uM; nigericin, 5 MM.
protonmotive potential difference was also seen in experiments
where the membrane potential was shifted by addition of K+ to
the medium. With the initial membrane potential depolarized
from -26 to -10 mv, the plateau value of the MgATP-catalyzed
membrane potential was similar to control conditions.
There was considerable GDP and ADP hydrolytic activity on
the tonoplast, but these nucleotides had little or no effect on the
membrane potential and pH gradient. This supports previous
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PROTON TRANSLOCATION IN SUGARCANE VACUOLES
333
Table II. Internal pH of Sugarcane Vacuoles and Tonoplast Vesicles
Internal pH was determined as described in "Materials and Methods." MgATP and MgADP were added to
give a final concentration of 3 mm. Values are the mean of at least three determinations ± SD.
Plant
pH
Change of pH
PoeAdto
pH
Material
Probe
Addition
pH
by Addition
Gradient
Vacuoles
quinacrine
5.09 ± 0.28
1.41
0.22 ± 0.08
1.63
quinacrine
MgATP 4.87 ± 0.18
Tonoplast vesicles
quinacrine
quinacrine
acridine orange
quinacrine
MgATP
MgATP
MgADP
6.5
5.27 ± 0.30
5.61 ± 0.27
6.28 ± 0.23
1.23 ± 0.30
0.89 ± 0.27
0.22 ± 0.23
0
1.23
0.89
0.22
MgATP
Val + Nig
41
Val
+
I
Nig
MgATP
a
,*
|- F
=0.1
5 min
FIG. 5. Fluorescence quenching of acridine orange by tonoplast vesicles. Tonoplast vesicles (30 Ml internal volume, 300 Mg protein) were
suspended in 1.5 ml 25 mm Tricine-Mes (pH 6.5), 10 mm MgSO4, 5 mM
KCl, 250 mm mannitol, and 5 ,M acridine orange at pH 6.5. Final
concentration of MgATP was 3 mM; valinomycin, 5 Mm; nigericin, 5 MM.
findings that the proton translocator is specific for MgATP (24)
and confirms that GDPase and ADPase do not participate in
formation of the electrochemical gradient. The ATP-dependent
positive polarization of the membrane potential and the acidification of the internal space of both intact vacuoles and tonoplast
vesicles confirm previous suggestions that the ATPase ofvacuoles
is a proton-translocating device.
Acknowledgments-We would like to thank Professor G. Hauska and Dr. E.
Hurt (Universitat Regensburg) for the membrane potential probes and for the use
of the Amico-Chance dual wavelength spectrophotometer and Dr. H. Yamamoto
(University of Hawaii, Honolulu) for the use of the Perkin-Elmer dual wavelength
spectrophotometer.
LITERATURE CITED
1. BASHFORD CL 1981 The measurement of membrane potential using optical
indicators. Biosci Rep 1: 183-196
2. BATES GW, MHM GOLDSMITH, TH GOLDSMITH 1982 Separation of tonoplast
and plasmalemma potential and resistance in cells of oat coleoptile. J Membr
Biol 66: 15-23
3. BENNETF AB, RM SPANSWICK 1983 Optical measurements of ApH and AO, in
corn root membrane vesicles: Kinetic analysis of Cl- effects on a protontranslocating ATPase. J Membr Biol 71: 95-107
4. BENNETT AB, SD O'NEILL, RM SPANSWICK 1984 H+-ATPase activity from
storage tissue of Beta vulgaris. I. Identification and characterization of an
anion-sensitive H -ATPase. Plant Physiol 74: 538-544
5. BOLLER T 1982 Enzymatic equipment of plant vacuoles. Physiol Veg 20: 247-
FIG. 6. Fluorescence quenching of quinacrine by tonoplast vesicles.
Tonoplast vesicles (20 Ml internal volume, 200Mug protein) were suspended
in buffer as described in Figure 5. Final concentration of quinacrine was
10OM.
257
6. CHURCHILL KA, H SZE 1983 Anion-sensitive, H'-pumping ATPase in membrane vesicles from oat root. Plant Physiol 71:610-617
7. CRETIN H 1982 The proton gradient across the vacuo-lysosomal membrane of
lutoids from the latex of Hevea brasiliensis. I. Further evidence for a protontranslocating ATPase on the vacuo-lysosomal membrane of intact lutoids. J
Membr Biol 65: 175-184
8. DOLL R HAUER 1981 Determination of the membrane potential of vacuoles
isolated from red beet storage tissue. Evidence for an electrogenic ATPase.
Plant 152: 153-158
9. GUILLERMO A 1941 The Cytoplasm of the Plant Cell. Chronica Botanica Co.,
Waltham, MA
10. KAKINUMA Y, Y OHSUMI, Y ANRUKU 1981 Properties of H'-translocating
adenosine triphosphatase in vacuolar membranes of Saccharomyces cerevi5,
siae. J Biol Chem 256: 10859-10863
11. KOMOR E, M THOM, A MARETZKI 1982 Vacuoles from sugarcane suspension
cultures. III. Protonmotive potential difference. Plant Physiol 69: 1326-1330
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1985 American Society of Plant Biologists. All rights reserved.
334
THOM ANC) KOMOR
12. LEE HC, JG FORTE, D EPEL 1982 The use of fluorescent amines for the
measurement of pH: Application of lysosomes, gastric microsomes, and sea
urchin gametes. In R Naccitelli, DW Deamer, eds, Intracellular pH: Its
Measurement, Regulation and Utilization in Cellular Functions. Liss Incorporation, New York, pp 135-160
13. MARIN B, M MARIN-LANZA, E KOMOR 1981 The protonmotive potential
difference across the vacuo-lysosomal membrane of Hevea brasiliensis (rubber tree) and its modification by a membrane-bound adenosine triphosphatase. Biochem J 198: 365-372
14. METTLER IJ, S MANDALA, L TAIZ 1982 Characterization of in vitro proton
pumping by microsomal vesicles isolated from corn coleoptiles. Plant Physiol
70: 1738-1742
15. MILLER AJ, JJ BRIMELOW, P JOHN 1984 Membrane-potential changes in
vacuoles isolated from storage roots of red beet (Beta vulgaris L.). Plant
160: 59-65
16. MITCHELL P 1968 Chemiosmotic coupling and energy transduction. Glynn
Res. Ltd., Bodmin, England
17. NICKELL LS, A MARETZKI 1969 Growth of suspension cultures of sugarcane
cells in chemically defined media. Physiol Plant 22: 117-125
18. OKOROKOV LA, LP LICHKO 1983 The identification of a proton pump on
19.
20.
21.
22.
23.
24.
25.
26.
Plant Physiol. Vol. 77, 1985
vacuoles of the yeast Saccharomyces carlbergensis. ATPase is electrogenic
H+-translocase. FEBS Letts 155: 102-106
STOUT RG, RE CLELAND 1982 Evidence for a Cl-stimulated MgATPase
proton pump in oat root membranes. Plant Physiol 69: 798-803
SzE H 1980 Nigericin-stimulated ATPase in microsomal vesicles of tobacco
callus. Proc Natl Acad Sci USA 77: 5904-5908
THOM M, A MARETZKI, E KOMOR 1982 Vacuoles from sugarcane suspension
cultures. I. Isolation and partial characterization. Plant Physiol 69: 13151319
THOM M, E KOMOR, A MARETZKI 1982 Vacuoles from sugarcane suspension
cultures. II. Characterization of sugar uptake. Plant Physiol 69: 1320-1325
THOM M, J WILLENBRINK, A MARETZKI 1983 Characteristics of ATPase from
sugarcane protoplast and vacuole membranes. Physiol Plant 58: 497-504
THOM M, E KOMOR 1984 Role of ATPase of sugarcane vacuoles in energization
of the tonoplast. Eur J Biochem 138: 93-99
WAGNER GJ 1979 Content and vacuole/extravacuole distribution of neutral
sugars, free amino acids and anthocyanins in protoplasts. Plant Physiol 64:
88-93
WAGNER GJ, W LIN 1982 An active proton pump of intact vacuoles isolated
from Tulipa petals. Biochim Biophys Acta 689: 261-266
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1985 American Society of Plant Biologists. All rights reserved.