Proton Transport in Isolated Vacuoles from Corn Coleoptiles

Plant Physiol. 1985) 78, 104-1090032-0889/85/78/0 104/06/$0 1.00/0

Proton Transport in Isolated Vacuoles from Corn Coleoptiles'Received for publication April 27, 1984 and in revised form January 18, 1985

SUZANNE MANDALA AND LINCOLN TAIZ*Biology Department, Thimann Laboratories, University of California, Santa Cruz, Santa Cruz,California 95064

ABSTRACT

Vacuoles were isolated from corn coleoptile protoplasts and ATP-dependent proton transport was measured by quinacrine fluorescencequenching or by the uptake of l'4Clmethylamine. Intact vacuoles werejudged to be free of a surrounding plasma membrane based on fluorescentstaining with fluoroscein-diacetate. Essentially all of the detectable ATP-stimulated methylamine uptake and a-mannosidase activities present inintact protoplasts were recovered in isolated vacuoles. In contrast, theactivities of marker enzymes for plasma membranes, Golgi, endoplasmicreticulum, and mitochondria were reduced to 5 to 17% in vacuolarpreparations. The characteristics of proton pumping by isolated vacuoleswere compared to those of light microsomal membranes possibly derivedfrom the tonoplast. ATP-dependent proton pumping by both isolatedvacuoles and light microsomal vesicles was stimulated by Cl1, andinhibited by NO3-, carbonyl cyanide-m-chlorophenylhydrazone, N,N'-dicyclohexylcarbodiimide, N-ethylmaleimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, diethylstilbestrol, and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but not by vanadate. Both activities also showed sub-strate specificity for Mg-ATP. Finally, proton transport activities ofvacuolar and microsomal fractions exhibited similar profiles after flota-tion in linear dextran gradients. We conclude that the microsomal protonpump previously charcterized in com coleoptiles (Mettler et al. 1982Plant Physiol 70: 1738-1742) is derived from the tonoplast.

The importance of electrogenic proton pumping ATPases inthe transport of solutes across the plasma membrane and tono-plast of plant cells is being increasingly recognized. A cation-stimulated Mg2+-ATPase on the plasma membrane is thought tobe an electrogenic proton pump (25), and recent attempts topurify and reconstitute plasma membrane proteins have sup-ported this idea (28). The presence of a proton-pumping ATPaseon the tonoplast has been more controversial, although recentstudies with isolated, intact vacuoles have led to the identificationof a tonoplast ATPase in several systems (2-4, 7, 17, 19, 29-31).Investigations of the transport properties of sealed microsomalmembranes have indicated that vesicles capable of in vitro ATP-dependent proton transport can be isolated from a number ofdifferent plant tissues (26 and references therein). The bulk ofthis activity appears to be due to a novel ATPase which isdistinguishable from other known ATPases (plasma membrane,mitochondrial, and chloroplast), but is similar to the recentlycharacterized tonoplast ATPase.We have previously shown that microsomal membranes from

corn coleoptiles have ATP-dependent proton pumping activitywhich is stimulated by Cl-, has a slightly alkaline pH optimum,

'Supported by grant PCM-8301995 from the National Science Foun-dation.

and is inhibited by DCCD2 and CCCP, but not by vanadate oroligomycin (21, 22). In addition, we found that on linear sucroseand dextran gradients the proton pumping vesicles formed asingle peak of activity at low density ( 1.1 1 g/m3 in sucrose) ( 18,21). These characteristics led us to propose that the vesicles maybe derived from the tonoplast (18, 21) based on the similaritiesto the beet root tonoplast ATPase (4, 23, 31), the proton-pumping activity in Hevea latex lutoids (2, 3, 19), and the lightdensity of vacuolar membranes (6, 8, 20). Recent characteriza-tion of a proton-pumping ATPase of Tulipa petal vacuolesprovides additional evidence which supports this idea (29, 30),and several other groups who have detected anion-sensitive pro-ton-pumping activity in vesicles of low density have attributed itto the tonoplast (26). Intact vacuoles and microsomal mem-branes have been isolated from the storage tissue of red beets (4),and proton-pumping activity is similar in several respects. How-ever, proton-pumping activity has not been directly comparedin microsomal membranes and isolated vacuoles of any othertissue. To test the hypothesis that the 'light' fraction of proton-pumping vesicles in corn coleoptiles is derived from the tono-plast, we have studied proton transport in isolated vacuoles andhave compared it to the proton-pumping activity in microsomalvesicles.

MATERIALS AND METHODS

Plant Material. Corn (Zea mays L. cv Golden Cross Bantam)seeds were soaked for 5 h in tap water, and sown in trays layeredwith vermiculite and perlite. The germinating seedlings weremaintained in the dark at 20 to 22°C with a 15-s pulse of mistsprayed every 30 min.Vacuole Isolation. Coleoptiles from 5-d-old seedlings were

chopped into I-mm pieces on ice and vacuum infiltrated with1.5 ml/g fresh weight of 2% Cellulysin (Calbiochem), 0.5%Macerozyme (Yakult, Japan), 0.1% Pectolyase Y23 (Seishin,Japan) in 0.6 M mannitol, 0.25% BSA, 2 mm DTT, 1 mmspermidine, 0.25 M choline chloride, and 20 mM Mes/Tris (pH5.6). Following incubation for 2.5 to 3 h at 22°C in a water bathshaker (60 rpm), the tissue was filtered through nylon mesh,rinsed, and the remaining tissue vortexed gently in the digestbuffer. The protoplasts from the combined filtrates were thenpelleted several times at 350g for 10 min. The procedure ofThom et al. (27) was modified to prepare isolated vacuoles.Protoplasts were resuspended in 6 ml of 0.45 M sorbitol, 0.25%BSA, 1 mm DTT, 1 mM EDTA, and 20 mm Tris/Mes (pH 7.5),and 1 ml was layered onto six separate 8% Ficoll cushions inresuspension buffer (0.6 M sorbitol, 0.25% BSA, 1 mM DTT, 1mM EDTA, and 20 mM Tris/Mes, pH 7.5). The step gradients

2 Abbreviations: DCCD, N,N'-dicyclohexylcarbodiimide; CCCP, car-bonyl cyanide-m-chlorophenylhydrazone; DES, diethylstilbestrol; DIDS,4,4'-diisothiocyano-2,2'-stilbene disulfonic acid; MeA, methylamine;FDA, fluoroscein-diacetate; NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; NEM, N-ethylmaleimide; BTP, bis-Tris-propane.

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PROTON TRANSPORT BY CORN VACUOLES

were centrifuged at 2 10,000g for I h at 4°C in a Beckman SW60rotor. Vacuoles were collected from the interface and mixed with1.5 volumes of 4% Ficoll in resuspension buffer. The mixturewas overlaid with 0.25 ml of resuspension buffer and centrifugedin a 9-ml test tube (Pyrex No. 9820) at 12,000g for 15 min in aSorvall SS-34 rotor. Intact vacuoles floated to the top and werecollected at the Ficoll interface.

Microsomal Membrane Preparation. Microsomal membraneswere prepared as previously described (22). Briefly, 5-d-old corncoleoptiles were ground twice in an ice cold mortar and pestle in0.25 M sorbitol, I mM DTT, 2 mM EDTA, 0.1% BSA, and 50mM Tris/Mes (pH 7.8). After filtering through nylon mesh, thehomogenate was centrifuged at 5OOg for 5 min in a Sorvall SS-34 rotor. The supernatant was centrifuged at 12,000g for 10 minto remove mitochondria. A microsomal pellet was obtained bycentrifuging the 12,000g supernatant at 80,000g for 1 h in aBeckman SW28 rotor. The microsomal pellet was resuspendedin 0.25 M sorbitol, I mm DTT, 1 mm EDTA, and 10 mm Tris/Mes (pH 7.5), and layered onto a dextran step gradient composedof 4 ml 8% dextran (bottom) and 4 ml 1% dextran (top) in thesame buffer. After centrifugation at 120,000g for 2 h in a Beck-man SW28.1 rotor, light microsomal membranes were collectedfrom the 1/8% dextran interface. They were frozen in liquid N2and stored at -70°C.

Linear Density Gradient Centrifugation. A microsomal pelletwas prepared as described above except that all buffers contained0.4 M sorbitol. The pellet was resuspended in gradient buffer (0.4M sorbitol, I mm DTT, I mM EDTA, 25 mM KCI, and 5 mMTris/Mes, pH 7.5) and mixed with 15% dextran in gradientbuffer to give a density of approximately 10.5% dextran. Vacu-oles removed from the Ficoll flotation gradient were lysedthrough a 25-gauge needle and mixed with 15% dextran to givethe appropriate density. Three ml of either microsomes or vac-uoles were added to the bottom of two separate tubes, and 14 mlof a linear 1 to 10% dextran gradient (in gradient buffer) waspoured on top. The gradients were centrifuged at 120,000g for3.5 h in a SW28.1 rotor and 1.5 ml fractions were collected fromthe top.Enzyme Assays. Enzyme assays were carried out as previously

described (18, 22). Briefly, ['4C]MeA uptake was performed in a0.1-ml volume containing 2.5 mm ATP-MgSO4 or ADP-MgSO4(control), 18 uM MeA (0.77 PCi/mliassay, 44 mCi/mmol inethanol), 50 mM KCI, sorbitol (0.6 M for vacuoles and 0.25 M formicrosomal vesicles), and 5 mM Tris-Mes (pH 7.5). Protoplastswere lysed through a 25-gauge needle before incubation. Ap-proximately 5,000 vacuoles or protoplasts per assay were incu-bated for 5 min at 30C, followed by Millipore filtration. Quin-acrine fluorescence quenching was performed in a 0.6-ml volumecontaining 10 jM quinacrine, 50 mM KCI, sorbitol (0.6 M forvacuoles and 0.25 M for microsomal vesicles), and 5 mM Tris-Mes (pH 7.5). The reaction was initiated with the addition of 3mM MgSO4 and 3 mm ATP. Quinacrine fluorescence was meas-ured with excitation and emission wavelengths of 420 and 495,respectively, using a Hitachi Perkin Elmer MPF-2A fluorescencespectrophotometer.ATPase and latent UDPase were measured with 2.5 mm sub-

strate, 2.5 mM MgSO4, 50 mM KCI, and 25 mm Tris-Mes. Aftera 30-min incubation at 37°C, released Pi was determined (13).Vanadate-inhibited ATPase was assayed at pH 6.5 in the presenceof 1 mm Na-molybdate to inhibit phosphatase activity (2, 14).Azide-inhibited ATPase was determined at pH 8.5. LatentUDPase, measured at pH 6.5, was defined as the difference inactivity in the presence and absence of 0.1% digitonin (24).NADPH-Cyt c reductase and Cyt c oxidase activities were deter-mined spectrophotometrically as described by Hodges and Leon-ard (16), and a-mannosidase activity was measured according toBoller and Kende (6).

Microscopy. Numbers of vacuoles and protoplasts were esti-mated using a Neubauer Spot lite counting chamber with an

Olympus microscope. Fluorescence microscopy was performedby staining vacuoles and protoplasts with FDA (1), and exam-ining them with an Olympus Vanox fluorescence microscopeusing a blue filter.

Materials. Cellulysin, ATP, DIDS, monensin, and DTT werepurchased from Calbiochem. Dextran was obtained from Phar-macia (Sweden), Macerozyme from Yakult Pharmaceuticals (Ja-pan), Pectolyase Y23 from Seishin Pharmaceuticals (Japan),sodium orthovanadate from Fisher, DCCD from Aldrich, and['4C]MeA from New England Nuclear. The salts were all analyticreagent grade from Mallinckrodt. All other chemicals were ob-tained from Sigma.

RESULTS

Light micrographs of corn coleoptile protoplasts (a) and vac-uoles (b) stained with neutral red are shown in Figure 1. Toinsure that the vacuoles were not surrounded by a plasma mem-brane, vacuoles and protoplasts were stained with FDA andexamined under a fluorescence microscope (1). The hydrolysisof FDA to fluorescein resulted in intense staining of the corncoleoptile protoplasts (c) while the vacuoles failed to fluoresce(d).The extent of contamination of the vacuoles by membranes

from other organelles was assessed using marker enzymes asshown in Table I. All of the ATP-dependent proton pumping inprotoplasts, as measured by MeA uptake, was recovered in thevacuolar fraction. MeA uptake could only be measured in pro-toplasts after they had been lysed by passage through a 25-gaugeneedle or by freeze-thawing (data not shown). To duplicate theseconditions, vacuoles were also lysed in this experiment wherethe activity was compared with that of protoplasts. In theseexperiments, 93% of the a-mannosidase activity, which has beenshown to be a soluble vacuolar enzyme (6), was recovered in thevacuolar fraction. All of the activities of the markers for othermembranes, however, were significantly reduced in the vacuolarfraction. Azide-sensitive ATPase and Cyt c oxidase, both innermitochondrial membrane markers, showed 4.5% and 5.6% con-tamination, respectively. Vanadate-inhibited ATPase has beenused as a plasma membrane marker when molybdate is presentas an inhibitor of phosphatase (2, 14). In these experiments,17.4% of the plasma membranes were present in the vacuolarfraction. Latent UDPase, a Golgi marker, and NADPH-Cyt creductase, an ER marker, showed 7% and 16.5% contaminationof the vacuolar fraction, respectively.To compare the vacuolar ATP-driven proton pump with the

previously described activity from corn coleoptile microsomalvesicles (18, 21, 22), several characteristics were examined inboth preparations of membranes. Table II shows the effects of avariety of inhibitors on ATP-stimulated MeA uptake in intactvacuoles and microsomal membranes. Vanadate, at 100 Mm, didnot inhibit MeA uptake in vacuoles or microsomal vesicles.Molybdate, which inhibits phosphatase (2, 14), also had littleeffect. Oligomycin, a mitochondrial inhibitor, decreased MeAuptake by 31% in both microsomes and tonoplasts at a highconcentration. However, the complete inhibition of protonpumping by DES, and the low level of azide-sensitive ATPase(Table I), are inconsistent with a mitochondrial origin for eitheractivity (15). Other effective inhibitors were two ionophores:monensin, which exchanges Na+ or K+ for a proton, and CCCP,which is a protonophore, as well as DCCD (a proton channelblocker), NEM (a sulftydryl reagent), DIDS (an anion channelblocker), and NBD-Cl. An inhibitor which has been suggested tobe relatively specific for low density vesicles and tonoplasts isKNO3 (26). KNO3 resulted in 89% and 93% inhibition of thevacuolar and microsomal proton pumps, respectively. The sim-ilar effects of all of these inhibitors on vacuolar and microsomalproton transport indicates that they closely resemble each other,and are both distinguishable from plasma membrane and mito-

105

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MANDALA AND TAIZ

0

FIG. 1. Photomicrographs of corn coleoptile pro-_* toplasts (a, c) and vacuoles (b, d). Neutral red was the

stain used in a and b. For c and d, protoplasts andvacuoles were stained with FDA and examined undera fluorescence microscope with a blue filter. Bar = 30

/ m.

Table I. Enzyme Marker Activity in Protoplasts and VacuolesActivity is expressed as nmol/min- I 0 vacuoles or protoplasts ± SE,

except for MeA uptake which is calculated as the ATP stimulation abovethe ADP control and expressed in cpm/10' vacuoles or protoplasts.

Treatment Protoplasts VacuolesActivity Activity (%)

nmol/min- 104MeAuptake 1443a± 160 1645a± 274 (114)a-Mannosidase 0.099b + 0.017 0.092b + 0.018 (93)Van-inhibited ATPase(I00 JAM) 4.77c ± 0.38 0.83b ± 0.08 (17.4)

Azide-inhibited ATPase(I mM) 3.13c± 0.45 0.14b 0.04 (4.5)

Cyt c oxidase 14.16b + 1.0 0.79b + 0.09 (5.6)Cyt c reductase 3.64b + 0.73 0.60b ± 0.08 (16.5)Latent UDPase 6.1OC ± 0.89 0.43b + 0.08 (7)

an =lO bn = 6. Cn = 9-

chondrial proton pumping activities.Previous characterization of the substrate specificity of the

corn coleoptile microsomal proton pump as measured by MeAuptake, indicated that Mg-ATP was the preferred substrate (22).This was confirmed in Figure 2 using quinacrine fluorescencequenching in tonoplast vesicles from isolated vacuoles and mi-crosomal vesicles. Only Mg-ATP was able to stimulate protontransport under the normal assay conditions. GTP, UTP, ADP,UDP, and AMP resulted in a very slight initial quench whichwas not reversible by monensin. PPi, at 3 mm in the presence of50 mM KCl, did not stimulate proton pumping in either vacuolesor microsomes. However, at I mm Mg-PPi and with 50 mMKNO3, a significant amount of proton pumping activity wasmeasured (Fig. 2).

Table II. Effect ofInhibitors on MeA Uptake by Isolated Vacuoles andMicrosomes

Twenty gl ofvacuoles (-3000 vacuoles) or 20 ,d ofmicrosomal vesicleswere incubated with 2.5 mM Mg-ATP or Mg-ADP, 50 mM KCI, ±inhibitor, and filtered after 5 min as described in "Materials and Meth-ods." Activity, in cpm, is expressed as ATP-stimulation above the ADPcontrol.

Treatment Vacuoles Microsomes

cpm % control cpm % controlControl 1084 100 5191 100Ethanol (1%) 1200 111 4908 92Vanadate (100Mm) 1208 112 4968 96Na-molybdate(I mM) 943 87 5110 98Oligomycin (10 gg/ml) 743 69 3606 69DES (100l m) 100 9 84 2NEM(I mM) 23 2 0 0NBD-CI (100 M,) 54 5 182 3Monensin (5 Mm) 108 10 0 0CCCP (IlOMm) 125 1 1 0 0DCCD (I0l M) 16 1 797 15DIDS(100Mm) 71 6 0 0KNO3(100mM) 114 11 355 7

In general, the response of the vacuolar ATP-driven protonpump to salts was similar to that of the microsomal pump asshown in Table III. In both cases, proton pumping was stimulated3- to 4-fold by Cl- salts with only minor differences attributableto the cation, although BTP-Cl was more stimulatory than otherC1- salts in microsomes. Some differences between the twomembrane preparations were observed. In the absence of addedmonovalent ions, the intact vacuoles showed more proton pump-ing activity than the microsomal vesicles, especially when ex-

b

S^

S

106 Plant Physiol. Vol. 78, 1985




5%l

C31 I~~mi< _ \~~ ~~~'a0I mmn Monensin 5gM

c Substrate B0n -

0

-: b,c,d,e,f,g

h

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5%J \ IlImin t

Monensin 5jtMFIG. 2. Substrate specificity of quinacrine fluorescence quenching in

tonoplast vesicles (A) and microsomal vesicles (B). Vacuoles and micro-somes, which had been previously frozen, were incubated with 50 mMKCI, sorbitol (0.6 M for tonoplasts and 0.25 M for microsomes), and 5mM Tris-Mes until a stable baseline was reached. Fluorescence quenchingwas initiated by the addition of 2.5 mm MgSO4 and 2.5 mM ATP (a),GTP (b), UTP (c), ADP (d), UDP (e), AMP (fl, or PPi (g). In (h), 50 mmKN03 was added, and the reaction was initiated with I mM MgSO4 andI mM PPi.

pressed as the total quench (A Q). Salts with less permeant anions,such as KMes and K2SO4 failed to stimulate proton pumpingsignificantly above the minus salt control in both vacuoles andmicrosomes. NaHCO3 was somewhat more stimulatory in intactvacuoles than in microsomal vesicles. The activity of both prep-arations was totally dependent on the presence of Mg2+, andCa2+ could not substitute for the Mg2' requirement.

Figure 3 compares the densities of the vacuolar proton pumpwith the microsomal pump after flotation in linear dextrangradients. The distribution of proton-pumping activity in tono-plast vesicles was similar to that of the microsomal fraction, andwas clearly separable from markers for other organelles in micro-somal membranes. ATP-stimulated MeA uptake was located atthe top of both gradients at 9 to 10% equivalent sucrose. Thepeak of NADPH-Cyt c reductase, the ER marker, was found at1 % equivalent sucrose. Small amounts of enzyme activities forthe plasma membrane (vanadate-inhibited ATPase), Golgi (la-tent UDPase), and mitochondrial membranes (Cyt c oxidase)were found in the middle of the gradient although most of theactivity remained at the bottom.

DISCUSSION

ATP-driven proton-pumping activity has now been detectedin microsomal membranes from a variety of plant tissues (re-viewed in 26). In some systems, two populations of proton

Table III. Effect ofSalts on Quinacrine Fluorescence Quenching byIntact Vacuoles and Microsomal Vesicles

All treatments included 2.5 mM MgS04 except for the -MgSO4 andthe CaCI2 experiments which were performed in the presence of 50 mmKCI. AQ is defined as the per cent of quench that is reversible by 5 gMmonensin after 6 min. Initial control (50 mM KCI) rates for the vacuoleand microsomal vesicles were 2.04 and 4.42 %/min, respectively, whileAQ were 9.5 and 14.6%, respectively.

Vacuoles MicrosomesTreatment Initial Initial

rate AQ rate A

% control50 mM KCI (control) 100 100 100 100No monovalent salt 36 16 27 150 mM BTP/C1 104 79 112 12950 mM Choline Cl 107 81 92 9450 mM NaCl 104 100 90 8650 mM K/MES 40 23 22 025 mM K2SO4 43 10 24 050 mM NaHCO3 36 44 14 3-MgSO4 12 0 9 02.5 mM CaC12 0 0 NT NT

*NT, not tested.

pumping vesicles can be distinguished (4, 23, 26). The moreactive pump is lighter in density, is stimulated by anions, and isinhibited by KNO3. The second pump (reviewed in 26) has aheavier density, responds to cations more than anions, and canbe inhibited by vanadate. The second pump is thought to bederived from plasma membrane vesicles, based on its similaritiesto the well-characterized K+-stimulated plasma membrane ATP-ase (16, 25). In the cultivar of corn used in this and previousreports (Golden Cross Bantam), we have only been able to detecta single population of vesicles with the characteristics of the lightpump (I18, 21, 22). This also seems to be the case for other groupsworking with corn (12, 26), although recent work in our labora-tory using a different variety of corn and a modified membraneisolation procedure has resulted in the identification of a secondproton pump attributed to the Golgi (10).Our previous work with microsomal vesicles from corn co-

leoptiles demonstrated that the characteristics of the protonpump and the light density ofthe active vesicles were inconsistentwith a plasma membrane, ER, Golgi, or mitochondrial origin(18, 21, 22). We suggested that the light vesicle fraction may bederived from the tonoplast ( 18, 22). This presumes the presenceof an ATPase on the tonoplast, which has been controversial.Except for the highly specialized lutoid system, where an electro-genic proton-pumping ATPase is well established (2, 3, 19), ithas been difficult to demonstrate the presence of a specificATPase on the vacuolar membrane. Early reports did not distin-guish acid phosphatase activity from ATPase, making this workdifficult to interpret (I 1, 17). When the contribution made byacid phosphatase was evaluated in vacuoles from cultured to-bacco cells, the authors concluded that there was no detectablespecific ATPase on the tonoplast (8, 20). More recently, molyb-date has been used to inhibit phosphatase activity in red beetroot vacuoles, resulting in the identification of an ATPase whichis Mg2"-dependent, anion-sensitive, and having a slighltly alkalinepH optimum (31). A Neurospora tonoplast ATPase has also beenrecently characterized (7). In addition, Wagner and Lin (29)provided convincing evidence for a tonoplast proton-pumpingATPase using anthocyanin pigments within Tulipa petal vacu-oles to show ATP-dependent acidification of the vacuoles. TheTulipa tonoplast ATPase as well as a pyrophosphatase has beensolubilized and characterized (30).The evidence accumulating for the presence of a Mg-ATPase

rateA

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107

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MANDALA AND TAIZ

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M 100-

1EE 50X ~~~~~~200,@1

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50 100 0U

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*=300ra

j oj65Q200 - 40 X

0

o 15000. 0.*1

8 10 12 14 16% Sucrose Equivalence

FIG. 3. Tonoplast vesicles or microsomal membranes on linear 1%to 10% dextran flotation gradients. Vacuoles or microsomes were mixedwith 15% dextran, overlayed with a I toIO0O linear dextran gradient,and centrifuged at 120,000g for 3.5 h. Enzyme activities of microsomalmembranes are expressed as nmol/min*-fraction for NADPH-Cyt c re-ductase (0), Cyt c oxidase (A), vanadate-inhibited ATPase(O), and latentUDPase (A). ATP-stimulated MeA uptake in microsomes ( ) is ex-pre-osed as cpm/fraction x 10-3, and cpm/fraction x 10'2for vacuoles(O).

on the tonoplast, and the close correspondence between theproperties of the tonoplast ATPase and the microsomal protonpump, supports the idea that the light fraction ofproton pumpingvesicles is vacuolar in origin. However, for a definitive demon-stration it is important to compare the proton pump found inisolated vacuoleswith microsomal membranes ofthe same tissue.A comparison of proton-pumping activity in isolated vacuolesand microsomal membranes has been performed in beet roots,and the activities were shown to be very similar, but data on thepurity of the vacuolar preparation were not presented (4). Corncoleoptiles, which have a well characterized microsomal pump(I18,21l,22), proved to be a difficult system from which to isolatevacuoles because of the low amount of starting material. Inaddition, proton-pumping activity was more labile than otherenzyme activities, probably because it requires a sealed mem-brane as well as a functional ATPase. Pectolyase Y-23 during

cell wall digestion enhanced the numbers of protoplasts, andhigh speed centrifugation of protoplasts onto a Ficoll cushion torelease the vacuoles resulted in approximately a 16% yield-of

vaulsfo4 rtpat.Tepeec fahg ocnrto

vacuoles from protoplasts. The presence of a high concentrationof BSA (0.25%) throughout the isolation procedure, and cholinechloride (0.25M) during the period of cell wall digestion appeared

to greatly enhance proton-pumping activity in protoplasts andvacuoles. These methods provided a high enough yield of vacu-oles to detect proton transport, but also resulted in a significantcontamination of the vacuoles by other membranes. Neverthe-less, the level of contamination we measured (5-17%) is similarto other reports on isolated vacuoles. Boller summarized theresults of a number of different preparations of isolated vacuoles,which ranged between <1% and 35% contamination (5). In thesestudies, no attempt was made to estimate the amount of contam-inating plasma membranes, which we have found to be the mostpersistent of all nonvacuolar membranes. Admon and Jacoby(1) have proposed the use of FDA to assess the purity of isolatedvacuoles. FDA is hydrolyzed to fluorescein and accumulates inthe cytosol resulting in intensely fluorescent protoplasts. Vacu-oles which are surrounded by a plasma membrane show a haloof fluorescence while naked vacuoles appear as dark, nonflu-orescing organelles. In Figure 1, c and d, we tested FDA on corncoleoptile protoplasts and vacuoles. While the protoplasts (c)were intensely fluorescent, the vacuoles (d) remained unstained.Marker enzymes were a second method used to estimate the

amount of contamination by other organelles. Table I shows thatin the vacuolar fraction, activities of enzyme markers for the ER,Golgi, mitochondria, and plasma membranes were between 5and 17% of that found in an equal number of protoplasts. Mostof the enzyme activities associated with these membranes sedi-mented to the bottom ofthe Ficoll step during ultracentrifugation(data not shown), while the intact vacuoles remained at theinterface. Correlated with the intact vacuoles was the solublevacuolar marker, a-mannosidase (6), and all of the detectableATP-dependent proton-pumping activity. These data indicatethat there is a proton-pumping ATPase associated with thetonoplast of corn coleoptiles.The characteristics of the proton pump are almost identical to

those previously described for microsomal vesicles in corn co-leoptiles (18,21,22), and are in good agreement with descriptionsof other vacuolar ATPases (2-4, 7, 17, 19, 23, 29-31). Table IIshows that ATP-stimulated MeA uptake in intact vacuoles andmicrosomal vesicles was similarly affected by a large variety ofinhibitors. Proton pumping was inhibited by DES which hasbeen used as a plasma membrane ATPase inhibitor (15). This isin agreement with our previous results (21), although DES didnot inhibit the ATPase of Hevea latex vacuoles (19). Inhibitionby DES does not indicate that the proton-pumping activity isdue to plasma membranes since vanadate, a potent and specificinhibitor of plasma membrane ATPases of fungi, plants, andanimals (15), did not affect microsomal or vacuolar activity(Table II). In addition, KNO3 was very effective at preventingproton pumping in vacuoles and microsomes. KNO3 has beenused as a relatively specific inhibitor of the ATPase found intonoplast vesicles and low density microsomal vesicles (26). At ahigh concentration of oligomycin, some inhibition was observed,possibly due to a nonspecific effect at these concentrations ordue to contamination by mitochondria. The complete inhibitionof proton pumping by DIDS (the anion channel blocker) wasexpected since we have previously shown that36C1 is transportedinto the light microsomal vesicles in response to the pH gradient(18, 22). DIDS may also have a direct effect on the ATPase (26).Many of the other compounds tested were almost completelyinhibitory, including NEM, NBD-Cl, monensin, CCCP, andDCCD. The similar effects of all of these inhibitors on bothvacuoles and microsomal vesicles supports the conclusion thatproton-pumping activity in light microsomal membranes is de-rived from the tonoplast.

Previous studies on tonoplasts and low density microsomalvesicles have indicated that proton pumping is greatly enhancedby anions, and in particular, by Cl- (26). Table III shows thatCl- stimulated proton pumping 3- to 4-fold in corn coleoptilevacuoles and microsomal vesicles, while the cation appeared tohave only minor effects. Small differences in the extent of salt

108 Plant Physiol. Vol. 78, 1985




stimulation were observed between vacuoles and microsomalvesicles, although the overall pattern was similar. Relative to theKCI control, more proton pumping activity by vacuoles wasdetected in the presence of KMes, K2SO4, and NaHCO3, and inthe absence of monovalent ions, than was measured in micro-somal vesicles. This may be expected since the vacuoles wereintact, presumably with most of their original contents, while themicrosomal vesicles have been purified away from the solublematerial. Alternatively, the vacuoles may have contained residualcholine chloride left over from the protoplast isolation procedure.Table III also indicates that MgSO4 was required for proton-pumping activity. Ion-translocating ATPases have been shownto use Mg-ATP as the substrate (15).The substrate specificity of the proton pump in tonoplast and

microsomal vesicles was examined by quinacrine quenching inFigure 2. Under the normal conditions for the assay (3 mm Mg-substrate, 50 mm KCI), only ATP was able to promote protonpumping. There have been several reports on the presence ofpyrophosphatase activity in tonoplast or microsomal vesicles (30,31), which is able to pump protons (9). Chanson et al. haveshown that this enzyme is distinct from the ATPase (9). At alower substrate concentration (1 mM) and with 50 mM KNO3,conditions which would prevent ATP-driven proton transport,PPi did stimulate quinacrine quenching in both tonoplast andmicrosomal vesicles.To compare the density of the vacuolar proton pump with the

microsomal pump, both methods were used to prepare themembranes for 1 to 10% dextran gradients. Sedimentation inlinear dextran gradients was used previously to separate protonpumping from other membrane markers in corn coleoptiles (18,21). In the present experiments, the results were complicated bythe presence of a high concentration of sorbitol (0.6 M) used toosmotically stabilize the vacuoles. The best results were obtainedusing a flotation gradient. Under these conditions, the peak ofATP-dependent proton pumping was found at the top of bothgradients at 9 to 10% equivalent sucrose (Fig. 3). Enzymemarkers for the ER, Golgi, mitochondrial, and plasma mem-branes were concentrated in the middle of the gradient, resultingin a clear separation from proton pumping activity. Flotationgradients made with sucrose have been used to estimate thedensity of tonoplasts from tobacco (8). The tonoplast vesiclesfloated to a density of 1.12 g/cc, while other enzyme markersremained at the bottom of the gradient. Our previous work withcorn coleoptile microsomes gave an estimated density for theproton pump of 1. I g/cm3 in sucrose gradients (18, 21). This isin good agreement with the tonoplast ATPase of Tulipa petalsmeasured at 1.11 to 1.13 g/cm3 (30), Neuirospora at 1.11 g/cm3(7), red beets at 1.09 to 1.10 g/cm3 (4, 23), as well as tobaccotonoplast vesicles measured by sedimentation or flotation gra-dients (6, 8, 20).

In summary, a comparison of proton-pumping activities as-sociated with intact vacuoles and microsomal vesicles from corncoleoptiles has provided strong evidence that the light micro-somal pump is tonoplast in origin. Properties of the tonoplastproton pump include Cl- stimulation and KNO3 inhibition, anda low density distribution on linear gradients. These attributesare also diagnostic for the proton pump on light microsomalvesicles, providing convenient assays for the tonoplast in mixedmembrane populations.Note Added in Proof. Hager and Biber (Z Naturforsch 39c:

927-937, 1984) recently demonstrated a small amount of vana-date-sensitive proton pumping in corn coleoptile microsomalmembranes following an osmotic shock. The bulk of the protontransport activity was attributed to the tonoplast.

Acknowledgment-We would like to thank Dr. L. Goff for her generous assist-ance in the fluorescence microscopy experiments.

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21. METrLER IJ, S MANDALA, L TAIZ 1982 Proton gradients produced in vitrofrom microsomal vesicles from corn coleoptiles: Tonoplast origin? In DMarme, E Marre, R Hertel, eds, Plasmalemma and Tonoplast: Their Func-tions in the Plant Cell. Elsevier-North Holland, Amsterdam, pp 395-400

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23. POOLE RJ, DP BRISKIN, Z KRATKY, RM JOHNSTONE 1984 Density gradientlocalization of plasma membrane and tonoplast from storage tissue ofgrowing and dormant red beet. Plant Physiol 74: 549-556

24. RAY PM 1979 Maize coleoptile cellular membranes bearing different types ofglucan synthetase activity. In E Reid, ed, Plant Organelles, MethodologicalSurveys, Biochemistry, Vol 9. Halstead Press, Chichester, pp 135-146

25. SPANSWICK RM 1981 Electrogenic ion pumps. Annu Rev Plant Physiol 32:267-289

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27. THOM M, A MARETZKI, E KoMOR 1982 Vacuoles from sugarcane suspensioncultures. 1. Isolation and partial characterization. Plant Physiol 69: 1315-1319

28. VARA F, R SERRANO 1982 Partial purification and properties of the proton-translocating ATPase of plant plasma membranes. J Biol Chem 257: 12826-12830

29. WAGNER GJ, W LIN 1982 An active proton pump of intact vacuoles isolatedfrom Tulipa petals. Biochim Biophys Acta 689: 261-266

30. WAGNER GJ, P MULREADY 1983 Characterization and solubilization of nu-cleotide-specific Mg2+-ATPase and Mg'+-pyrophosphatase of tonoplast.Biochim Biophys Acta 728: 267-280

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Proton Transport in Isolated Vacuoles from Corn Coleoptiles