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Affinity purification of sucrose binding proteins from the plant plasma membrane
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Biochi~ic~a et Biophysica A~ta ELSEVIER
Biochimica et Biophysica Acta 1219 (1994) 389-397
Affinity purification of sucrose binding proteins from the plant plasma membrane Ze-Sheng
Li, A b d e l M a j i d N o u b h a n i ,
Andr~e Bourbouloux,
Serge Delrot *
UA CNRS 574, Laboratoire de Biologie et Physiologie V[g~tales, Universitg de Poitiers, 25 Rue du Faubourg Saint-Cyprien, 86000 Poitiers, France Received 28 February 1994
Abstract
Purified plasma membranes from sugar beet leaves were solubilized by 1% 3-((3-cholamidopropyl)dimethylammonio)-l-propanesulfonate and loaded on a sepharose 6 B column substituted with sucrose. Elution with sucrose at pH 5.2 yielded a peak that represented 0.2% of the loaded protein. This peak did not appear when the samples were pretreated with either 0.5 mM N-ethylmaleimide (NEM) or 0.5 mM para-chloromercuribenzenesulfonicacid. It was also absent when palatinose, a sucrose analogue not recognized by the sucrose transporter, was used as the affinity ligand. The peak specifically eluted by sucrose from the sucrose-Sepharose column exhibited sucrose transport activity after reconstitution into proteoliposomes. This peak was further fractionated by ion-exchange chromatography on a Mono-Q column, and the different fractions obtained were differentially labeled by [3H]NEM in the presence of sugars recognized (sucrose, maltose) or not recognized (palatinose) by the sucrose transporter. The data allowed to identify two fractions that were enriched with two polypeptides (56 and 41 kDa) differentially labeled by NEM in the presence of sucrose.
Keywords:Affinity chromatography; Sucrose transport;
Plasmalemma; Plasma membrane; Reconstitution; (Plant); (Sugar beet)
1. Introduction
Sugar transport in plant controls the distribution of reduced carbon available as a metabolic source and as a structural precursor for cell growth. In addition, a growing body of evidence shows that the expression of many genes may be controlled by sugars or by their metabolic products [1-5]. Long distance transport of sugars between donor organs (leaves) and receiving organs (fruits, roots, tubers) results from a combination of apoplastic and of symplastic exchanges [6,7]. Symplastic exchanges occur via the plasmodesmata that cross the cell wall between two neighbouring cells. Considerable progress has recently been made in the understanding of the function and regulation of plas-
Abbreviations: CHAPS, 3-((3-cholamidopropyl)-dimethylammonio)-l-propanesulfonate; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis; PCMBS, para-chloromercuribenzenesulfonic acid; SDS, sodium dodecyl sulfate. * Corresponding author. Fax: + 33 49 559374. 0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 4 ) 0 0 1 0 5 - C
modesmatal activity [8,9]. Apoplastic transport of sugars involves their release from the assimilating cell into the cell wall, which is thought to be carrier-mediated [10], and their uptake across the plasma membrane of the neighbouring cells, which occurs with protoncotransport [11]. Uptake of sugars across the plasma membrane concerns sucrose, the main form of long distance transport in plants, but also hexoses [11]. Hexose uptake occurs with proton cotransport in algae and higher plants [12-15]. Proton-driven hexose uptake in purified plasma membrane vesicles from sugar beet leaves has been reported [16] and the hexose transporter from Chlorella kessleri [17] and Arabidopsis thaliana [18] have been cloned. From its sequence, the Chlorella hexose transporter gene (HUP1) is predicted to encode a protein 533 amino acids in length, with twelve transmembrane spanning regions. The c D N A encoding the hexose transporter of Chlorella has been functionally expressed in the fission yeast Schizosaccharomyces pombe, allowing protondriven uptake of hexose [19], and the c D N A encoding the hexose transporter of Arabidopsis has been func-
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tionally expressed in Xenopus oocyte, resulting in high rates of hexose uptake and in hexose-induced plasma membrane depolarization [20]. Kinetics of sucrose uptake in plant tissues reveal the existence of two saturable phases, with a high affinity phase mediating proton sucrose cotransport, and a low affinity phase whose functioning seems less dependent on protons [13,21]. Both phases of sucrose uptake also differ by their sensitivity to chemical reagents [22]. Proton-driven sucrose uptake has been demonstrated in plasma membrane vesicles from several species [2326]. Biochemical approaches have identified several candidates as putative sucrose carriers. A 62 kDa polypeptide involved in sucrose uptake by soybean cotyledons has been identified by photoaffinity labeling of microsomal fractions [27]. Immunolocalization shows that this polypeptide is associated with the plasma membrane, and more particularly the plasma membrane of phloem cells [28,29], but its sequence shows neither homology with animal or bacterial sugar transporters nor the expected transmembrane domains [29]. Expression of the corresponding transcript shows some correlation with sucrose transport activity, although the correlation is not tight. It is still possible that this protein is a peripherical protein involved in the regulation a n d / o r in the transport as part of a multimeric complex [29]. Differential labeling with NEM indicates the presence of a sucrose-protected 42 kDa polypeptide in plasma membrane fractions from broad bean [30] and sugar beet leaves [31]. Antibodies raised against the 42 kDa region of the plasma membrane from sugar beet leaf cells inhibit sucrose transport in broad bean protoplasts [32] and in sugar beet plasma membrane vesicles [33] but they do not affect amino acid transport [32,33]. Separation of plasma membrane proteins from sugar beet leaf by gel filtration and by ion-exchange chromatography, followed by reconstitution in proteoliposomes also showed that the highest sucrose transport activity was associated with a fraction enriched in the 42 kDa band [34]. The sink/source transition in sugar beet leaf is accompanied by the appearance of a NEM-sensitive sucrose transporter, and of additional bands in the 42 kDa region of the plasma membrane
[35]. A cDNA (pS21) encoding a sucrose transporter from spinach leaf was recently identified by functional complementation of a yeast [36]. The cDNA encodes a protein of 55 kDa, mediating sucrose uptake, and exhibiting the same substrate specificity and inhibitor sensitivity as those described in physiological and biochemical studies [6,11]. However, the relationship between this cDNA and the putative sucrose transporters identified by biochemical approaches in other plant species (see above) is still unknown. Ageing (flotation of leaf tissues for several hours on
a simple medium) induces the appearance of a new sucrose uptake system whose localization and sensitivity to chemical reagents differs from that existing in fresh tissues [37]. Although the mechanism of sucrose effiux is still unclear, recent studies with plasma membrane vesicles suggest that the protein mediating efflux is not the same as the protein mediating active uptake
[lO]. In conclusion, results from physiological, biochemical and molecular biology approaches make it possible that the plasma membrane of plant cells contains several populations of sucrose transporters differing by their kinetics, their tissue localization and their sensitivity to thiol reagents. This led us to develop a novel approach for the purification of sucrose-binding proteins by affinity chromatography on a sucrose-column. The potential interest of this approach is a rather wide selectivity, because it may allow the recovery of various sucrose transporters, but also of other sucrose-binding proteins. Concerning the transporters, the method is less selective than cloning because it may allow the recovery of several unrelated populations of transporters, if several transporter populations exist, and it may allow the recovery of multimeric transport proteins.
2. Materials and methods
2.1. Preparation of plasma membranes Sugar beet plants (Beta l,ulgaris L. var. Aramis) were grown in a greenhouse as described previously [31]. Mature exporting leaves (4-5 weeks old) were harvested for the isolation of plasma membrane vesicles by phase partition between Dextran T 500 and polyethylene glycol 3350 [31,38].
2. 2. Affinity resin preparation Sucrose was covalently coupled to Epoxy-activated Sepharose 6B according to the following procedure. 2 g of Epoxy-activated Sepharose 6B (Pharmacia) were swollen for 1 h and washed extensively with deionized water. After washing with 200 ml of coupling buffer (0.1 M NaOH, pH 12.5), the wet gel (6 ml) was incubated in a solution of 50 mM sucrose in 0.1 M N a O H (50 ml) at 40°C for 48 h on a heating shaker. The coupled gel was washed to remove uncoupled sucrose and incubated with a 1 M ethanolamine solution for 4 h to prevent further cross-linking. Ethanolamine was eliminated by successive swellings with a solution (200 ml) containing 0.5 M NaC1 in 0.1 M sodium borate (pH 8.0) alternatively with a solution containing 0.5 M NaCl in 0.1 M sodium acetate (pH 4.0). Treatment of the gel
Z.-S. Li et aL / Biochimica et Biophysica Acta 1219 (1994) 389-397
by invertase and subsequent enzymatic measurement of the hexoses released in the elution buffer indicated that the gel contained about 0.35 mg bound sucrose ml- 1. The coupled gel was stocked (up to 1 week) in 50 mM Tris-HCl buffer (pH 7.5) with 0.1 M sucrose and 0.01% sodium azide at 4°C until further use. Control experiments were made with a gel for which palatinose (6-O-a-D-glucopyranosyl-D-fructofuranose), a sucrose analogue not recognized by the sucrose transporter [39] was used as the bound ligand. The preparation of the gel was the same as described above for sucrose.
2.3. Membrane solubilization and chromatography procedures Plasma membrane proteins (100 to 150 mg) were resuspended (2 mgm1-1) and solubilized with 1% CHAPS (= 16 mM, final concentration). After acidification with a few drops of saturated NaHzPO 4 (pH 5.2), 3 ml of CHAPS supernatant (about 0.7 mg proteinm1-1) were used for affinity purification, and loaded on a column (1 × 10 cm, Pharmacia) containing 6 ml of coupled gel. Unbound proteins were then washed with buffer A (50 mM sodium phosphate, pH 5.2, 0.1% CHAPS, 0.5 mM CaCI 2 and 0.25 mM MgC12) at a flow rate of 0.2 mlmin -1 for 30 rain and 0.5 ml min-l for 15 min. Bound proteins were eluted with buffer B (buffer A + 0.1 M sucrose) for 30 min at 0.5 ml min-1. In some experiments, 0.1 M lactose was used instead of sucrose in buffer B to test the specificity of the elution. Non specifically bound proteins were removed by washing the gel 30 min with 0.5 M NaC1 in 50 mM sodium phosphate (pH 7.8) (buffer C). Fractions were collected at 5 rain intervals, and pooled as described in Results. The samples were frozen at -20°C, lyophilised, and resuspended in 50 mM potassium phosphate (pH 7.5) for dialysis against 50 mM potassium phosphate + 0.1% CHAPS, using a membrane dialysis with a cut-off size of 6000-8000. This dialysis removed the phosphate and decreased the CHAPS concentration to 0.1%. The samples were then frozen at -20°C, lyophilised, and resuspended in 0.5 ml 50 mM potassium phosphate pH 7.5 for reconstitution experiments. In some experiments, sodium phosphate was replaced with 50 mM Tris-HCl, and the protein samples recovered from the sucrose affinity column were further separated on a Mono-Q ion exchange column (HR 5/5 Pharmacia). The corresponding fractions from 6 to 12 injections on the sucrose-affinity column were pooled, dialyzed and concentrated as described above. About 500 /xg protein (0.5 ml) were loaded on the column. The different peaks separated on the Mono-Q column were recovered for SDS-PAGE analysis [31] and differential labeling.
391
2.4. Differential labeling [3H]NEM labeling experiments were modified from [31] and carried out as follows. Ten /~g of Mono-Q purified proteins were incubated with [3H]NEM (518 kBq, 3.2 /zM) and 20 mM sucrose in 500 mM phosphate buffer (pH 5.5) for 30 rain at 25°C. Sucrose was used as a protecting sugar against NEM inhibition [31]. Free [3H]NEM was neutralized by addition of 10 foldexcess of dithiothreitol. The effect of sucrose was compared with that of maltose, poorly recognized by the sucrose transporter and palatinose, not recognized by the sucrose transporter [39]. Control samples were also prepared in the absence of any protecting sugar. The [3H]NEM labeled samples were then solubilized 20 min at 37°C and separated by SDS-PAGE [31]. After silver staining [40], each lane was cut into 2 ram-thick slices, which were digested overnight with perchloric a c i d / H 2 0 2 (1:2, v/v). After addition of 4 ml AquaLuma Plus (Amersham), the radioactivity of the samples was measured in a Packard 1900TR scintillation counter. For each slice, a labeling index was calculated according to the formula: 3H of the slice (control) total 3H of the lane (control) 3H of the slice (sugar) total 3H of the lane (sugar)
2.5. Reconstitution and transport assays Using affinity purified proteins, proteoliposomes were obtained according to the method previously described [34,41]. Soybean asoleetin (12 rag) (Sigma IV-S) was sonicated in potassium phosphate buffer (pH 7.5) with 1 mM DT/'. CHAPS and glycerol were added to the final concentration of 0.5 and 20%, respectively. Proteoliposomes were formed by dilution of plasma membrane proteins (125 /~g) from affinity column or from CHAPS supernatant into 25 ml of potassium phosphate buffer (pH 7.5). After centrifugation (1 h at 100000 × g), the pellet recovered was resuspended in 100/xl of potassium phosphate buffer (pH 7.5). Uptake experiments were carried out by diluting 2 /xl of the proteoliposomes suspension into 400 /xl of incubation medium containing 0.3 M sorbitol, 50 mM sodium phosphate (pH 5.5), 0.5 mM CaCI2, 0.25 mM MgCI2, 0.5 mM DTr, 5 /zM valinomycin and 1 mM [6,6'(n)3H]sucrose (26 kBq) [34]. The artificial proton motive force thus created combined a transmembrane pH gradient (pH 7.5 inside, pH 5.5 outside), and a transmembrane electrical gradient, inside negative, due to valinomycin-induced diffusion of potassium outside the proteoliposomes. Active uptake was measured after 1
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Step I
min as the difference between proteoliposomes energized in this way, and non-energized proteoliposomes [34]. Proteins were assayed according to [42].
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3. R e s u l t s
3.1. Affinity purification and reconstitution of sucrose transport activity
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of the plant plasma membrane was not retained by the gel coupled with sucrose and was eluted with buffer A (Fig. 1A, peak I). Addition of 0.1 M sucrose in this buffer at pH 5.2 resulted in the elution a small peak (0.22_+ 0.03% of loaded proteins, mean + S.E. of 4 measurements) (Fig. 1A, peak II). Washing the column with a sucrose-free buffer at pH 7.8 allowed the recovery of a third peak (0.28 _+ 0.07% of loaded proteins, Fig. 1A, peak III). When the sucrose-coupled column was eluted with lactose, a similar pattern was obtained concerning the appearance of peak I and peak III, but lactose did not cause the elution of a peak at pH 5.2 (Fig. 1B). The peak eluted at pH 5.2 by sucrose (peak II in Fig. 1A) therefore seemed the more specific for sucrose-binding proteins. Because at least part of the sucrose transporters are sensitive to thiol reagents [6,11,25,26,37], the elution pattern of the solubilized proteins was studied with samples pretreated either with 0.5 mM PCMBS (Fig. 2A) or 0.5 mM N E M (Fig. 2B). In both cases, pretreatment of the samples with the thiol reagents prevented
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the appearance of p e a k II normally eluted by sucrose at p H 5.2, while peak I I I eluted at p H 7.8 was still present. Chromatography of the C H A P S supernatant on a gel where palatinose was used as the ligand also failed to yield a p e a k eluted by sucrose at p H 5.2 (Fig. 3). Altogether, the data presented abbve suggest that affinity chromatography on a sucrose ligand allow the recovery of a peak selectively eluted by sucrose at p H 5.2. The binding of this fraction to the ligand is prevented by N E M and PCMBS, as expected for the proton-sucrose cotransporter. The different fractions recovered after separation of the C H A P S supernatant on a sucrose-affinity column were assayed for active transport of sucrose after reconstitution into proteoliposomes. The data presented in Fig. 4 give the initial rate of the transport energized by the artificial proton-motive force, measured after a 1 min incubation, and expressed on a weight basis (mg protein actually reincorporated into the liposomes). Very weak activity was associated with peak I, whereas p e a k II exhibited a 4-fold increase in transport activity, compared to the C H A P S supernatant. The activity of peak III was similar to that of the C H A P S supernatant. Fig. 5 presents the S D S - P A G E analysis of the different fractions recovered after affinity chromatography on a sucrose ligand, as described in Fig. 1. Most of the injected proteins are recovered in peak I, and there is hardly any difference between lane B (CHAPS supernatant, S) and lane C (peak I). Peaks II and III were characterized by the absence of any band above 60 kDa. Although theoretically a similar amount of protein was loaded in each lane, peaks II and III only gave faint bands in silver staining, due either to a poor
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Fig. 4. Active transport of sucrose into proteoliposomes reconstituted with the different fractions obtained in Fig. 1. Active uptake was measured as the difference between uptake in the presence of imposed ApH+ A~F minus the uptake measured in the absence of imposed A + Ag'. Means of 12 measurements (3 independent experiments) + S.E.
staining with the method used or inaccuracy in assaying dilute protein samples. C o m p a r e d to peaks I and III, peak II was enriched in three bands (marked with dots), at 23 kDa and at 41 kDa (absent in peak I and III), and at 57 kDa (absent in peak III). C o m p a r e d to peak II, peak III was enriched with a 33 kDa band. Since peak II is selectively eluted by sucrose, exhibits sensitivity to N E M and to PCMBS, as well as the highest rate of sucrose transport after reconstitution, further attempts to characterize the sucrose transporters were run with this peak. After dialysis, and lyophilisation, this peak was resuspended in 50 m M
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Fraction Fig. 3. Separation of plasma membranes proteins on a palatinoseSepharose column. A, the plasma membrane proteins solubilized by 1% CHAPS were loaded on the column and eluted by buffer A (step 1, peak I), buffer A+0.1 M sucrose (step 2) and buffer C (step 3, peak III).
24
.......
Fig. 5. Silver-staining of the different peaks recovered from a sucrose-affinity column as described in Fig. 1A. (A) Molecular weight standards; (S) CHAPS supernatant; peaks I, II and III. The same amount of protein (about 10/~g) was loaded in each lane.
394
Z.-S. Li et al. /Biochimica et Biophysica Acta 1219 (1994) 389-397
-~ 0"10/
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Tris-HCl (pH 7.5) and loaded on a Mono-Q column for separation by ion-exchange chromatography. Fractionation of peak II on the Mono-Q column by a 0 to 0.5 M NaC1 gradient yielded 4 major fractions (B, C, D, E) which were collected as shown in Fig. 6. On an absorbance basis, fractions B, C, D and E accounted for 10, 19.5, 10 and 15% of the loaded proteins, respectively. Rinsing of the column with 1 M NaC1 gave another peak, which was not collected for further experiments.
3.2. Differential labeling Reconstitution of transport activity into proteoliposomes with the fractions recovered after Mono-Q
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Fig. 7. Differential labeling patterns obtained after separation by S D S - P A G E of the fractions recovered on a M o n o - Q column as described in Fig. 6. (A) peak C; (B) peak D. After dialysis and concentration, the fractions recovered from the M o n o - Q column were differentially labeled by [3H]NEM in the presence of various sugars, and separated by SDS-PAGE. A differential labeling index representative of sugar protection was calculated as detailed in Materials and methods. Hatched areas shows bands exhibiting the labeling expected for a sucrose transporter.
z.-s. Li et al. / Biochimica et Biophysica Acta 1219 (1994) 389-397
fractionation was not possible, due to their very low content in protein. Yet, we analyzed their pattern of labeling by [3H]NEM in the presence of sucrose, maltose (recognized by the sucrose transporters) and palatinose (not recognized). Unlike in previous experiments [34], it was not possible to conduct a double s t e p / d o u b l e labeling procedure combining unlabeled NEM, and [3H]NEM/[14C]NEM, because the present experiments were run with a low amount of solubilized proteins, while the former one were made directly on large amounts of native plasma membrane. According to the procedure developed here, any polypeptide protected from N E M by sucrose should be distinguished by a higher [3H], and hence a higher labeling index. The general pattern of labeling of peaks C and D are clearly different (Fig. 7) and also differ from the patterns found with peaks B and E (data not shown). Possible candidates for a sucrose transporter may be differentially labeled in the presence of sucrose and maltose, but not in the presence of palatinose. The data were therefore analyzed by searching peaks of differential labeling that would meet these criteria. No such peak was found in fraction B, or E (data not shown), whereas proteins ranging with apparent molecular mass of 38-41 kDa in fraction D (Fig. 7B), and a peak at 56 kDa in fraction C (Fig. 7A), fulfilled the expected requirements. Among different experiments, the differential labeling index in the presence of maltose for the 38-41 kDa region of fraction D represented about 70% of the index found in the presence of sucrose, while for the 56 kDa protein of fraction C, the differential labeling index in the presence of maltose never exceeded 30% of the index found in the presence of sucrose.
kDa
66
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29 Fig. 8. SDS-PAGE and silver-stainingof fractions C and D separated in Fig. 6. About 10 /zg were loaded in each lane. (A) Molecular weight standards.
395
Silver-staining of these different fractions showed that they still contain a number of different bands in spite of the different procedures used for protein separation (Fig. 8). Most of the protein bands found in fraction C are also found in fraction D, but fraction C contains a faint band at 57 kDa (marked wit dots) which is not found in fraction D. This band may correspond to the peak of differential labeling found in the same region of this fraction (Fig. 7A). Another band at 70 kDa was much more visible in fraction C than in fraction D. Compared to fraction C, fraction D is characterized by the presence of an additional band at 41 kDa. It is likely that the additional 41 kDa is associated with the peak of differential labeling found in this region (Fig. 7B).
4. Discussion
Various biochemical approaches conducted with different plant species have resulted in the identification of various polypeptides as putative sucrose transporters (42 kDa in sugar beet, 62 kDa in soybean). These polypeptides were identified after denaturating SDSPAGE. In a recent review, Bush [11] also reported that a radiolabeled phenyl derivative of forskolin specifically labeled a 55 kDa polypeptide, which may also be regarded as another possible sucrose transporter. Whether these differences are due to the different species investigated, to the different techniques used, or to the presence of several sucrose transporters is not known. Furthermore, these data do not easily reconcile with the results from molecular cloning, which predict a molecular mass of 55 kDa for the spinach sucrose transporter [36]. Due to their high hydrophobicity, membrane proteins often show a smaller molecular weight in SDS-PAGE than their actual molecular weight. For example, the triose-phosphate translocator of the inner chloroplastic membrane (molecular mass = 35.6 kDa) appears as a 29 kDa polypeptide in SDSP A G E [43], the hexose transporter from Chlorella kessleri (molecular mass = 57.4 kDa) appears as a 47 kDa band [17], and the lac permease of Escherichia coli (molecular mass = 46 kDa) appears as a 33 kDa band [44]. In several papers, the possibility of the sucrose transporter being a multimeric protein has also been suggested [29,34]. Altogether, these data point to the interest of non destructive techniques aimed at characterizing a n d / o r purifying sucrose transporters. In an attempt to purify sucrose transporters (and sucrose-binding proteins) by a non destructive approach, the present work combines affinity chromatography on a new support (sucrose-Sepharose gel) and the possibility to reconstitute sucrose transport activity into proteoliposomes made from plant membrane extracts [34].
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Z.-S. Li et al. / Biochimica et Biophysica Acta 1219 (1994) 389-397
In our previous work [34], the first step used to purify the sucrose transporter was a gel filtration on a TSK SW 3000 column, and the eluate was monitored by ELISA with anti-42 kDa antibodies to purify specifically the 42 kDa polypeptide. This step yielded a 120 kDa peak which represented 40% of the CHAPS supernatant, and it resulted in a 5-fold increase in specific activity of sucrose transport after reconstitution in proteoliposomes. Silver-staining showed that the fraction thus recovered still contained almost all the proteins of the CHAPS supernatant [34]. In the present work, the initial step of the purification was not aimed specifically at purifying the 42 kDa polypeptide, but all the polypeptides interacting strongly enough with sucrose. This step allowed the recovery of a fraction (peak II), that represented only 0.2% of the CHAPS supernatant, and exhibited the properties expected for a sucrose transporter. This fraction was not eluted by lactose (Fig. 1B), not retained on a palatinose-sepharose column (Fig. 3), sensitive to NEM and PCMBS (Fig. 2), and exhibited transport activity (Fig. 4). The affinity chromatography step resulted in a 4-fold increase in sucrose transport activity, compared to the initial CHAPS supernatant (Fig. 4). The rise in specific activity of transport was considerably lower than that expected from the protein yield (a theoretical increase of 500-fold might have been expected), but numerous reasons already discussed [34,45] may explain this resuit. In addition to the usual loss of activity during dialysis, concentration, lyophilisation, it is not necessarily valid to compare directly transport activity of proteoliposomes made from different membrane fractions, because their size and permeability to protons and potassium may be different, which would considerably affect the rate and the time course of uptake. The rate of transport may also be limited by thermodynamical reasons including the actual proton motive force created by the ion gradients used, and the density and the orientation of proteins in the proteoliposome. The highest specific activity measured in the present paper (70 n m o l / m g protein per min) was about twice that reported before using the gel filtration/ion exchange purification [34]. Unlike the gel filtration step used before [34], the use of affinity chromatography as the first step of purification only yielded a limited amount of polypeptides in the present work (Fig. 1), and peak II was characterized by a limited number of polypeptides, including a band at 41 kDa and at 56 kDa (Fig. 5). Because this peak exhibited the highest activity of transport, it may be assumed that one or both of these polypeptides are involved in sucrose transport. Due to the low recovery of proteins, when peak II was further fractionated on a Mono-Q column, reconstitution experiments were not possible. In order to characterize these fractions, we therefore developed a
differential labeling procedure which was simpler than the former one, and concerned solubilized proteins instead of native plasma membrane vesicles. The value of differential affinity labeling by N E M to identify the sucrose transporter has been questioned, on the basis that sucrose uptake into plasma membrane vesicles has sometimes been reported to be NEM-insensitive [11]. We have shown that N E M sensitivity of sucrose transport depends on experimental conditions and on plant material, and that the differential labeling approach was therefore valid ([37], Sakr S., Gaillard C., Roblin G. and Delrot S., unpublished data). The differential labeling procedure developed in the present paper allowed us to distinguish two (groups of) polypeptides which were differentially labeled in the presence of sucrose or maltose, but not in the presence of palatinose. A fraction eluted at 0.17 M NaCI (Fig. 6, fraction C) included a 56 kDa polypeptide which was differentially labeled (Fig. 7A), and apparent after silver-staining (Fig. 8). A fraction eluted at 0.30 M NaCI (Fig. 6, fraction D) contained a 41 kDa band that was differentially labeled (Fig. 7B) and was apparent after silverstaining (Fig. 8). Within the limits of experimental errors, we assume that this polypeptide is the same than the one identified before at 42 kDa, that was differentially labeled by NEM and eluted at 0.31 M NaC1 on a Mono-Q column [34]. Although peak II did not contain polypeptides above 60 kDa upon silver-staining, some polypeptides up to 100 kDa appear when this peak is further resolved on a Mono-Q column, after dialysis and concentration. The appearance of high molecular weight bands may result from agregation phenomena during the concentration of proteins before ion-exchange chromatography. Silver-staining of the fractions recovered after affinity chromatography followed by ion exchange (Fig. 8) still shows the presence of several polypeptide bands, although lane C and D clearly differ by several bands. In addition to possible agregation or degradation artefacts which can hardly be dismissed, the presence of multiple bands in these fractions is also a result of the strategy of purification. Indeed, the procedure used theoretically allows the recovery not only of sucrose transporters, but also of any protein binding sucrose with enough affinity, as well as regulatory proteins which may be associated with the sucrose transporters and the sucrose binding proteins. The data therefore provide another line of evidence suggesting that a 42 kDa polypeptide may be involved in sucrose transport in sugar beet leaf. However, we were also able to identify a 56 kDa polypeptide which also exhibits some properties of a sucrose transporter. For reasons already discussed above (hydrophobicity), it is not likely that this polypeptide may be encoded by cDNA similar to the one encoding a 55 kDa protein in spinach. The relationship between the 56 kDa polypep-
Z.-S. Li et al. / Biochimica et Biophysica Acta 1219 (1994) 389-397
tide identified here and this cDNA, as well as the relationship between this polypeptide and the 55 kDa labeled by forskolin [11] also remain to be established.
Acknowledgements This work was supported in part by the EEC under the Bridge Programme (Contract BIOT-0175-C).
References [1] Salanoubat, M. and Belliard, G. (1989) Gene 84, 181-185. [2] Rocha-Sosa, M., Sonnewald, U., Frommer, W.B., Stratmann, M. and Willmitzer, L. (1989) EMBO J. 8, 23-29. [3] Hattori, T., Fukumoto, H., Nagakawa, S. and Nakamura, K. (1991) Plant Cell Physiol. 32, 79-86. [4] Tsukaya, H., Ohshima, T., Naito, S., Chino, M. and Komeda, Y. (1991) Plant Physiol. 97, 1414-1421. [5] Kim, S.-R., Kim, Y., Costa, M.A. and An, G. (1991) Plant Physiol. 98, 1479-1483. [6] Delrot, S. (1989) In Transport of Photoassimilates (Baker, D.A. and Milburn, J.A., eds.), pp. 167-205, Longman, Harlow. [7] Van Bel, A.J.E. (1993) Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 253-281. [8] Robards, A.W. and Lucas, W.J. (1990) Annu. Rev. Plant Physiol. Plant Mol. Biol. 41, 369-419. [9] Wolf, S., Deom, C.M., Beachy, R. and Lucas, W.J. (1991) Plant Cell 3, 593-604. [10] Laloi, M., Delrot, S. and M'Batchi, B. (1993) Plant Physiol. Biochem. 31, 731-742. [11] Bush, D.R. (1993)Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 513-542. [12] Komor, E. (1973) FEBS Lett. 38, 16-18. [13] Delrot, S. (1981) Plant Physiol. 68 706-711. [14] Felle, H. and Bentrup, F.W. (1980) Planta 147, 471-476. [15] Reinhold, L. and Kaplan, A. (1984) Annu. Rev. Plant Physiol. 35, 45-83. [16] Tubbe, A. and Buckhout, T.J. (1992) Plant Physiol. 99, 945-951. [17] Sauer, N. and Tanner, W. (1989) FEBS Lett. 259, 43-46. [18] Sauer, N., Friedl~inder, K. and Gram-Wicke, U. (1990) EMBO J. 9, 3045-3050. [19] Sauer, N., Caspari, T., Klebl, F. and Tanner, W. (1990) Proc. Natl. Acad. Sci USA 87, 7949-7952.
397
[20] Boorer, K.J., Forde, B.G., Leigh, R.A. and Miller, A.J. (1992) FEBS Lett. 302, 166-168. [21] Delrot, S. and Bonnemain, J.L. (1981) Plant Physiol. 67, 560-564. [22] Maynard, J.W. and Lucas, W.J. (1982) Plant Physiol. 70, 14361443. [23] Bush, D.R. (1989) Plant Physiol. 89, 1318-1323. [24] Buckhout, T.J. (1989) Planta 178, 393-399. [25] Lemoine, R. and Delrot, S. (1989) FEBS Lett. 249, 129-133. [26] Williams L.E., Nelson, S.J. and Hall, J.L. (1990) Planta 182, 540-545. [27] Ripp, K.G., Viitanen, P.V., Hitz, W.D. and Franceschi, V.R. (1988) Plant Physiol. 88, 1435-1445. [28] Warmbrodt, R.D., Buckhout, T.J. and Hitz, W.D. (1989) Planta 180, 105-115. [29] Grimes, H.D., Overwoode, P.J., Ripp, K., Franceschi, V.R. and Hitz, W.D. (1992) Plant Cell 4, 1561-1574. [30] Pichelin-Poitevin, D. and Delrot, S. (1987) CR Acad. Sci. Paris 304, 371-374. [31] Gallet, O., Lemoine, R., Larsson, C. and Delrot, S. (1989) Biochim. Biophys. Acta 978, 56-64. [32] Lemoine, R., Delrot, S., Gallet, O. and Larsson, C. (1989) Biochim. Biophys. Acta 78, 65-71. [33] Gallet, O., Lemoine, R., Gaillard, C., Larsson, C. and Delrot, S. (1992) Plant Physiol. 98, 17-23. [34] Li, Z.-S., Gallet, O., GaiUard, C., Lemoine, R. and Delrot, S. (1992) Biochim. Biophys. Acta 1103, 259-267. [35] Lemoine, R., Gallet, O., Gaillard, C., Frommer, W. and Delrot, S. (1992) Plant Physiol. 100, 1150-1156. [36] Riesmeier, J.W., Willmitzer, L and Frommer, W.B. (1992) EMBO J. 11, 4705-4713. [37] Sakr, S., Lemoine, R., GaiUard, C. and Delrot, S. (1993) Plant Physiol. 103, 49-58. [38] Lemoine, R., Bourquin, S. and Delrot, S. (1991) Physiol. Plant. 82, 377-384. [39] M'Batchi B. and Delrot, S. (1988) Planta 174, 340-348. [40] Guevara, J., Jr., Johnston, D.A., Ramagali, L.S., Martin, B.A., Capetillo, S. and Rodriguez, L.V. (1982) Electrophoresis 3, 197-205. [41] Schumaker, K.S. and Sze, H. (1990) Plant Physiol. 2, 340-345. [42] Bearden, J.C., Jr. (1978) Biochim. Biophys. Acta 533, 525-529. [43] Wallmeier, H., Weber, A., Gross, A. and Fliigge, U.-I. (1992) In Transport and receptor Proteins of Plant Membranes. Molecular Structure and Function (Cooke, D.T. and Clarkson, D.T., eds.), pp. 77-89, Plenum Press, New York. [44] Kaback, H.R. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 279-319. [45] Fredrikson, K., Kjellbohm, P. and Larsson, C. (1991) Physiol. Plant. 81, 289-294.
Biochi~ic~a et Biophysica A~ta ELSEVIER
Biochimica et Biophysica Acta 1219 (1994) 389-397
Affinity purification of sucrose binding proteins from the plant plasma membrane Ze-Sheng
Li, A b d e l M a j i d N o u b h a n i ,
Andr~e Bourbouloux,
Serge Delrot *
UA CNRS 574, Laboratoire de Biologie et Physiologie V[g~tales, Universitg de Poitiers, 25 Rue du Faubourg Saint-Cyprien, 86000 Poitiers, France Received 28 February 1994
Abstract
Purified plasma membranes from sugar beet leaves were solubilized by 1% 3-((3-cholamidopropyl)dimethylammonio)-l-propanesulfonate and loaded on a sepharose 6 B column substituted with sucrose. Elution with sucrose at pH 5.2 yielded a peak that represented 0.2% of the loaded protein. This peak did not appear when the samples were pretreated with either 0.5 mM N-ethylmaleimide (NEM) or 0.5 mM para-chloromercuribenzenesulfonicacid. It was also absent when palatinose, a sucrose analogue not recognized by the sucrose transporter, was used as the affinity ligand. The peak specifically eluted by sucrose from the sucrose-Sepharose column exhibited sucrose transport activity after reconstitution into proteoliposomes. This peak was further fractionated by ion-exchange chromatography on a Mono-Q column, and the different fractions obtained were differentially labeled by [3H]NEM in the presence of sugars recognized (sucrose, maltose) or not recognized (palatinose) by the sucrose transporter. The data allowed to identify two fractions that were enriched with two polypeptides (56 and 41 kDa) differentially labeled by NEM in the presence of sucrose.
Keywords:Affinity chromatography; Sucrose transport;
Plasmalemma; Plasma membrane; Reconstitution; (Plant); (Sugar beet)
1. Introduction
Sugar transport in plant controls the distribution of reduced carbon available as a metabolic source and as a structural precursor for cell growth. In addition, a growing body of evidence shows that the expression of many genes may be controlled by sugars or by their metabolic products [1-5]. Long distance transport of sugars between donor organs (leaves) and receiving organs (fruits, roots, tubers) results from a combination of apoplastic and of symplastic exchanges [6,7]. Symplastic exchanges occur via the plasmodesmata that cross the cell wall between two neighbouring cells. Considerable progress has recently been made in the understanding of the function and regulation of plas-
Abbreviations: CHAPS, 3-((3-cholamidopropyl)-dimethylammonio)-l-propanesulfonate; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis; PCMBS, para-chloromercuribenzenesulfonic acid; SDS, sodium dodecyl sulfate. * Corresponding author. Fax: + 33 49 559374. 0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 4 ) 0 0 1 0 5 - C
modesmatal activity [8,9]. Apoplastic transport of sugars involves their release from the assimilating cell into the cell wall, which is thought to be carrier-mediated [10], and their uptake across the plasma membrane of the neighbouring cells, which occurs with protoncotransport [11]. Uptake of sugars across the plasma membrane concerns sucrose, the main form of long distance transport in plants, but also hexoses [11]. Hexose uptake occurs with proton cotransport in algae and higher plants [12-15]. Proton-driven hexose uptake in purified plasma membrane vesicles from sugar beet leaves has been reported [16] and the hexose transporter from Chlorella kessleri [17] and Arabidopsis thaliana [18] have been cloned. From its sequence, the Chlorella hexose transporter gene (HUP1) is predicted to encode a protein 533 amino acids in length, with twelve transmembrane spanning regions. The c D N A encoding the hexose transporter of Chlorella has been functionally expressed in the fission yeast Schizosaccharomyces pombe, allowing protondriven uptake of hexose [19], and the c D N A encoding the hexose transporter of Arabidopsis has been func-
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tionally expressed in Xenopus oocyte, resulting in high rates of hexose uptake and in hexose-induced plasma membrane depolarization [20]. Kinetics of sucrose uptake in plant tissues reveal the existence of two saturable phases, with a high affinity phase mediating proton sucrose cotransport, and a low affinity phase whose functioning seems less dependent on protons [13,21]. Both phases of sucrose uptake also differ by their sensitivity to chemical reagents [22]. Proton-driven sucrose uptake has been demonstrated in plasma membrane vesicles from several species [2326]. Biochemical approaches have identified several candidates as putative sucrose carriers. A 62 kDa polypeptide involved in sucrose uptake by soybean cotyledons has been identified by photoaffinity labeling of microsomal fractions [27]. Immunolocalization shows that this polypeptide is associated with the plasma membrane, and more particularly the plasma membrane of phloem cells [28,29], but its sequence shows neither homology with animal or bacterial sugar transporters nor the expected transmembrane domains [29]. Expression of the corresponding transcript shows some correlation with sucrose transport activity, although the correlation is not tight. It is still possible that this protein is a peripherical protein involved in the regulation a n d / o r in the transport as part of a multimeric complex [29]. Differential labeling with NEM indicates the presence of a sucrose-protected 42 kDa polypeptide in plasma membrane fractions from broad bean [30] and sugar beet leaves [31]. Antibodies raised against the 42 kDa region of the plasma membrane from sugar beet leaf cells inhibit sucrose transport in broad bean protoplasts [32] and in sugar beet plasma membrane vesicles [33] but they do not affect amino acid transport [32,33]. Separation of plasma membrane proteins from sugar beet leaf by gel filtration and by ion-exchange chromatography, followed by reconstitution in proteoliposomes also showed that the highest sucrose transport activity was associated with a fraction enriched in the 42 kDa band [34]. The sink/source transition in sugar beet leaf is accompanied by the appearance of a NEM-sensitive sucrose transporter, and of additional bands in the 42 kDa region of the plasma membrane
[35]. A cDNA (pS21) encoding a sucrose transporter from spinach leaf was recently identified by functional complementation of a yeast [36]. The cDNA encodes a protein of 55 kDa, mediating sucrose uptake, and exhibiting the same substrate specificity and inhibitor sensitivity as those described in physiological and biochemical studies [6,11]. However, the relationship between this cDNA and the putative sucrose transporters identified by biochemical approaches in other plant species (see above) is still unknown. Ageing (flotation of leaf tissues for several hours on
a simple medium) induces the appearance of a new sucrose uptake system whose localization and sensitivity to chemical reagents differs from that existing in fresh tissues [37]. Although the mechanism of sucrose effiux is still unclear, recent studies with plasma membrane vesicles suggest that the protein mediating efflux is not the same as the protein mediating active uptake
[lO]. In conclusion, results from physiological, biochemical and molecular biology approaches make it possible that the plasma membrane of plant cells contains several populations of sucrose transporters differing by their kinetics, their tissue localization and their sensitivity to thiol reagents. This led us to develop a novel approach for the purification of sucrose-binding proteins by affinity chromatography on a sucrose-column. The potential interest of this approach is a rather wide selectivity, because it may allow the recovery of various sucrose transporters, but also of other sucrose-binding proteins. Concerning the transporters, the method is less selective than cloning because it may allow the recovery of several unrelated populations of transporters, if several transporter populations exist, and it may allow the recovery of multimeric transport proteins.
2. Materials and methods
2.1. Preparation of plasma membranes Sugar beet plants (Beta l,ulgaris L. var. Aramis) were grown in a greenhouse as described previously [31]. Mature exporting leaves (4-5 weeks old) were harvested for the isolation of plasma membrane vesicles by phase partition between Dextran T 500 and polyethylene glycol 3350 [31,38].
2. 2. Affinity resin preparation Sucrose was covalently coupled to Epoxy-activated Sepharose 6B according to the following procedure. 2 g of Epoxy-activated Sepharose 6B (Pharmacia) were swollen for 1 h and washed extensively with deionized water. After washing with 200 ml of coupling buffer (0.1 M NaOH, pH 12.5), the wet gel (6 ml) was incubated in a solution of 50 mM sucrose in 0.1 M N a O H (50 ml) at 40°C for 48 h on a heating shaker. The coupled gel was washed to remove uncoupled sucrose and incubated with a 1 M ethanolamine solution for 4 h to prevent further cross-linking. Ethanolamine was eliminated by successive swellings with a solution (200 ml) containing 0.5 M NaC1 in 0.1 M sodium borate (pH 8.0) alternatively with a solution containing 0.5 M NaCl in 0.1 M sodium acetate (pH 4.0). Treatment of the gel
Z.-S. Li et aL / Biochimica et Biophysica Acta 1219 (1994) 389-397
by invertase and subsequent enzymatic measurement of the hexoses released in the elution buffer indicated that the gel contained about 0.35 mg bound sucrose ml- 1. The coupled gel was stocked (up to 1 week) in 50 mM Tris-HCl buffer (pH 7.5) with 0.1 M sucrose and 0.01% sodium azide at 4°C until further use. Control experiments were made with a gel for which palatinose (6-O-a-D-glucopyranosyl-D-fructofuranose), a sucrose analogue not recognized by the sucrose transporter [39] was used as the bound ligand. The preparation of the gel was the same as described above for sucrose.
2.3. Membrane solubilization and chromatography procedures Plasma membrane proteins (100 to 150 mg) were resuspended (2 mgm1-1) and solubilized with 1% CHAPS (= 16 mM, final concentration). After acidification with a few drops of saturated NaHzPO 4 (pH 5.2), 3 ml of CHAPS supernatant (about 0.7 mg proteinm1-1) were used for affinity purification, and loaded on a column (1 × 10 cm, Pharmacia) containing 6 ml of coupled gel. Unbound proteins were then washed with buffer A (50 mM sodium phosphate, pH 5.2, 0.1% CHAPS, 0.5 mM CaCI 2 and 0.25 mM MgC12) at a flow rate of 0.2 mlmin -1 for 30 rain and 0.5 ml min-l for 15 min. Bound proteins were eluted with buffer B (buffer A + 0.1 M sucrose) for 30 min at 0.5 ml min-1. In some experiments, 0.1 M lactose was used instead of sucrose in buffer B to test the specificity of the elution. Non specifically bound proteins were removed by washing the gel 30 min with 0.5 M NaC1 in 50 mM sodium phosphate (pH 7.8) (buffer C). Fractions were collected at 5 rain intervals, and pooled as described in Results. The samples were frozen at -20°C, lyophilised, and resuspended in 50 mM potassium phosphate (pH 7.5) for dialysis against 50 mM potassium phosphate + 0.1% CHAPS, using a membrane dialysis with a cut-off size of 6000-8000. This dialysis removed the phosphate and decreased the CHAPS concentration to 0.1%. The samples were then frozen at -20°C, lyophilised, and resuspended in 0.5 ml 50 mM potassium phosphate pH 7.5 for reconstitution experiments. In some experiments, sodium phosphate was replaced with 50 mM Tris-HCl, and the protein samples recovered from the sucrose affinity column were further separated on a Mono-Q ion exchange column (HR 5/5 Pharmacia). The corresponding fractions from 6 to 12 injections on the sucrose-affinity column were pooled, dialyzed and concentrated as described above. About 500 /xg protein (0.5 ml) were loaded on the column. The different peaks separated on the Mono-Q column were recovered for SDS-PAGE analysis [31] and differential labeling.
391
2.4. Differential labeling [3H]NEM labeling experiments were modified from [31] and carried out as follows. Ten /~g of Mono-Q purified proteins were incubated with [3H]NEM (518 kBq, 3.2 /zM) and 20 mM sucrose in 500 mM phosphate buffer (pH 5.5) for 30 rain at 25°C. Sucrose was used as a protecting sugar against NEM inhibition [31]. Free [3H]NEM was neutralized by addition of 10 foldexcess of dithiothreitol. The effect of sucrose was compared with that of maltose, poorly recognized by the sucrose transporter and palatinose, not recognized by the sucrose transporter [39]. Control samples were also prepared in the absence of any protecting sugar. The [3H]NEM labeled samples were then solubilized 20 min at 37°C and separated by SDS-PAGE [31]. After silver staining [40], each lane was cut into 2 ram-thick slices, which were digested overnight with perchloric a c i d / H 2 0 2 (1:2, v/v). After addition of 4 ml AquaLuma Plus (Amersham), the radioactivity of the samples was measured in a Packard 1900TR scintillation counter. For each slice, a labeling index was calculated according to the formula: 3H of the slice (control) total 3H of the lane (control) 3H of the slice (sugar) total 3H of the lane (sugar)
2.5. Reconstitution and transport assays Using affinity purified proteins, proteoliposomes were obtained according to the method previously described [34,41]. Soybean asoleetin (12 rag) (Sigma IV-S) was sonicated in potassium phosphate buffer (pH 7.5) with 1 mM DT/'. CHAPS and glycerol were added to the final concentration of 0.5 and 20%, respectively. Proteoliposomes were formed by dilution of plasma membrane proteins (125 /~g) from affinity column or from CHAPS supernatant into 25 ml of potassium phosphate buffer (pH 7.5). After centrifugation (1 h at 100000 × g), the pellet recovered was resuspended in 100/xl of potassium phosphate buffer (pH 7.5). Uptake experiments were carried out by diluting 2 /xl of the proteoliposomes suspension into 400 /xl of incubation medium containing 0.3 M sorbitol, 50 mM sodium phosphate (pH 5.5), 0.5 mM CaCI2, 0.25 mM MgCI2, 0.5 mM DTr, 5 /zM valinomycin and 1 mM [6,6'(n)3H]sucrose (26 kBq) [34]. The artificial proton motive force thus created combined a transmembrane pH gradient (pH 7.5 inside, pH 5.5 outside), and a transmembrane electrical gradient, inside negative, due to valinomycin-induced diffusion of potassium outside the proteoliposomes. Active uptake was measured after 1
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Step I
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of the plant plasma membrane was not retained by the gel coupled with sucrose and was eluted with buffer A (Fig. 1A, peak I). Addition of 0.1 M sucrose in this buffer at pH 5.2 resulted in the elution a small peak (0.22_+ 0.03% of loaded proteins, mean + S.E. of 4 measurements) (Fig. 1A, peak II). Washing the column with a sucrose-free buffer at pH 7.8 allowed the recovery of a third peak (0.28 _+ 0.07% of loaded proteins, Fig. 1A, peak III). When the sucrose-coupled column was eluted with lactose, a similar pattern was obtained concerning the appearance of peak I and peak III, but lactose did not cause the elution of a peak at pH 5.2 (Fig. 1B). The peak eluted at pH 5.2 by sucrose (peak II in Fig. 1A) therefore seemed the more specific for sucrose-binding proteins. Because at least part of the sucrose transporters are sensitive to thiol reagents [6,11,25,26,37], the elution pattern of the solubilized proteins was studied with samples pretreated either with 0.5 mM PCMBS (Fig. 2A) or 0.5 mM N E M (Fig. 2B). In both cases, pretreatment of the samples with the thiol reagents prevented
393
Z.-S. Li et al. / Biochimica et Biophysica Acta 1219 (1994) 389-397
the appearance of p e a k II normally eluted by sucrose at p H 5.2, while peak I I I eluted at p H 7.8 was still present. Chromatography of the C H A P S supernatant on a gel where palatinose was used as the ligand also failed to yield a p e a k eluted by sucrose at p H 5.2 (Fig. 3). Altogether, the data presented abbve suggest that affinity chromatography on a sucrose ligand allow the recovery of a peak selectively eluted by sucrose at p H 5.2. The binding of this fraction to the ligand is prevented by N E M and PCMBS, as expected for the proton-sucrose cotransporter. The different fractions recovered after separation of the C H A P S supernatant on a sucrose-affinity column were assayed for active transport of sucrose after reconstitution into proteoliposomes. The data presented in Fig. 4 give the initial rate of the transport energized by the artificial proton-motive force, measured after a 1 min incubation, and expressed on a weight basis (mg protein actually reincorporated into the liposomes). Very weak activity was associated with peak I, whereas p e a k II exhibited a 4-fold increase in transport activity, compared to the C H A P S supernatant. The activity of peak III was similar to that of the C H A P S supernatant. Fig. 5 presents the S D S - P A G E analysis of the different fractions recovered after affinity chromatography on a sucrose ligand, as described in Fig. 1. Most of the injected proteins are recovered in peak I, and there is hardly any difference between lane B (CHAPS supernatant, S) and lane C (peak I). Peaks II and III were characterized by the absence of any band above 60 kDa. Although theoretically a similar amount of protein was loaded in each lane, peaks II and III only gave faint bands in silver staining, due either to a poor
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staining with the method used or inaccuracy in assaying dilute protein samples. C o m p a r e d to peaks I and III, peak II was enriched in three bands (marked with dots), at 23 kDa and at 41 kDa (absent in peak I and III), and at 57 kDa (absent in peak III). C o m p a r e d to peak II, peak III was enriched with a 33 kDa band. Since peak II is selectively eluted by sucrose, exhibits sensitivity to N E M and to PCMBS, as well as the highest rate of sucrose transport after reconstitution, further attempts to characterize the sucrose transporters were run with this peak. After dialysis, and lyophilisation, this peak was resuspended in 50 m M
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Fraction Fig. 3. Separation of plasma membranes proteins on a palatinoseSepharose column. A, the plasma membrane proteins solubilized by 1% CHAPS were loaded on the column and eluted by buffer A (step 1, peak I), buffer A+0.1 M sucrose (step 2) and buffer C (step 3, peak III).
24
.......
Fig. 5. Silver-staining of the different peaks recovered from a sucrose-affinity column as described in Fig. 1A. (A) Molecular weight standards; (S) CHAPS supernatant; peaks I, II and III. The same amount of protein (about 10/~g) was loaded in each lane.
394
Z.-S. Li et al. /Biochimica et Biophysica Acta 1219 (1994) 389-397
-~ 0"10/
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Tris-HCl (pH 7.5) and loaded on a Mono-Q column for separation by ion-exchange chromatography. Fractionation of peak II on the Mono-Q column by a 0 to 0.5 M NaC1 gradient yielded 4 major fractions (B, C, D, E) which were collected as shown in Fig. 6. On an absorbance basis, fractions B, C, D and E accounted for 10, 19.5, 10 and 15% of the loaded proteins, respectively. Rinsing of the column with 1 M NaC1 gave another peak, which was not collected for further experiments.
3.2. Differential labeling Reconstitution of transport activity into proteoliposomes with the fractions recovered after Mono-Q
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Fig. 7. Differential labeling patterns obtained after separation by S D S - P A G E of the fractions recovered on a M o n o - Q column as described in Fig. 6. (A) peak C; (B) peak D. After dialysis and concentration, the fractions recovered from the M o n o - Q column were differentially labeled by [3H]NEM in the presence of various sugars, and separated by SDS-PAGE. A differential labeling index representative of sugar protection was calculated as detailed in Materials and methods. Hatched areas shows bands exhibiting the labeling expected for a sucrose transporter.
z.-s. Li et al. / Biochimica et Biophysica Acta 1219 (1994) 389-397
fractionation was not possible, due to their very low content in protein. Yet, we analyzed their pattern of labeling by [3H]NEM in the presence of sucrose, maltose (recognized by the sucrose transporters) and palatinose (not recognized). Unlike in previous experiments [34], it was not possible to conduct a double s t e p / d o u b l e labeling procedure combining unlabeled NEM, and [3H]NEM/[14C]NEM, because the present experiments were run with a low amount of solubilized proteins, while the former one were made directly on large amounts of native plasma membrane. According to the procedure developed here, any polypeptide protected from N E M by sucrose should be distinguished by a higher [3H], and hence a higher labeling index. The general pattern of labeling of peaks C and D are clearly different (Fig. 7) and also differ from the patterns found with peaks B and E (data not shown). Possible candidates for a sucrose transporter may be differentially labeled in the presence of sucrose and maltose, but not in the presence of palatinose. The data were therefore analyzed by searching peaks of differential labeling that would meet these criteria. No such peak was found in fraction B, or E (data not shown), whereas proteins ranging with apparent molecular mass of 38-41 kDa in fraction D (Fig. 7B), and a peak at 56 kDa in fraction C (Fig. 7A), fulfilled the expected requirements. Among different experiments, the differential labeling index in the presence of maltose for the 38-41 kDa region of fraction D represented about 70% of the index found in the presence of sucrose, while for the 56 kDa protein of fraction C, the differential labeling index in the presence of maltose never exceeded 30% of the index found in the presence of sucrose.
kDa
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29 Fig. 8. SDS-PAGE and silver-stainingof fractions C and D separated in Fig. 6. About 10 /zg were loaded in each lane. (A) Molecular weight standards.
395
Silver-staining of these different fractions showed that they still contain a number of different bands in spite of the different procedures used for protein separation (Fig. 8). Most of the protein bands found in fraction C are also found in fraction D, but fraction C contains a faint band at 57 kDa (marked wit dots) which is not found in fraction D. This band may correspond to the peak of differential labeling found in the same region of this fraction (Fig. 7A). Another band at 70 kDa was much more visible in fraction C than in fraction D. Compared to fraction C, fraction D is characterized by the presence of an additional band at 41 kDa. It is likely that the additional 41 kDa is associated with the peak of differential labeling found in this region (Fig. 7B).
4. Discussion
Various biochemical approaches conducted with different plant species have resulted in the identification of various polypeptides as putative sucrose transporters (42 kDa in sugar beet, 62 kDa in soybean). These polypeptides were identified after denaturating SDSPAGE. In a recent review, Bush [11] also reported that a radiolabeled phenyl derivative of forskolin specifically labeled a 55 kDa polypeptide, which may also be regarded as another possible sucrose transporter. Whether these differences are due to the different species investigated, to the different techniques used, or to the presence of several sucrose transporters is not known. Furthermore, these data do not easily reconcile with the results from molecular cloning, which predict a molecular mass of 55 kDa for the spinach sucrose transporter [36]. Due to their high hydrophobicity, membrane proteins often show a smaller molecular weight in SDS-PAGE than their actual molecular weight. For example, the triose-phosphate translocator of the inner chloroplastic membrane (molecular mass = 35.6 kDa) appears as a 29 kDa polypeptide in SDSP A G E [43], the hexose transporter from Chlorella kessleri (molecular mass = 57.4 kDa) appears as a 47 kDa band [17], and the lac permease of Escherichia coli (molecular mass = 46 kDa) appears as a 33 kDa band [44]. In several papers, the possibility of the sucrose transporter being a multimeric protein has also been suggested [29,34]. Altogether, these data point to the interest of non destructive techniques aimed at characterizing a n d / o r purifying sucrose transporters. In an attempt to purify sucrose transporters (and sucrose-binding proteins) by a non destructive approach, the present work combines affinity chromatography on a new support (sucrose-Sepharose gel) and the possibility to reconstitute sucrose transport activity into proteoliposomes made from plant membrane extracts [34].
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Z.-S. Li et al. / Biochimica et Biophysica Acta 1219 (1994) 389-397
In our previous work [34], the first step used to purify the sucrose transporter was a gel filtration on a TSK SW 3000 column, and the eluate was monitored by ELISA with anti-42 kDa antibodies to purify specifically the 42 kDa polypeptide. This step yielded a 120 kDa peak which represented 40% of the CHAPS supernatant, and it resulted in a 5-fold increase in specific activity of sucrose transport after reconstitution in proteoliposomes. Silver-staining showed that the fraction thus recovered still contained almost all the proteins of the CHAPS supernatant [34]. In the present work, the initial step of the purification was not aimed specifically at purifying the 42 kDa polypeptide, but all the polypeptides interacting strongly enough with sucrose. This step allowed the recovery of a fraction (peak II), that represented only 0.2% of the CHAPS supernatant, and exhibited the properties expected for a sucrose transporter. This fraction was not eluted by lactose (Fig. 1B), not retained on a palatinose-sepharose column (Fig. 3), sensitive to NEM and PCMBS (Fig. 2), and exhibited transport activity (Fig. 4). The affinity chromatography step resulted in a 4-fold increase in sucrose transport activity, compared to the initial CHAPS supernatant (Fig. 4). The rise in specific activity of transport was considerably lower than that expected from the protein yield (a theoretical increase of 500-fold might have been expected), but numerous reasons already discussed [34,45] may explain this resuit. In addition to the usual loss of activity during dialysis, concentration, lyophilisation, it is not necessarily valid to compare directly transport activity of proteoliposomes made from different membrane fractions, because their size and permeability to protons and potassium may be different, which would considerably affect the rate and the time course of uptake. The rate of transport may also be limited by thermodynamical reasons including the actual proton motive force created by the ion gradients used, and the density and the orientation of proteins in the proteoliposome. The highest specific activity measured in the present paper (70 n m o l / m g protein per min) was about twice that reported before using the gel filtration/ion exchange purification [34]. Unlike the gel filtration step used before [34], the use of affinity chromatography as the first step of purification only yielded a limited amount of polypeptides in the present work (Fig. 1), and peak II was characterized by a limited number of polypeptides, including a band at 41 kDa and at 56 kDa (Fig. 5). Because this peak exhibited the highest activity of transport, it may be assumed that one or both of these polypeptides are involved in sucrose transport. Due to the low recovery of proteins, when peak II was further fractionated on a Mono-Q column, reconstitution experiments were not possible. In order to characterize these fractions, we therefore developed a
differential labeling procedure which was simpler than the former one, and concerned solubilized proteins instead of native plasma membrane vesicles. The value of differential affinity labeling by N E M to identify the sucrose transporter has been questioned, on the basis that sucrose uptake into plasma membrane vesicles has sometimes been reported to be NEM-insensitive [11]. We have shown that N E M sensitivity of sucrose transport depends on experimental conditions and on plant material, and that the differential labeling approach was therefore valid ([37], Sakr S., Gaillard C., Roblin G. and Delrot S., unpublished data). The differential labeling procedure developed in the present paper allowed us to distinguish two (groups of) polypeptides which were differentially labeled in the presence of sucrose or maltose, but not in the presence of palatinose. A fraction eluted at 0.17 M NaCI (Fig. 6, fraction C) included a 56 kDa polypeptide which was differentially labeled (Fig. 7A), and apparent after silver-staining (Fig. 8). A fraction eluted at 0.30 M NaCI (Fig. 6, fraction D) contained a 41 kDa band that was differentially labeled (Fig. 7B) and was apparent after silverstaining (Fig. 8). Within the limits of experimental errors, we assume that this polypeptide is the same than the one identified before at 42 kDa, that was differentially labeled by NEM and eluted at 0.31 M NaC1 on a Mono-Q column [34]. Although peak II did not contain polypeptides above 60 kDa upon silver-staining, some polypeptides up to 100 kDa appear when this peak is further resolved on a Mono-Q column, after dialysis and concentration. The appearance of high molecular weight bands may result from agregation phenomena during the concentration of proteins before ion-exchange chromatography. Silver-staining of the fractions recovered after affinity chromatography followed by ion exchange (Fig. 8) still shows the presence of several polypeptide bands, although lane C and D clearly differ by several bands. In addition to possible agregation or degradation artefacts which can hardly be dismissed, the presence of multiple bands in these fractions is also a result of the strategy of purification. Indeed, the procedure used theoretically allows the recovery not only of sucrose transporters, but also of any protein binding sucrose with enough affinity, as well as regulatory proteins which may be associated with the sucrose transporters and the sucrose binding proteins. The data therefore provide another line of evidence suggesting that a 42 kDa polypeptide may be involved in sucrose transport in sugar beet leaf. However, we were also able to identify a 56 kDa polypeptide which also exhibits some properties of a sucrose transporter. For reasons already discussed above (hydrophobicity), it is not likely that this polypeptide may be encoded by cDNA similar to the one encoding a 55 kDa protein in spinach. The relationship between the 56 kDa polypep-
Z.-S. Li et al. / Biochimica et Biophysica Acta 1219 (1994) 389-397
tide identified here and this cDNA, as well as the relationship between this polypeptide and the 55 kDa labeled by forskolin [11] also remain to be established.
Acknowledgements This work was supported in part by the EEC under the Bridge Programme (Contract BIOT-0175-C).
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