Substrate Specificity of the Arabidopsis thaliana Sucrose Transporter AtSUC2*

The Arabidopsis sucrose transporter AtSUC2 is expressed in the companion cells of the phloem (specialized vascular tissue) and is essential for the long distance transport of carbohydrates within the plant. A variety of glucosides are known to inhibit sucrose uptake into yeast expressing AtSUC2; however, it remains unknown whether glucosides other than sucrose could serve as transported substrates. By expression of AtSUC2 in Xenopus oocytes and two-electrode voltage clamping, we have tested the ability of AtSUC2 to transport a range of physiological and synthetic glucosides. Sucrose induced inward currents with a K0.5 of 1.44 mm at pH 5 and a membrane potential of –137 mV. Of the 24 additional sugars tested, 8 glucosides induced large inward currents allowing kinetic analysis. These glucosides were maltose, arbutin (hydroquinone-β-d-glucoside), salicin (2-(hydroxymethyl)phenyl-β-d-glucoside), α-phenylglucoside, β-phenylglucoside, α-paranitrophenylglucoside, β-paranitrophenylglucoside, and paranitrophenyl-β-thioglucoside. In addition, turanose and α-methylglucoside induced small but significant inward currents indicating that they were transported by At-SUC2. The results indicate that AtSUC2 is not highly selective for α-over β-glucosides and may function in transporting glucosides besides sucrose into the phloem, and the results provide insight into the structural requirements for transport by AtSUC2.

Plant sucrose transporters (SUTs, also named SUCs) 1 are integral membrane proteins within the glycoside-pentosidehexuronide cation symporter family. SUTs are expressed in the plasma membrane of companion cells and sieve elements within the phloem, a specialized tissue within the plant vasculature, and catalyze the H ϩ -coupled uptake of sucrose. Sucrose is the primary photosynthetic product that is transported by the phloem to heterotrophic tissues such as roots, developing leaves, and seeds (1,2). The Arabidopsis genome encodes nine SUT homologs (3). One of these, AtSUC2 (4), functions as the main phloem-loading transporter. AtSUC2 is highly expressed in the companion cells of source (photosynthetic) leaves (5) and is essential for the long distance transport of sucrose as evidenced by the severe phenotype of insertional mutants (6). As demonstrated by the [ 14 C]sucrose uptake of yeast expressing AtSUC2, the transporter has a high affinity for sucrose (K m ϭ 0.8 mM) (4) compared with Arabidopsis homologs AtSUT4 (7) and AtSUT2 (8).
In plant cell protoplasts or plasma membrane vesicles, sucrose uptake is inhibited by a variety of glucosides (9,10) providing insight into the structural requirements for binding to sucrose transporters. Results indicated that none of the fructosyl hydroxyls of sucrose interact specifically with the transporter (9). The ability of phenylthioglucoside to inhibit sucrose uptake and to serve as a transported substrate further indicated that the fructosyl moiety of sucrose presents a hydrophobic surface required for binding (9,10). Substitutions of the glucosyl hydroxyls 3, 4, and 6 of phenylthioglucoside greatly increased the K i indicating that hydroxyls at these positions are required for substrate recognition (9). It is now clear that plants express multiple sucrose transporters with differing affinities for sucrose (7,8) even within the same cells (11). Therefore, to understand the mechanism of sucrose recognition and uptake it is important to determine the substrate specificity of individual sucrose transporters.
Information on the substrate specificity of cloned plant sucrose transporters is limited and has been investigated primarily by expression in yeast and competition of [ 14 C]sucrose uptake, an approach that does not discriminate between substrate uptake and block of transport. In general, maltose, ␣-phenylglucoside, and ␤-phenylglucoside inhibit sucrose uptake (4,(12)(13)(14)(15); however, kinetic analyses were not performed. Of the potentially transported substrates other than sucrose, only the kinetics of maltose uptake by AtSUT4 has been analyzed in detail (7). To differentiate substrate transport from substrate block, AtSUC2 was expressed in Xenopus oocytes, and using two-electrode voltage clamping, the ability to transport a wide range of physiological and synthetic glucosides was tested.

EXPERIMENTAL PROCEDURES
Cloning-The coding region of AtSUC2 was amplified using ExTaq polymerase (Panvera, Madison, WI) and the following primers, 5Ј-ATGGTCAGCCATCCAATGGA and 5Ј-TCAATGAAATCCCATAG-TAGC. An A. thaliana ecotype Col-0 seedling library was used as a template. The PCR product was cloned into pCR2.1 (Invitrogen) and sequenced. AtSUC2 was subcloned into the EcoRI site of pOO2 (16), an expression vector containing Xenopus ␤-globin 5Ј-and 3Ј-untranslated regions and a 92-bp poly(A) tail. This construct was linearized using PmaCI (Panvera), and 1 g was used as template for cRNA synthesis using the mMessage mMachine kit (Ambion, Austin, TX).
Electrophysiological Methods-Oocytes were bathed in modified Ringer's solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl 2 , 1 mM NaHCO 3 , 10 mM HEPES, pH 5.0, and 10 mM MgCl 2 ) with continuous perfusion at 1 ml/min. Recording pipettes, filled with 1 M KCl, with resistances between 1.5 and 3 megaohms were used. Currents were measured using the two-electrode voltage clamp technique with a Dagan TEV 200A amplifier (Dagan Corp., Minneapolis, MN). Currents were filtered on line at 200 Hz and digitized at 2000 Hz using pClamp 5.5.1 (Axon Instruments, Inc., Union City, CA). The holding potential was Ϫ40 mV, and voltage pulses from Ϫ157 to 57 mV were applied for 150 ms. Steady-state currents are presented as the mean current between 110 and 140 ms following the onset of voltage pulses. Substratedependent currents were obtained by subtracting an average of background currents before and after substrate application.
To analyze the voltage dependence of sucrose-induced currents, voltage pulses of 150 ms were made from 57 to Ϫ157 mV. Steady-state currents were observed following capacitive transients and were averaged between 110 and 140 ms following the onset of voltage pulses. The current/voltage relation of steady-state currents before, during, and after perfusion with 10 mM sucrose is shown in Fig. 2A. Background currents averaged 33 Ϯ 2.1 nA (n ϭ 6 oocytes) at Ϫ40 mV in sodium-Ringer solution at pH 5.0. Sucrose-dependent currents, obtained by subtracting background currents (Fig. 2B), were inward at all potentials (between 57 and Ϫ157 mV).
For kinetic analysis, currents were measured at sucrose concentrations between 0.05 and 10 mM. Currents were saturable ( Fig. 2C) with an apparent affinity for sucrose (K 0.5 ) of 1.44 Ϯ 0.19 mM (n ϭ 3 oocytes) at Ϫ137 mV. K 0.5 values were voltagedependent ( Fig. 2D) with higher apparent affinity at more negative potentials and ranged between 1.44 Ϯ 0.19 mM at Ϫ137 mV and 6.13 Ϯ 1.9 mM at Ϫ1.1 mV.
Kinetic analysis was performed for substrates that induced large inward currents. The apparent affinities for all transported substrates were voltage-dependent (Fig. 4) with a lower K 0.5 observed at more negative membrane potentials. The voltage dependence of K 0.5 for all transported substrates was similar to that of sucrose (Fig. 2D) indicating a common binding site. K 0.5 values for transported substrates at Ϫ137 mV are presented in Table I. The K 0.5 for maltose transport was 7.8fold higher than for sucrose indicating that AtSUC2 is highly selective for sucrose over maltose. The apparent affinities for the plant glucosides salicin and arbutin were similar to that of sucrose. The ability of AtSUC2 to transport synthetic glucosides allows a determination of the effect of ␣versus ␤-linkage on substrate affinity (Table I). The K 0.5 for ␣-phenylglucoside (0.94 mM) was lower than for ␤-phenylglucoside (1.18 mM). Similarly, the K 0.5 for ␣-paranitrophenylglucoside (0.40 mM) was lower than for ␤-paranitrophenylglucoside (2.70 mM). These results indicate that AtSUC2 binds ␣-linked glucosides at higher affinity than ␤-linked glucosides. The apparent affinities for paranitrophenyl-␤-thioglucoside and ␤-paranitrophen- ylglucoside were similar indicating that AtSUC2 does not discriminate between O-linked and S-linked glucosides. DISCUSSION Previous studies have shown that glucosides such as maltose and ␣or ␤-phenylglucoside inhibit [ 14 C]sucrose uptake into yeast expressing plant sucrose transporters (4,7,(12)(13)(14)(15). To address the question of whether these and other glucosides are transported substrates or transport inhibitors, we expressed AtSUC2 in Xenopus oocytes and applied two-electrode voltage clamping to measure transport activity.
Sucrose and other glucosides were found to induce inward currents consistent with a H ϩ -coupled transport mechanism (17). The K m for sucrose transport at pH 5.0 by AtSUC2expressing yeast was reported previously to be 0.8 mM (4). This is significantly lower than the value obtained in this study (1.44 Ϯ 0.19 mM, at pH 5.0 and Ϫ137 mV). When the apparent affinity of AtSUC2 for sucrose was measured by uptake into yeast, it is likely that higher concentrations of sucrose caused membrane depolarization lowering the driving force for sucrose uptake, as well as the affinity of the transporter for sucrose, and therefore the observed V max . Under these conditions, the observed K m for sucrose would be lower than when the membrane potential is clamped, as in this study. This idea is supported by data from Sauer and Stolz (4) who found higher K m values for sucrose uptake into yeast expressing AtSUC2 in the presence of glucose than in the absence of glucose, which would support a more negative membrane potential.
The voltage dependence of the K 0.5 for sucrose of AtSUC2 (Fig. 2) was generally consistent with results for StSUT1 from potato (17) and AtSUC1 from Arabidopsis (18). For all of these transporters, the K 0.5 for sucrose is lower and less voltage-dependent at a lower pH. At pH 5.0, the K 0.5 for sucrose of StSUT1 is not significantly voltage-dependent (17). However, as shown in Fig. 2D, the K 0.5 for sucrose of AtSUC2 at pH 5.0 is lower at more polarized potentials. This represents a difference between StSUT1 and AtSUC2 activities. The dependence of the apparent affinity for sucrose on extracellular pH and membrane potential within the physiological range is impor-  Fig. 1. A, currents recorded before, during, and after application of 10 mM sucrose. Symbols for control currents before and after sucrose application overlap. B, sucrose-dependent currents recorded at different sucrose concentrations. Background currents before and after sucrose application (as in A) were averaged and subtracted from currents recorded during sucrose application to obtain sucrose-dependent currents. C, sucrose-dependent currents at a membrane potential of Ϫ137 mV (from B) plotted against the sucrose concentration. Line indicates a fit of the Michaelis-Menten equation to the data, and error bars are S.E. (n ϭ 3 oocytes). D, voltage dependence of AtSUC2 affinity for sucrose. K 0.5 was determined as in C at different membrane potentials, and error bars are S.E. (n ϭ 3 oocytes). tant for the function of sucrose transporters in plants. The results of this study and others (17,18) indicate that activation of the plasma membrane H ϩ -ATPase, which generates a negative membrane potential and acidifies the extracellular space relative to the cytoplasm, would enhance sucrose transport not only through increasing proton motive force but by lowering the K m of sucrose transporters.
Glucosides such as maltose and ␣and ␤-phenylglucoside, which had been demonstrated previously to inhibit [ 14 C]sucrose uptake into yeast expressing AtSUC2 (4), induced inward currents indicating that they serve as transported substrates. Furthermore, of the 25 sugars tested, 8 induced large inward currents (Fig. 3) in addition to sucrose allowing determination of apparent affinities (Table I and Fig. 4). In addition, both turanose and ␣-methylglucoside induced significant inward currents (Fig. 3) indicating that they were transported; however, currents were not large enough for kinetic analysis. The results show that AtSUC2 is capable of transporting a wide range of physiological and synthetic glucose conjugates.
Structural Requirements for Transport by AtSUC2-AtSUC2 transported glucosides with both ␣-linkage (sucrose and maltose) and ␤-linkage (arbutin and salicin). Cellobiose, which is similar to maltose except for a ␤-linkage, was not transported. This is consistent with the finding that AtSUC2 had a higher apparent affinity for ␣-linked glucosides (Table I). Both ␣and ␤-linked phenyl (and nitrophenyl) glucosides were transported by AtSUC2 with ␣-linked glucosides consistently showing lower K 0.5 values. It is particularly interesting that salicin and arbutin (␤-linked glucosides) are transported with a K 0.5 similar to sucrose. Although arbutin has not been detected in Arabidopsis, homologs of the plant glycosyltransferase arbutin synthase (19) exist in Arabidopsis (At4g01070 and At1g01420). This suggests that arbutin or related glucosides are present in Arabidopsis and potentially may be transported into the phloem by AtSUC2. There is evidence that the transport of glucosides other than sucrose by the phloem is physiologically important. For example, glucosinolates are transported in the phloem in Arabidopsis (20).
In uptake studies using isolated plant protoplasts, sucrose transporters were found to interact specifically with the glucosyl hydroxyls 3, 4, and 6 of sucrose (9). Similarly, phenyl-␤galactopyranoside did not induce inward currents, whereas ␤-phenylglucoside was transported with a K 0.5 of 1.18 mM indicating strong selectivity for the glucoside, which differs in side group orientation at position 4 compared with the galactoside. The conclusion by Hitz et al. (9) that the fructosyl moiety of sucrose does not interact specifically with the transporter but presents a hydrophobic surface that interacts with the binding site is also supported by our results. Although glucose did not induce inward currents in oocytes expressing AtSUC2, ␣-methylglucoside served as a substrate (Fig. 3) indicating that even small hydrophobic groups at position 1 of glucose are sufficient for interaction with the transporter.
For compounds with larger side groups, orientation or linkage position was critical in determining whether interaction with AtSUC2 occurred. For example, trehalose, which is an ␣-glucose [131] ␣-glucoside and is similar to maltose except that glucose is linked at position 1, was not transported. Along the same lines, palatinose was not transported although it is similar to sucrose except that fructose is ␣-linked at position 6 (compared with position 2 in sucrose). It is widely assumed that the sucrose isomers turanose and palatinose are not transported into plant cells (21), although this has not been tested directly. Our results show that turanose, but not palatinose, serves as a substrate for AtSUC2 (Fig. 3). This is significant because turanose and palatinose both induce extracellular invertase expression similarly to sucrose, and although sucrose inhibits expression of photosynthetic genes, turanose and palatinose do not (21). Differences in the perception of turanose and palatinose in comparison to sucrose have been explained in terms of the inability of plant cells to transport the sucrose analogs, and this conclusion may need to be reevaluated in light of the current results. However, it should be pointed out that plants encode multiple sucrose transporters, and differences in transport activity have been reported. Although substrate specificity has not been analyzed for other plant sucrose transporters, in Arabidopsis AtSUT2 and AtSUT4 show a low affinity for sucrose (7,8) compared with AtSUC2. It is possible that plant sucrose transporters have different substrate specificities. For example, for yeast expressing AgSUT1, a sucrose transporter from celery, raffinose competed more effectively than maltose for [ 14 C]sucrose uptake (15). This was not the case for AtSUC2 (4), and raffinose did not induce inward currents in oocytes (Fig. 3).
In conclusion, the results show that AtSUC2 has a weak selectivity for ␣-linked glucosides but will transport ␤-glucosides such as arbutin and salicin with a K 0.5 equal to or lower than for sucrose. AtSUC2 did not show selectivity between thio-and O-linked glucosides. This is potentially of physiological significance because glucosinolates (S-linked glucosides) are known to be transported in the phloem (20). The broad specificity of AtSUC2 indicates that glucosides other than sucrose may be transported as physiological substrates. For example, several plant hormones, such as auxins and cytokinins, are glucosylated (22,23), and the ability to transport low concentrations within the phloem may be important. Insertional mutants of AtSUC2 show severe growth phenotypes (6) that have been assumed to be caused by an inability to load sucrose into the phloem. Additional work will be required to determine whether AtSUC2 function in the plant includes transport of glucosides in addition to sucrose.