Planta

Planta (1989) 178:393-399

9 Springer-Verlag 1989

Sucrose transport in isolated plasma-membrane vesicles from sugar beet (Beta vulgaris L.) Evidence for an electrogenic sucrose-proton symport T h o m a s J. Buckhout Plant Photobiology Laboratory, Beltsville Agricultural Research Center, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA

Abstract. An analysis of the molecular mechanism

of sucrose transport across the plasmalemma was conducted with isolated plasma-membrane (PM) vesicles. Plasma membrane was isolated by aqueous two-phase partitioning from fully expanded sugar beet (Beta vulgaris L.) leaves. The isolated fraction was predominantly PM vesicles as determined by marker-enzyme analysis, and the vesicles were oriented right-side-out as determined by structurally linked latency of the PM enzyme, vanadate-sensitive Mg 2 +-ATPase. Sucrose uptake was investigated by equilibrating P M vesicles in pH 7.6 buffer and diluting them 20-fold into pH 6.0 buffer. Using this pH-jump technique, vesicles accumulated acetate in a pH-dependent, protonophore-sensitive manner, which demonstrated the presence of a pH gradient (ApH) across the vesicle membrane. Addition of sucrose to pH-jumped PM vesicles resulted in a pH-dependent, protonophoresensitive uptake of sucrose into the vesicles. Uptake was sucrose-specific in that a 10-fold excess ofmannose, glucose, fructose, mannitol, melibiose, lactose or maltose did not inhibit sucrose accumulation. The rate of pH-dependent uptake was saturable with respect of sucrose concentration and had an apparent Km of 0.45 mM. Sucrose uptake was stimulated approximately twofold by the addition of valinomycin and K +, which indicated an electrogenic sucrose-H + symport. Membrane potentials (A 0) were imposed across the vesicle membrane using valinomycin and K +. A membrane potential, negative inside, stimulated pH-dependent sucrose uptake while a AO, positive inside, Abbreviations and symbols: CCCP = carbonyl cyanide m-chlorophenylhydrazone; cyt = cytochrome; PM = plasma-membrane(s); AO = electrical potential difference The mention of vendor or product does not imply that they are endorsed or recommended by U.S. Department of Agriculture over vendors of similar products not mentioned

inhibited uptake. Conditions that produce a negative A 0 in the absence of a pH gradient supported, although weakly, sucrose uptake. These data support an electrogenic sucrose-H + symport as the mechanism of sucrose transport across the P M in Beta leaves. Key words: Assimilate partitioning - B e t a P h l o e m loading - Plasma membrane - Proton gra-

dient - Symport - Sucrose transport - Transport (sucrose)

Introduction

Sucrose is the principal chemical form used by plants to distribute photosynthetically derived energy and carbon to nonphotosynthetic organs. Movement of sucrose through the plant occurs by a series of reactions that range from simple diffusion to active transport against concentration gradients. Experimental evidence for carrier-mediated sucrose transport across the plasmalemma of higher plants has been obtained in a number of plant organs and cells (Giaquinta 1983). Of particular interest to this study are the reports of carriermediated sucrose transport in beet leaves (Sovonick et al. 1974; Maynard and Lucas 1982a, b) and tap roots (Lemoine et al. 1988; see Wyse 1979). Analyses of the concentration dependence of sucrose uptake into leaf discs and intact leaves indicate the presence of two pathways of uptake, one saturable and obeying Michaelis-Menten kinetics, and a second linear component obeying first-order kinetics. Initially, the saturable component of sucrose uptake into leaf discs was thought to represent only phloem loading; however, Fondy and Geiger (1977) and later Maynard and Lucas (1982a, b) showed that sucrose uptake into leaf

394

T.J. Buckhout: Sucrose transport in isolated PM vesicles of Beta vulgaris leaves

discs may not solely represent phloem loading. Thus, it is likely that a saturable sucrose-uptake system is localized on the plasmalemma of cell types other than those involved in phloem loading. The mechanism of carrier-mediated sucrose transport across the plasmalemma of higher-plant cells has been widely accepted as a sucrose-H + symporter (Giaquinta 1983). Experiments testing the sucrose-H + symport hypothesis were based on the initial work by Komor and Tanner (1974a, b) who reported the simultaneous uptake of glucose and alkalization of the media by Chlorella. Examples where evidence for a sucrose-H + symport in higher plants has been obtained include Ricinus cotyledons and petioles, Viciafaba leaf tissues, lily pollen, maize scutella and maize leaves (reviewed by Giaquinta 1983). The nature of the sucrose-H + symport has been investigated using electrophysiological techniques. Using microelectrodes to measure membrane potential and extracellular pH simultaneously, Racusen and Galston (1977) reported a sucrose-induced membrane depolarization concomitant with alkalization of the extracellular medium in Samanea pulvini. Both Wright and Fisher (198/) in Salix phloem and Lichtner and Spanswick (1981) in soybean cotyledon cells reported a sucrose-induced depolarization of the membrane potential. In both cases, the sucrose-induced depolarization showed sucrose saturation kinetics with apparent Km values in the low millimolar range. Lin (1985), using membrane potential and pH-sensitive probes, demonstrated sucrose-dependent acidification of the cytoplasm, alkalization of the external medium, and transient depolarization of the membrane potential in soybean cotyledon protoplasts. Thus, in a variety of experimental systems, there is evidence that the H+-sucrose symport catalyzes an electrogenic transport of sucrose across the plasmalemma. The understanding of the molecular mechanism of sucrose transport and of the sucrose-H + stoichiometry has been hindered by the complex composition of intact organs used in uptake studies. Attempts at establishing a cell-free sucrose-transport system have been for the most part unsuccessful. The recent report by Bush (1989) is, to the best of our knowledge, the first successful characterization of sucrose transport in isolated plasma-membrane (PM) vesicles. Using PM isolated from sugar beet leaves, Bush (1989) demonstrated a pH-dependent accumulation of sucrose in PM vesicles. Uptake was sensitive to the sulfhydral reagent pchloromercuribenzenesulfonic acid (PCMBS) and displayed saturable, Michaelis-Menten-like kinet-

ics. We report a similar pH-dependent uptake of sucrose into PM vesicles of sugar beet leaves and demonstrate that the coupled sucrose-H + transport is electrogenic. Material and methods Plant material and growth conditions. Sugar beets (Beta vulgaris L. cv. Mono HYE4; Michigan Sugar Co., Saginaw, Mich., USA) were grown from seed in growth chambers at 25 ~ C and 60% relative humidity. Lights-on was at 7:00 local time, and lights-off was at 19:00. Illumination was provided by a combination of high-intensity, cool-white, fluorescent lamps (F96TI2/CW/VHO ; North American Philips, Bloomfield, N.J., USA) with supplemental incandescent lights giving a photon fluence rate at canopy height of 0.5 m m o l . m - 2 - s - t . Plants were fertilized daily with a modified Hoagland's solution (Robinson et al. 1988). Fully expanded leaves from 6- to 12-weekold plants were harvested for membrane isolation 1 h after the beginning of the light period.

Isolation of PM. For membrane isolation, the leaf midrib was removed and the remaining blade cut into 1-crn strips. The strips were further chopped with a razor blade to approx, l-ram segments and homogenized in four volumes (g F W . m l - i ) of buffer consisting of 50 m M 4-(2-hydroxyethyl)-l-piperazineethane Sulfonic acid (Hepes)-l,3-bis[tris(hydroxymethyl)methylamino]propane (BTP), pH 7.8, 0.25 M sorbitol, 1 mM ethylene glycol-bis-(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM KC1, 0.5% (w/v) bovine serum albumin and 5 mM dithiothreitol (DTT) using a Polytron homogenizer (Brinkmann Instruments, Westbury, New York, USA) at 60% maximum speed for two 30-s runs. Microsomal pellets and PM were obtained from the homogenate by differential centrifugation and aqueous two-phase partitioning as described by Buckhour and Hrubec (1986). For the phase partitioning, four 36-g phase systems were constructed containing 6.4% (w/w) dextran T500 (Pharmacia, Uppsala, Sweden), 6.4% (w/w) polyethylene glycol with an average molecular mass of 3350 daltons (Da) (Sigma Chemical Co., St. Louis, Mo., USA), 0.048% phosphate buffer titrated to pH 7.8 with KOH, 0.037% KC1 and 8.56% sorbitol, all calculated by weight. Ten grams of membranes suspended in the 0.048% phosphate buffer containing 8.56% sorbitol solution plus 5 mM DTT were added to a fifth phase system to give a 36-g system of the composition described above. All systems were equilibrated to 2 ~ and partitioned by repeated inversion for 20 s. The phases were separated by centrifugation at 2000"gm, x for 10 min at 2 ~ (Sorvall HB-4 rotor; Du Pont, Wilmington, Deta., USA). The partitioning procedure was repeated for a total of four partitions. Upper phases were diluted with five volumes of 2 mM Hepes-BTP, pH 7.6, 350 mM sorbitol, / m M K-BTP, K-sulfate or KC1 (as indicated) and I mM DTT (dilution buffer), and pelleted at ll3000.gma x for 45 min. The PM pellets were resuspended in dilution medium and pelleted as above. Membranes were resuspended at a protein concentration of approx. 20 rag- m l - 1 and frozen at - 8 0 ~ C. Membranes used for the analysis of membrane marker enzymes were diluted and washed in dilution medium without DTT, and enzyme activity analyzed without freezing.

Marker-enzyme analysis. Marker enzymes for endoplasmic reticulum, Golgi apparatus and mitochondria were, respectively, antimycin A-insensitive, NADH-cytochrome (cyt)-c reductase (Hodges and Leonard 1972), Triton X-100-stimulated uridine 5'-diphosphatase (Nagahashi and Nagahashi 1982) and cyt-c

T.J. Buckhout: Sucrose transport in isolated PM vesicles of Beta vulgaris leaves

395

Table 1. Analysis of marker enzymes in membrane fractions from Beta vulgaris leaves isolated by the aqueous two-phase partitioning procedure. Numbers in parentheses are specific activities/rag protein. Marker enzymes were: Triton X-100-stimulated uridine 5'-diphosphatase (UDPase) for Golgi apparatus, NADH-cyt-c reductase for endoplasmic reticulum, cyt-c oxidase for mitochondria. NADH-cyt-e reductase was assayed in the presence of 10 gg. m l - 1 antimycin A. N D = not detected Fraction

Microsomal fraction Recovery (%) Plasma membrane Recovery (%)

Protein

Chlorophyll

(rag)

(rag)

130.6 100.0

3.3 100.0

6.2 4.7

0.005 0.1

oxidase (Hodges and Leonard 1972) performed by the modifications of Buckhout et al. (1982). Vanadate-inhibited MgZ+-AT Pase activity, a marker enzyme for PM, and chloride-stimulated Mg2+-ATPase, a marker enzyme for tonoplast membranes, were assayed with and without 0.0125% (w/v) Triton X-100 as described by Robinson et al. (1988). Protein was determined by the dye-binding assay of Bradford (1976).

Triton X-100-stimulated UDPase (nmoi- min - z)

NADH cyt-c reductase (mmol- m i n - ~)

278.5 (2.13)

4.7 (0.04)

lOO.O

lOO.OO

14.8 (2.4) 5.3

Cyt-e oxidase (mmol. min - 1) 4.9 (0.04)

1oo.oo

0.01 (0.001) 0.2

ND 0.0

Table 2. Analysis of ATPase activity in membrane fractions

from Beta vulgaris leaves isolated by the aqueous two-phase partitioning procedure. Vanadate-sensitive Mg z +-ATPase was assayed as a marker for the PM and chloride-stimulated and nitrate-inhibited MgZ+-ATPases were markers for the tonoplast. Activity is expressed as gmol.min-1, and numbers in parentheses are specific activities/mg protein. N D = n o t detected

Assays for sucrose, thiocyanate and acetate uptake. Analysis of sucrose and acetate uptake in isolated PM vesicles was conducted by generating transmembrane pH gradients with the pH-jump method slightly modified from the method of (Bush 1989). The typical transport assay was conducted at 10~ C and consisted of 484.5 gt acidic buffer (dilution buffer titrated to pH 6.0 with solid Hepes), 1 gl carrier-free sucrose (503.2 GBq. mmo1-1, 3 7 M B q - m l - 1 ; N E N Products, Boston, Mass., USA), 1 gl 50 mM sucrose (or as indicated, +_1 gl valinomycin (0.5 m M stock solution in ethanol; Sigma Chemical Co.) and 12.5 gl PM in dilution buffer added at time zero. Uptake dependent on a pH gradient across the vesicle membrane was calculated by subtracting sucrose uptake in the absence of a pH gradient, i.e. assays conducted in the presence of 1 gM CCCP or with 484.5 gl dilution buffer at pH 7.6. Transport assays were run for 0.25-30 min, and sucrose uptake determined by sampling 0.1-ml reaction volume and collecting vesicles on 0.2gm nitrocellulose filters (Schleicher & Schuell, Keene, N.H., USA). Filters were washed three times with 1-ml each of pH 6.0 or 7.6 dilution buffer, dried, and the radioactivity was determined by scintillation spectroscopy. Uptake rates were calculated by least-squares linear regression analysis. Values reported are the average of at least three replications. Because the experimental protocol varied slightly in the experiments reported in Figs. 1 and 2, values are from single experiments but are quantitatively similar to results obtained from three or greater replications. Acetate uptake was conducted by a similar procedure as for sucrose uptake with the exclusion of sucrose and the inclusion of 2 p l carrier-free acetate (2.1GBq.mmol 1, 3.7 MBq. ml-1; N E N Products). Thiocyanate uptake was assayed in PM vesicles equilibrated in 2 mM Hepes-BTP, pH 7.6, 350 mM sorbitol, 1 mM Ksulfate and 1 mM DTT. Vesicles were diluted 20-fold into the above buffer with 4 mM, 1 mM or without K + in the presence or absence of valinomycin. The typical assay consisted of 486.3 gl of 2 m M Hepes-BTP buffer, pH7.6, containing 350 m M sorbitol, 1 m M K-suifate and 1 mM DTT, 0.2 ~tl carrier-free Na-thiocyanate (555 M B q . m m o l - 1, 92.5 M B q . m l 1 ; ICN Radiochemicals, Irvine, Cal., USA),_+ 1 Ixl valinomycin (0.5 mM stock solution in ethanol) and 12.5 lxl PM. The assay was initiated with the addition of PM, and uptake into the vesicles was determined by the filtration assay described above.

Fraction

VanadateChlorideNitrate-sensitive -stimulated -inhibited Mg 2+-ATPase Mg 2 +-ATPase Mg 2 +-ATPase

Microsomal fraction Recovery

165.2 (1.3)

Plasma membrane Recovery

48.5 (7.8)

(%)

(%)

I00.00

29.3

53.8 (0.4)

0.3 (0.05)

100.0

100,0

ND

ND

0.0

0.0

Results

Plasma-membrane isolation. Plasma-membranes were isolated by the aqueous two-phase partitioning method described by Larsson (1983) and modified by Buckhout and Hrubec (1986). This method generally provides membrane fractions composed of >90% PM vesicles of a defined, right-side-out orientation (Buckhout and Hrubec 1986). Analysis of membrane-marker enzymes showed that contamination of the PM fraction by endoplasmic reticulum (antimycin A-insensitive, NADH cyt-c reductase) and Golgi apparatus (latent uridine 5'diphosphatase) was, respectively, 0.2 and 5.3% of the total marker-enzyme activities in the microsomal fraction recovered in the PM fraction (Table 1). Mitochondrial cyt-c oxidase (Table 1), and tonoplast C1--stimulated or NO3-inhibited Mg z+ATPase (Table 2) were not detected in the PM fraction. The chlorophyll content of the PM was 0.1% of that found in the microsomal fraction (Table 1). In contrast, approx. 4.7% of the total pro-

396

T.J, Buckhout: Sucrose transport in isolated PM vesicles of Beta vulgar& leaves

rein (Table 1) and nearly 30% of the total PM as determined by vanadate-sensitive Mg2+-AT Pase were recovered in the PM fraction (Table 2). The vanadate-sensitive ATPase was stimulated approximately sevenfold by the addition of 0.0125% (v/v) Triton X-100, indicating that the vesicles were primarily oriented with the ATP-hydrolysis site on the inside surface of the vesicles (data not shown; Larsson et al. 1984). Taken together the data indicate that the membrane fraction used in this study was predominantly right-side-out PM vesicles.

Acetate uptake in P M vesicles. Initial experiments were conducted to establish the presence of a p H gradient across the PM vesicle. Vesicles were equilibrated at p H 7.6 and diluted into a p H 6.0 buffer. Acetic acid was used as a pH-sensitive probe to confirm p H gradient, alkaline inside. Weak, lipophilic acids accumulate in compartments that are more basic than the bulk solution. Analysis of acetate uptake into PM vesicles demonstrated a pHdependent accumulation (Fig. 1). The pH-dependent uptake was sensitive to the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that acetate was accumulated in the vesicles and not bound to the membrane. The pH-dependent component of acetate uptake was similar in the presence or absence of valinomycin and K § ; however, in the absence of valinomycin and K +, acetate accumulation was only partially reversed by addition of CCCP or gramicidin (data not shown). Although we have not investigated this phenomenon in any detail, the result may indicate the trapping of acetate in the interior of vesicles, possible by a Donnan potential induced by the imposed p H gradient. The inclusion of K § salts may have masked these charges and allowed free mobility of acetate in response to the p H gradient. Sucrose uptake in P M vesicles. To circumvent complications inherent in uptake experiments in complex tissues, a cell-free PM system to assay sucrose transport was developed. The P M vesicles were equilibrated in pH 7.6 buffer and diluted into pH 6.0 (pH-jump) or pH 7.6 (control) buffer in the presence of 100 gM sucrose. Vesicles diluted into pH 6.0 buffer showed a pH-dependent uptake of sucrose (Fig. 2, open squares), The pH-dependent uptake was linear over the 15-min assay and was sensitive to CCCP. A pH-independent component of sucrose uptake was also observed (Fig. 2, open triangles), which was linear for the 15-min assay but was insensitive to CCCP (Fig. 2, solid triangles). Sucrose uptake was specific for sucrose in

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2 3 Time (min)

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Fig, 1. Accumulation of acetate in pH-jumped vesicles from Beta vulgaris leaves. Acetate accumulation was assayed by diluting PM equilibrated at pH 7.67 20-fold into I ml of pH 5.91 or 7.67 buffer. Following dilution, a portion of the reaction mixture was removed and the PM vesicles recovered by filtration. The protonophore, CCCP, was added to one set of samples at 2 rain (arrow). The treatments were: vesicles diluted into pH 6.0 buffer ( o - - o ) , pH 6.0 plus CCCP after 2 min ( e - - o ) or vesicles diluted into pH 7.6 (rn--rn). The final acetate concentration was 3.5 ~tM. Assuming a vesicle Volume of 2 lal. (rng protein) 1 the Henderson-Hasselbach equation predicts a pH gradient of approx. ~.6 in vesicIes diluted into pH 5.91 buffer

0.6 ~0.4

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Fig. 2. pH-dependent uptake of sucrose into PM vesicles from Beta vulgaris leaves. The PM vesicles were equilibrated in pH 7.6 buffer and diluted 20-fold into pH 6,0 ( o - - o , D - - n ) or pH 7.6 ( A - - A ) buffer. Sucrose uptake was assayed in the presence (circles) or absence of Valinomycin (squares). No effect of valinomycin was observed with pH-independent sucrose uptake (data not shown). After 7.5 min of sucrose uptake, samples were divided and the protonophore, CCCP, was add to one-half of the samples (solid symbols)

that none of the four monosaccharides and three disaccharides tested significantly inhibited the sucrose uptake, although the competing sugars were present at 10 times the concentration of sucrose (Table 3). An analysis of sucrose-uptake activity versus sucrose concentration, conducted in the presence of valinomycin and K § (see below) and a p H gradient, was biphasic (Fig. 3 A, open circles), while a similar analysis in the absence of a p H gradient demonstrated only a linear, first-order uptake (Fig. 3 A, solid circles). The pH-dependent component of sucrose uptake was saturable (Fig. 3A,

T.J. Buckhout: Sucrose transport in isolated PM vesicles of Beta vulgaris leaves Table 3. Specificity of sucrose transport into PM vesicles isolated from leaves of Beta vulgaris. Sucrose-transport activity was assayed in the presence of the mono- and disaccharides listed in the table. The final sucrose concentration in the transport assay was 0.1 mM, and competing sugars were tested at 1 mM. Results are the average of four separate determinations and rates are pmol. rain - 1. (rag protein)- 1 +_1 SE in parentheses

A

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~ 400

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Sucrose uptake

Sucrose, pH 6.0 Sucrose, pH 7.6 Mannose Glucose Fructose Mannitol Melibiose Lactose Maltose

47.9 12.3 43.9 43.2 43.3 42.7 46.2 45.6 44.1

200 0

(7.7) (2.6) (10.4) (9.5) (3.9) (7.2) (4.6) (6.2) (7.9)

397

'~ .~

0.03

N

0.02

~

0.01

0

1

2

3

4

5

6

o

0.00

open squares) and produced a linear Hanes-Woolf plot (r 2 > 0.99) with an apparent half-maximal concentration of sucrose (Kin) equal to 0 . 4 5 m M (Fig. 3 B). These data support the hypothesis that pH-dependent sucrose uptake is a carrier-mediated process. The fact that vesicle-associated sucrose decreased following the addition of CCCP is supporting evidence that the sucrose concentration inside the vesicle was greater than that outside (Fig. 2, solid circles). In order to calculate the sucrose concentration inside the PM vesicles directly, vesicles were equilibrated in the reaction mixture containing labeled sucrose at pH 7.6 for 60 min. Under these conditions, the pH-independent uptake of sucrose reaches equilibrium (data not shown). Membrane vesicles were recovered from solution by filtration as describe in the Material and methods and the internal volume of the recovered vesicles determined assuming that the recovered sucrose on the filters had the same specific activity as the sucrose in the bulk solution. The results of this study indicated that the PM vesicle contained approx. 2 nl(gg protein)-1. Using this value, the internal concentration of sucrose in the PM vesicles at the time of CCCP addition in Fig. 2 was 2.7-fold greater than the sucrose concentration in the reaction mixture. Thus, the sucrose was transported against a concentration gradient.

Effect of valinomycin and membrane potential on sucrose uptake. Physiological evidence indicates an electrogenic H+-sucrose symport as the mechanism of sucrose transport across the PM. If pHdependent sucrose transport is electrogenic, dissipation of the electrical potential (A~9) across the

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Sucrose (mM) Fig. 3A, B. Analysis of the effect of sucrose concentration on sucrose uptake in isolated PM vesicles from Beta vulgaris leaves. A Sucrose-uptake assays were performed in the presence of valinomycin and 1 m M KC1. Uptake was measured in the presence ( o - - o ) or absence ( o - - o ) of a pH gradient and the pH-dependent component was determined ( D - - n ) . Vertical bars represent 4-1 SE. B Replotting the pH-dependent component of sucrose uptake by the method of Woolf-Hanes resulted in a linear plot (r2>0.99) giving an apparent K~ of 0.45 mM and a VmaXof 153 p m o l - m i n - 1. (mg protein)- 1

PM should result in an increase in the sucroseuptake rate, since the proton-motive force would transport sucrose and not charge. Our experimental findings support this hypothesis. Inclusion of valinomycin and K + in the sucrose-transport assay resulted in an approx. 2.5-fold increase in the uptake rate (Fig. 2, open circles). Valinomycin-stimulated sucrose uptake was pH-dependent and sensitive to CCCP (Fig. 2, solid circles). The data presented support an electrogenic H+-sucrose symport as the mechanism of sucrose movement across the PM. If a sucrose-H § symport mechanism is electrogenic, the rate of sucrose uptake should be affected by the membrane potential (A ~). Artificial membrane potentials were generated across the vesicle membrane with a combination of valinomycin and K § Three conditions were tested: I m M K § inside the vesicles without K § in the dilution buffer, resulting in a negative A ~ inside the vesicle; 1 m M K § inside the vesicle and in the dilution buffer, a condition which should hold AO near zero; and 1 m M K + inside the vesicle with 4 m M K § in the dilution buffer, resulting in a positive A O inside ~

T.J. Buckhout: Sucrose transport in isolated P M vesicles of Beta vulgaris leaves

398

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"Fable 4. Effect of membrane potentials (A0) on sucrose transport in plasma-membrane vesicles isolated from Beta vulgaris leaves. Plasma membranes were prepared in buffer containing l m M K § and diluted into buffer at pH 7,6 or pH 6.0 in the presence of 0, 1 or 4 m M K § supplied as K2SO4. Sucrose transport was assayed as described in Material and methods in the presence or absence of valinomycin. Rates of sucrose uptake were determined by linear regression analysis, and the values reported are averages from three membrane preparation 4-1 SE in parentheses. Values are expressed as p m o l . m i n - t . (mg protein) - 1

6

Time (min) Fig. 4. A 0-driven thiocyanate accumulation in P M vesicles isolated from Beta vulgaris leaves. The P M vesicles were equilibrated in pH 7.6 buffer containing 1 m M K § as K2SO4 and diluted 20-fold into pH 7.6 buffer containing 4 m M ( n - - n ) , 1 m M ( e - - e ) or without K + ( o - - o ) . The total concentration of SCN 1 - was 67 IxM. Assuming a vesicle volume of 2 pl. (rag protein)-1, the Nernst equation predicts a membrane potential (A 0) of approx. 48 mV, positive inside, in vesicles diluted into 4 m M K § . Data reported as valinomycin-dependent thiocyanate uptake

the vesicles. Thiocyanate was used as a probe to detect a A ~b positive inside the vesicle. In the presence of a A~b, positive inside, the lipophilic thiocyanate anion should accumulate. Analysis of thiocyanate uptake in vesicles equilibrated in I m M K § and diluted into 4 m M K § demonstrated a valinomycin-dependent accumulation of thiocyanate in the vesicles. The P M vesicles equilibrated in 1 m M K + and diluted into buffer with 1 m M or without K § showed no valinomycin-dependent thiocyanate uptake (Fig. 4). In fact, under these conditions, valinomycin treatment resulted in a slight decrease in vesicle-associated thiocyanate. These data verify the presence of a valinomycin + K§ A ~b across the PM. Using this experimental system it could be shown that the rate of sucrose uptake in the presence of valinomycin and a p H gradient was increased 38% in the presence of a A0, negative inside, and reduced 21% by a A~b, positive inside (Table 4). Altering the K § concentration in the absence of valinomycin did not greatly affect pHdependent or pH-independent sucrose uptake (Table 4). If the sucrose-H § symport is electrogenic, generation of A 0, negative inside, in the absence of a p H gradient should also drive sucrose uptake. The rate of sucrose uptake in the absence of a p H gradient and with a negative potential was increased 54% compared to uptake in the absence of a membrane potential or in the presence of a A 0 positive inside (Table 4). Superficially, this result supports a potential-driven uptake of sucrose. However, the magnitude of the potential-driven uptake was approx~ 10% of the pH-dependent su-

External Expected Valinomycin KC1 membrane (mM) potential -- (pH)

+ (pH)

(A0) 6.0

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6.0

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18.1 (/0.4) 6.1 (2.3) 41.4 (8.2) 7.4 (1.7)

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16.3 (4.5) 5.8 (1.4) 30.1 (6.1) 4.8 (1.4) 14.2 (8.7) 4.7 (2.3) 24.3 (4.3) 5.3 (2.1)

crose uptake, drawing into question the role of this relatively small component of sucrose uptake. Nevertheless, the data presented support an electrogenic sucrose-H § symport as the mechanism of carrier-mediated sucrose transport across the PM of beet leaf cells. Discussion

Our data demonstrate the presence of carrier-mediated sucrose transport in isolated P M vesicles from beet leaf. The carrier was shown to be pHdependent, specific for sucrose (Table 3), saturable (Fig. 3), and electrogenic (Fig. 2, Table 4). Bush (1989) also has reported a pH-dependent sucrosetransport system in PM vesicles from beet leaves. A sucrose-uptake versus sucrose-concentration analysis in that study also indicated biphasic kinetics with a saturable component conforming to Michaelis-Menten-like kinetics. The apparent Km of the enzyme for sucrose was approx. I mM. The results presented here and by Bush (1989) are in agreement. The kinetics of sucrose uptake were biphasic, indicating both saturable and nonsaturable components. Similar kinetics have been reported for sucrose uptake into intact tissues (Giaquinta 1983) and can be interpreted as deriving from two separate transport pathways. In fact, Schmitt et al. (/984) suggested three pathways of sucrose movement into soybean cotyledon cells: a saturable, carrier-mediated sucrose-H + symport, a carrier-mediated diffusion pathway; and a non-carrier-mediated diffusive pathway. The cellular localization of the sucrose-transport systems remains uncertain. Initially, the saturable component of sucrose

T.J. Buckhout: Sucrose transport in isolated PM vesicles of Beta vulgaris leaves

uptake was thought to be located on the companion-cell and-or sieve-element PM where it functioned in phloem loading (Giaquinta 1983). In support of this hypothesis Daie (1987) found only a single, saturable pathway of sucrose uptake in isolated vascular strands from celery petioles. Conversely, Maynard and Lucas (1982a, b) presented evidence that both saturable and non-saturable sucrose uptake are present in all cell types, although the relative contributions of each pathway to total sucrose transport may vary with the tissue investigated. Since the PM population in our preparations is heterogeneous, likely containing PM from all leaf cell types, results from this study can do little to resolve this controversy. The unique contribution of this report is the demonstration that the H+-sucrose symport is electrogenic. The electrogenic nature of the transport was indicated by the stimulatory effects of valinomycin plus K + (Fig. 2) and membrane potentials, negative inside (Table 4), on sucrose transport and the inhibitory effect of membrane potentials, positive inside (Table 4). Perplexing is the relatively small component of potential-driven sucrose uptake in the absence of a pH gradient. It is, however, likely that electrogenically driven sucrose uptake is also pH-dependent since movement of sucrose is coupled to H + movement. At a pH value significantly greater than the pK~ of the amino acid involved in H § binding, little binding and hence little transport could occur. However, the characteristics of the sucrose transport reported here and by Bush (1989) are similar to those determined in intact tissues, and we conclude that the sucrose transport into isolated PM vesicles is identical to the pH-dependent sucrose transport measured in intact tissues. The author gratefully acknowledges timely and stimulating discussions with Dr. Daniel Bush (USDA-Agricultural Research Service, Urbana, Ill., USA) and thanks him for making available his unpublished methods and results.

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