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Phloem-localized, Proton-coupled Sucrose Carrier ZmSUT1 Mediates Sucrose Efflux under the Control of the Sucrose Gradient and the Proton Motive Force *

Phloem-localized, Proton-coupled Sucrose Carrier ZmSUT1 Mediates Sucrose Efflux under the Control... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 22, Issue of June 3, pp. 21437–21443, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Phloem-localized, Proton-coupled Sucrose Carrier ZmSUT1 Mediates Sucrose Efflux under the Control of the Sucrose Gradient and the Proton Motive Force* Received for publication, February 16, 2005, and in revised form, March 31, 2005 Published, JBC Papers in Press, April 1, 2005, DOI 10.1074/jbc.M501785200 Armando Carpaneto‡§ , Dietmar Geiger§ , Ernst Bamberg**, Norbert Sauer‡‡, Jo¨rg Fromm§§, ¶¶ and Rainer Hedrich From the Julius-von-Sachs-Institute for Biosciences, Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Platz 2, D-97082 Wu¨rzburg, Germany, the ‡Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, I-16149 Genova, Italy, the **Max-Plank-Institute for Biophysics, Marie Curie Strasse 15, D-60439 Frankfurt am Main, Germany, the ‡‡University Erlangen-Nu¨rnberg, Molecular Plant Physiology, Staudtstrasse 5, D-91058 Erlangen, Germany, and the §§Technical University Mu¨nchen, Holzforschung, Winzererstrasse 45, D-80797 Mu¨nchen, Germany ing vascular cells. The hydrostatic pressure difference between The phloem network is as essential for plants as the vascular system is for humans. This network, assembled source and sink tissues drives the mass flow of water and by nucleus- and vacuole-free interconnected living cells, nutrients in the phloem vessels (1). In sink tissues, which are represents a long distance transport pathway for nutri- dependent on carbon supply via the phloem, a symplasmic ents and information. According to the Mu¨ nch hypoth- unloading of sucrose along its concentration gradient has been esis, osmolytes such as sucrose generate the hydrostatic shown for many plant species (2). Interestingly, however, su- pressure that drives nutrient and water flow between crose/H symporter transcripts and proteins have also been the source and the sink phloem (Mu¨ nch, E. (1930) Die localized in sink tissues, suggesting a role in sink loading/ Stoffbewegungen in der Pflanze, Gustav Fischer, Jena, retrieval or unloading of sucrose via these transporters (see Germany). Although proton-coupled sucrose carriers Ref. 2 for review). SUT1 from the potato, for example, has been have been localized to the sieve tube and the companion detected in the sieve elements of mature source leaves as well cell plasma membrane of both source and sink tissues, as in developing sink leaves, roots (3), and tubers (4, 5). Using knowledge of the molecular representatives and the a sink-specific antisense inhibition for SUT1 under the control mechanism of the sucrose phloem efflux is still scant. We of a tuber-specific promoter, Ku¨hn et al. (2003) (4) could dem- expressed ZmSUT1, a maize sucrose/proton symporter, onstrate the involvement of SUT1 in early tuber development in Xenopus oocytes and studied the transport character- istics of the carrier by electrophysiological methods. and, thus, phloem unloading. Further evidence for a sucrose Using the patch clamp techniques in the giant inside-out export system was added by the localization of sucrose/H patch mode, we altered the chemical and electrochemi- symporters expressed in symplasmically isolated tissues such cal gradient across the sucrose carrier and analyzed the as developing embryos (6, 7) and growing pollen tubes (8). currents generated by the proton flux. Thereby we could Furthermore Evert and Russin (1993) could show, for example, show that ZmSUT1 is capable of mediating both the that symplasmic unloading in maize is unlikely because of the sucrose uptake into the phloem in mature leaves lack of plasmodesmata in the protophloem and metaphloem of (source) as well as the desorption of sugar from the developing leaves (9). Although proton-coupled sucrose carriers phloem vessels into heterotrophic tissues (sink). As pre- have been localized to the sieve tubes and companion cell dicted from a perfect molecular machine, the ZmSUT1- plasma membranes of both source and sink tissues, the molec- mediated sucrose-coupled proton current was reversi- ular representatives and mechanism of the sucrose phloem ble and depended on the direction of the sucrose and pH efflux is still scant. gradient as well as the membrane potential across the In the present study we tested the biophysical properties and transporter. thermodynamics of ZmSUT1, a maize sucrose carrier ex- pressed at a high level in Xenopus laevis oocytes. Specific To ensure adequate partitioning of sucrose throughout the sucrose transport inhibitors are not available, but Xenopus plant body, sucrose has to be translocated from the mesophyll oocytes, like all other creatures apart from plants, do not trans- cells to the sieve element-companion cell complex. Because of port sucrose. Therefore oocytes are well suited for sucrose the energy-dependent sucrose/H symporter in apoplasmic transport studies. Using this sucrose-insensitive system we loading plant species, the transport sugar accumulates at con- could demonstrate that this sugar carrier mediates both su- centrations of several hundred mM to 1 molar in the conduct- crose uptake and release. Upon a drop in membrane potential and/or pH gradient, ZmSUT1 would release sucrose from, for example, sink phloem and thus seem to represent the molecu- * This work was funded in part by Deutsche Forschungsgemeinschaft grants (to R. H.). The costs of publication of this article were defrayed in lar equivalent for the sucrose efflux carrier. Based on our part by the payment of page charges. This article must therefore be biophysical characterization of ZmSUT1, the “source and sink hereby marked “advertisement” in accordance with 18 U.S.C. Section mode” of this transporter is discussed in respect to in planta 1734 solely to indicate this fact. § These authors contributed equally to this work. phloem loading and unloading conditions. In principle, each This author’s stay in Wu¨ rzburg, Germany was funded by an SFB individual transporter should be reversible. But, in contrast to 487 guest scientist stipend and a research fellowship of the Von Hum- the reversible transporters from the animal field and from boldt Foundation. bacteria that have been described to date, ZmSUT1 is the first ¶¶ To whom correspondence should be addressed. Tel.: 49-931-8886101; Fax: 49-931-8886157; E-mail: hedrich@botanik.uni-wuerzburg.de. that works in both directions under physiological conditions. This paper is available on line at http://www.jbc.org 21437 This is an Open Access article under the CC BY license. 21438 Reversibility of the Sucrose Transporter ZmSUT1 MATERIALS AND METHODS Patch Clamp Measurements—Giant patch recording (12) was per- formed in inside-out configuration on ZmSUT1 expressing Xenopus Aphid Breeding—Aphids of the species Rhopalosiphum padi were oocytes. Borosilicate glass pipettes were pulled and fire-polished to bred on barley and maize grown in a climate chamber under a 14-h have a final tip with a diameter between 25 and 30 m. Oocytes were photoperiod. bathed in an external solution consisting of 30 mM KCl, 1 mM CaCl , 1.5 Experimental Setup—Plant aphid cages were applied to the mature mM MgCl ,1mM GdCl , 145 mM sorbitol, and 10 mM MES/Tris, pH 5.6. 2 3 leaves of a 4-week-old potted maize. Aphids feeding on a leaf were After the seal was obtained, the external solution was changed (30 mM dissected from their stylets using a laser as described previously (10). KCl, 1 mM EGTA, 2 mM MgCl , 145 mM (or 500 mM) sorbitol, and 10 mM The recording electrodes were brought in contact with the phloem Tris/MES, pH 7.5), and the patch was excised. The recording pipette exudate appearing at the cut end of the stylet. The leaf was cut 15 cm was then placed in front of a polyethylene tube in connection with the proximal to the tip, and the cut end was incubated with artificial pond desired ionic solutions that were driven by gravity. The standard cyto- water containing the reference electrode (silver/silver chloride) and 1 1 solic solution contained 30 mM KCl, 1 mM EGTA, and 2 mM MgCl . The mM NaCl, 0.1 mM KCl, 0.1 mM CaCl , 100 mM sorbitol, and 1 mM MES, cytosolic sucrose concentration ranged from 0 to 500 mM, as indicated in adjusted to pH 6.0 with Tris. Sucrose pulses were applied by perfusion the remaining text where noted; sorbitol was appropriately added to of artificial pond water solution. Phloem potential measurements were each cytosolic solution to have a total sugar concentration of 500 mM. recorded according to (10). Cytosolic pH was 7.5 or 5.6 (with 10 mM Tris/MES or MES/Tris). The Two-electrode Voltage Clamp (TEVC) Analysis in Xenopus Oocytes— TM standard pipette solution was 30 mM KCl, 1 mM CaCl , 1.5 mM MgCl , 2 2 ZmSUT1 cRNA was prepared using the mMESSAGE mMACHINE 145 mM sorbitol, and 10 mM MES/Tris, pH 5.6; sucrose was added at RNA transcription kit (Ambion Inc., Austin, TX). Oocyte preparation concentrations of 0.5, 5, and 50 mM as indicated in the text where noted. and cRNA injection have been described elsewhere (11). In TEVC stud- Currents, filtered at 10 or 100 Hz and sampled at 200 or 400 Hz, were ies, oocytes were perfused with a standard solution containing 30 mM recorded with an EPC9 amplifier using Pulse 8.3 software (Heka Ele- KCl, 1 mM CaCl , and 1.5 mM MgCl based on Tris/MES buffers for pH 2 2 ktronic GmbH, Lembrecht, Germany). Data were analyzed by custom- values from 5.6 to 8.0 or based on citrate/Tris buffers for the pH values made programs using Igor (Wavemetrics; Lake Oswego, OR). 4.5 and 5.0. The sucrose concentrations and pH values are indicated in Figs. 2–4 and 6 (and the corresponding legends) and throughout the RESULTS text where noted. All solutions were adjusted to 220 mosmol kg using D-sorbitol. Steady state currents were obtained by stepping the mem- ZmSUT1 was isolated from maize and expressed in source brane potential from the holding potential of 0 mV to a series of 500-ms and sink tissues such as mature leave blades and sheaths as test pulses from 60 to 130 mV in 10-mV decrements. Difference well as pedicels and seeds (13). High sequence homologies to currents were calculated by subtracting the currents in the absence of the rice sucrose transporter OsSUT1 (14) and to known sucrose sucrose from the currents in its presence. The sucrose-induced steady state currents were measured in respect to ligand concentrations and transporters from the dicot species group ZmSUT1 into the membrane potential. At each test potential the currents were fitted to SUT2 subfamily of sucrose transporters with high sucrose af- the Michaelis-Menten equation shown in Equation 1, finity (for review, see Ref. 15). ZmSUT1 is a member of a large S S family of membrane proteins mediating the transport of sug- I  I [S]/([S]  K ) (Eq. 1) max m ars, amino acids, and osmolytes across membranes. These car- where the substrate (S) is either [sucrose] or [H ]. These fits yielded in riers share the typical 12-transmembrane-spanning -helix S H the maximal currents I for sucrose and I for H and the max max structure (16, 17). In most eukaryotic cells these transporters H  S half-maximal ligand concentrations K for H and K for sucrose. m m couple the uptake of their substrates to electrochemical ion Intracellular pH Measurements—PH-sensitive microelectrodes were gradients generated by the H -orNa /K -ATPase. pulled from borosilicate capillary (TW100F-3; WPI, Sarasota, FL) using a laser puller (P2000; Sutter Instruments, Novato, CA) and silanized Using the aphid stylet technique on maize leaf blades, it could with dimethyldichlorosilane (Fulka, Steinheim, Germany) at 200 °C for be shown that the addition of sucrose reversibly depolarized the 15 min. The tips of the pH microelectrodes were filled with hydrogen phloem potential (Fig. 1). To elucidate the transport characteris- ionophore I mixture B (Fulka) and then back-filled with a buffer con- tics of the underlying sucrose/H transporter activity with re- taining 40 mM KH PO ,23mM NaOH, and 150 mM NaCl (pH 6.8). Only 2 4 spect to sucrose affinity gradients and proton motive force, we electrodes with a linear slope of 55–60 mV/pH unit over the calibration heterologously expressed ZmSUT1 in Xenopus laevis oocytes. range before and after measurement were used. Signals were recorded with an electrometer (Model FD 223; WPI) in parallel to the currents in Functional analysis was performed using both the TEVC tech- the voltage clamp mode of a TEVC amplifier (Turbo TEC 10CD; npi nique and the patch clamp technique. Oocytes expressing Zm- electronic GmbH, Tamm, Germany). On the basis of the calibration SUT1 efficiently imported radio-labeled sucrose with uptake curve for the pH microelectrodes, the internal pH (pH ) of the oocytes rates of 6 nmol per hour and oocyte, whereas non-injected oocytes was calculated in consideration of the membrane potential. 14 did not accumulate sucrose in detectable amounts (Fig. 2A). To C Sucrose Uptake Experiments—In each experiment, 10 ZmSUT1- monitor the movement of protons accompanying the sucrose injected oocytes or 10 control oocytes were incubated in 0.05 Ci/ml C sucrose with a final sucrose concentration of 5 mM in the standard transport, we simultaneously recorded sucrose-induced ionic cur- solution at pH 5.6. At defined time points the oocytes were rapidly rents and changes in cytoplasmic pH by TEVC and proton- washed three times in ice-cold standard solution and transferred to selective microelectrodes (18). Upon the addition of sucrose to the liquid scintillation vials containing scintillation mixture (Emulsifier- external solution, large inward currents were elicited (Fig. 2B, TM 14 Safe ; Packard, Meriden, CT). The C radioactivity was counted in a upper trace). Inward currents were accompanied by a decrease in liquid scintillation analyzer (Model 1900CA; Packard), and the sucrose pH by up to 0.5 units within 10 min (Fig. 2B, lower trace). After uptake per oocyte was calculated from three independent experiments for each time point. the removal of sucrose from the bath medium, the inward cur- [ C]Sucrose Release Experiments—Control oocytes and ZmSUT1- rents returned to the pre-sucrose level again, whereas the recov- injected oocytes were loaded with 0.5 Ci of radiolabeled sucrose with a ery of pH was delayed. Control oocytes showed neither sucrose- TM final sucrose concentration of 50 mM by injection (Picospritzer II; induced currents nor sucrose-dependent changes in pH . General Valve Co., Fairfield, NJ). After a 10-min washing period in Stepwise increases in sucrose concentrations resulted in a grad- ice-cold ND96, each single oocyte was transferred into 200 lofthe ual rise in ZmSUT1-mediated currents (Fig. 2C). In the current standard solution at pH 5.6 or pH 5.6 in the presence of 10 mM acetate. After2hthe C radioactivity of the incubation-solution was measured clamp mode, membrane depolarization in response to different in a scintillation counter. The oocytes were rapidly washed in ice-cold sucrose concentrations could be recorded as well (Fig. 2D). Like standard solution and transferred to the scintillation mixture for count- the current response in Fig. 2C, the degree of membrane depo- ing the C radioactivity in the liquid scintillation analyzer. The per- larization depended on the sucrose concentration applied (up to centage of sucrose release was calculated. 50 mV with 10 mM sucrose). When the steady-state currents recorded in presence of extracellular sucrose concentrations be- tween 0.5 and 50 mM were plotted against the membrane poten- The abbreviations used are: MES, 4-morpholineethanesulfonic acid; pH , internal pH; TEVC, two-electrode voltage clamp. tial, ZmSUT1 currents increased upon hyperpolarization and i Reversibility of the Sucrose Transporter ZmSUT1 21439 FIG.1. Phloem potential measure- ments. Top, side-view of R. padi in feed- ing position on the upper side of a maize leaf (32). Bottom left, front view of R. padi sucking on maize with its stylet inserted into a sieve element of a vascular bundle. Bottom right, after the aphid is separated from its stylet by a laser pulse, the stylet stump exuded sieve tube sap to which the tip of a microelectrode was at- tached (400). Application of sucrose via the apoplast depolarizes phloem poten- tial, pointing to a proton-coupled cotrans- porter. Upon removal of sucrose, the membrane potential repolarized. FIG.2. ZmSUT1 is a sucrose/H symporter. A, uptake of C sucrose (5 mM final concentration) into ZmSUT1-in- jected and non-injected Xenopus oocytes over a time scale of 60 min at pH 5.6. B, parallel measurements of sucrose- dependent inward currents (upper trace) and the cytosolic pH (lower trace)ofa ZmSUT1-injected oocyte in response to 5 mM sucrose at an external pH of 5.6 and a holding potential (V )of 60 mV. Su- crose-induced currents are accompanied byadecreaseincytosolicpH.C,thesucrose- dependent inward currents were moni- tored in response to a stepwise increase in sucrose concentrations. V 60 mV. D, sucrose concentration-dependent mem- brane depolarization caused by a series of different sucrose concentrations at pH 5.6. 21440 Reversibility of the Sucrose Transporter ZmSUT1 pH 6.5 the sucrose affinity was reduced. Both K voltage curves could be fitted with a single exponential function, allowing us to extrapolate K to measured phloem potentials of up to 180 S S mV (20). A K of 3.7 mM at pH 5.6 and a K of 12.4 mM at pH m m 6.5 were calculated. The maximal carrier current I values max were found to be voltage-dependent also (not shown), decreasing linearly with negative-going membrane potentials. To study the proton coupling of ZmSUT1-mediated sucrose transport, the steady-state currents were measured as a func- tion of voltage and pH in the presence of 5 mM sucrose (not shown). As predicted for a proton-coupled transport process, in the pH range between 6.5 and 4.5 ZmSUT1 currents increased with increasing proton concentration and hyperpolarization. At pH values 7.0 no significant inward currents could be de- tected. The currents at selected voltages were plotted against the H concentration (not shown) and fitted by a single Michae- H H lis-Menten equation to calculate K and I (not shown). m max The proton affinity K of ZmSUT1 exponentially increased with hyperpolarizing membrane potentials (Fig. 3C). This be- havior is in line with the results for the sucrose affinities K (compare Fig. 3B). Thus, both the apparent affinity constants and the I values for sucrose as well as for protons decrease max upon hyperpolarization. To study the inverse transport mode of ZmSUT1 and its affinity toward cytosolic sucrose, we applied the giant patch clamp technique to ZmSUT1-expressing oocytes. In the inside- out configuration we varied the “cytosolic” sucrose concentra- tion in the presence of either 0.5, 5, or 50 mM extracellular (pipette) sucrose. Upon a stepwise increase in cytosolic sucrose from 0 to 50, 100, 200, and 500 mM in the presence of 50 mM in the pipette, a progressive decrease in inward current was measured (Fig. 4A). This effect was completely reversible; in- ward currents reached their pre-stimulus levels after the re- moval of cytosolic sucrose. Non-injected oocytes, however, did not respond to variations in the cytosolic sucrose concentration. When plotting the average currents shown in Fig. 4A as a function of the cytosolic sucrose concentration, data could be fitted by a Michaelis-Menten equation (Fig. 4A, continuous line) characterized by an apparent K of 160 mM (Fig. 4D). The inset of Fig. 4D depicts the extrapolation of the sucrose-induced currents from 2 to 3 M, a concentration range in which ZmSUT1 FIG.3. Voltage-, sucrose-, and pH-dependence of ZmSUT1. A, currents would reverse direction (I  0 at 2.38 M sucrose). steady-state, sugar-dependent, inward currents (mean  S.D.; n  4) at When the extracellular sucrose concentration was decreased to different potentials at pH 5.6 were plotted as a function of the external 5mM or even 0.5 mM, the ZmSUT1-mediated currents reversed sucrose concentration. Steady-state currents (currents in the absence of sucrose were subtracted) were normalized to the current induced by 10 direction at physiological cytosolic sucrose levels (Fig. 4, B and mM sucrose and a membrane potential of 100 mV. Curves were fitted C). In the presence of 5 mM external sucrose, a K of 278 mM with a Michaelis-Menten function. B, apparent affinity constants of was calculated (Fig. 4E). A rise in cytosolic sucrose concentra- ZmSUT1 K (deduced from panel A) as a function of the membrane tion above 314 mM even inverted the current direction. Upon a potential. K decreases exponentially upon hyperpolarization. Data were fitted with a single exponential function ([S]  [S ] exp (V/ ), further decrease in extracellular sucrose concentration to 0.5 0 0 where S is substrate) and extrapolated to more positive and more mM and the absence of cytosolic sucrose, only very small inward negative voltages. The fitting parameters at pH 5.6 were S  16.1 currents remained (Fig. 4C). Under these conditions, however, mM 0.7 mM and   122 mV  8 mV, and at pH 6.5 S  67 mM 3 0 0 a rise in cytosolic sucrose concentration to just 50 mM inverted mM and   108 mV  10 mV. C, the half-maximal proton concentra- the ZmSUT1 current already. From the Michaelis-Menten fit a tion K , was determined from the Michaelis-Menten fit (not shown) S H and plotted against the membrane potential. Like K , K was voltage- m m K of 362 mM and a zero current value at 31 mM was obtained dependent and could be fitted with a single exponential function as in (Fig. 4F). When plotting the K values versus the external panel B, with S  15.4 M 0.3 M and   98 mV  5 mV. 0 0 sucrose concentration, a decrease in K with the rise in exter- nal sucrose concentration became evident (not shown). were saturated at 30 mM sucrose (not shown). Plotting the cur- Likewise, the cytosolic sucrose concentration causing the rents as a function of the sucrose concentration a single Michae- ZmSUT1 current to change direction was plotted as a function lis-Menten function could be fitted to the individual, voltage-de- of external sucrose (Fig. 5). Under the equilibrium conditions pendent sucrose saturation curves (Fig. 3A). These current- depicted in Equation 2, concentration curves are hyperbolic in shape, suggesting that just one sucrose molecule binds to the transporter. The apparent n   n   0(Eq.2) suc suc H H affinity constant of ZmSUT1, K , exhibited pronounced voltage- and pH-dependence (Fig. 3B; compare also Ref. 19). Hyperpolar- where n and n are the number of moles of sucrose and suc H cyt ext izing voltages increased the apparent affinity to sucrose from 16 protons transported through the membrane,      is suc suc mM at0mVto7.2mM at 100 mV and pH 5.6. Upon a change to the difference between the cytosolic and external chemical poten- Reversibility of the Sucrose Transporter ZmSUT1 21441 FIG.4. Changes in cytosolic sucrose feedback on the magnitude and direction of ZmSUT1 currents. A–C, ZmSUT1 currents recorded in inside-out giant patches in the presence of 50 mM (A),5mM (B), and 0.5 mM external sucrose (C). Schematic representations above each graph depict the proton and sucrose concentrations; cytosolic and external pH was 7.5 and 5.6, respectively, and cytosolic sucrose concentrations were elevated from 0 to 50, 100, 200, and 500 mM as indicated. The membrane potential was clamped to 0 mV. D–F, averaged currents gained from experiments shown in panels A–C were plotted versus the corresponding cytosolic sucrose concentrations. Data were fitted by the Michaelis-Menten equation I  I ([Suc] /[Suc]  K )  I , where I  11.0 pA, K  161 mM, and I  10.3 pA (E); I  4.83 pA, K  278 mM, and I  2.56 1 cyt cyt m 0 1 m 0 1 m 0 pA (F); and I  536 fA, K  362 mM, and I  42.7 fA (G). The inset of panel D shows the current extrapolation for cytosolic sucrose concentrations 1 m 0 ranging from 2 to 3 M. FIG.5. Stoichiometry between H and sucrose of ZmSUT1. The cytosolic sucrose concentration that induces zero current, obtained by experiments as shown in Fig. 3, is plotted against the external sucrose concentration. The con- tinuous lines were obtained by the equi- librium equation (Equation 3) with V 0, pH  pH  1.9, and different val- cyt ext ues for n and n (the thicker line corre- suc H sponds to a 1:1 stoichiometry of the ZmSUT1 transporter). The same experi- mental conditions as those in Fig. 3 were used. cyt ext tial (or molar free energy) of sucrose, and      is revealed that the ZmSUT1 transporter has a 1 Suc/1 H stoi- H H H the difference between the cytosolic and external electro-chemi- chiometry (compare Refs. 19 and 21). cal potential of protons. Therefore, Equation 3, shown here, In agreement with a perfectly coupled thermodynamic ma- chine, the positive current in Fig. 4 represents the sucrose n FV H m (pHcyt  pHext  ) n 2.303RT [Suc]  [Suc] 10 suc (Eq. 3) cyt ext gradient-driven efflux of protons against the proton gradient. To study the two transport modes of ZmSUT1 in the absence of is another way in which Equation 2 can be written. The con- the proton motive force, in Fig. 6A we stepped the cytosolic tinuous lines in Fig. 5 are obtained by Equation 2 with V  0 mV and using different values for n and n . This analysis sucrose concentration from 0 to 500 mM (5 mM sucrose in the suc H 21442 Reversibility of the Sucrose Transporter ZmSUT1 FIG.7. Model for apoplasmic sucrose loading and unloading by sucrose/H symporters, modified after Ache et al. (32). The source site of the sieve element (SE)-companion cell (CC) complex is characterized by an outward-directed sucrose and an inward-directed H gradient. The membrane potential is hyperpolarized because of the FIG.6. Inward and inverse transport mode of ZmSUT1. A, activity of the H -ATPases localized in the companion cells. Under change in direction of ZmSUT1 currents upon changes in cytosolic these conditions, sucrose is accumulated in the phloem cells by su- sucrose concentration from 0 to 500 mM recorded in inside-out giant crose/H symporters like ZmSUT1. In the sink phloem the apoplastic patches. Note that the pH was a symmetrical 5.6 on both sides of the concentrations of sucrose is reduced, and the membrane potential is membrane. External sucrose was 5 mM, and the membrane voltage was depolarized to values around 60 mV. In this region the membrane 0 mV. B, similar experiment as in panel A, but performed in the potential mainly depends on the potassium conductance because of the presence of 50 mM external sucrose. This recording was chosen because reduced size (or even absence) of the energy-supplying companion cells. a fast rundown of the current was apparent. The decay of both positive Thus, the proton motive force is decreased. This regime directs Zm- and negative currents could be fitted by single exponentials with the SUT1 into the inverse transport mode, and sucrose is released. F, same time constant (  40 s). C, percentage of sucrose release from fructose; G, glucose; S, sucrose. [ C]sucrose-injected, ZmSUT1-expressing, and control oocytes. The percentage of sucrose release was measured after2hof oocyte incuba- tion ina1mM sucrose solution at pH 5.6 or pH 5.6 plus 5 mM acetate. in Fig. 4B, the ZmSUT1 currents were subject of a fast “run- down,” most likely due to the loss of regulatory cytosolic factors. pipette) in the absence of a pH gradient. With [Suc]  0mM cyt Interestingly, in Fig. 6B the decay of both inward and outward and the absence of a membrane potential, we recorded an currents could be fitted by single exponential functions (dashed inward current as expected from the steep inward-directed lines) with the same time constant. This indicates that both sucrose gradient. Inverting the sucrose gradient by increasing transport modes of ZmSUT1 are perfectly coupled via the su- [Suc] to 500 mM, the carrier current reversed direction. In the cyt crose gradient and proton motive force. presence of an inward-directed pH gradient, however, the mag- Under the conditions of the sink phloem, the sucrose gradient nitude of outward currents was smaller (compare Fig. 4). In- drives the efflux of protons and sucrose. To mimic this situation ward currents could be re-established again upon removal of in the oocyte system in Fig. 6C, ZmSUT1-expressing oocytes were the disaccharide. Following a rise in the extracellular sucrose injected with [ C]sucrose (final concentration of 50 mM), and the concentration from 5 to 50 mM and the absence of cytosolic release of the radioactively labeled sucrose was measured. In sucrose, carrier currents remained inward (Fig. 6B). During ZmSUT1-oocytes, but not in water-injected control-oocytes, pro- bath perfusion to [Suc]  500 mM, currents changed direc- cyt nounced sucrose release was measured. As expected from our tion. These experiments indicate that the sucrose gradient can thermodynamic assumptions, the sucrose-release was enhanced drive the proton flux and vice versa. In the experiment depicted when the cytosol was acidified by acetate treatment. Reversibility of the Sucrose Transporter ZmSUT1 21443 DISCUSSION on the potassium conductance mediated by K channels (32). Direct measurements in these sink phloem cells revealed mem- Because of the localization of a sucrose/H transporter in brane potentials of only 60 mV. At an apoplasmic sucrose sink tissues, it has previously been speculated that phloem concentration of 1 mM, a phloem sap sucrose concentration of unloading may be mediated by the same sucrose-H symport- 0.85 M, and a pH gradient of 1 unit of sucrose release would ers that are responsible for phloem loading (for example, Ref. occur at membrane potentials positive from 115 mV (accord- 22). The direct demonstration that ZmSUT1, a member of the ing to Equation 3 with n /n  1). This regime directs Zm- phloem sucrose carrier family, acts either in the source mode or H suc SUT1 into the inverse transport mode, and sucrose is released. sink mode for the life-maintaining uptake, and the adsorption The present work revealed the functional asymmetry of the of sucrose is underpinned by genetic evidence. Arabidopsis phloem sucrose carrier ZmSUT1. Our data, for the first time, mutants, which lack the ZmSUT1 homologue AtSUC2, are demonstrate the “sink mode” of this pivotal carrier type, pro- strongly impaired in phloem loading and unloading of sucrose, vide for the molecular mechanism of phloem sucrose release, which results in stunted growth, retarded development, and and explain the severe phenotype of phloem H /sucrose carrier sterility (23). Phloem unloading of sucrose is required for loss-of-function mutants and antisense-repression plants. In starch formation in storage tissues, such as the grains of cere- contrast to symplasmic unloading, this sucrose/H symporter- als or potato tubers. When the copy number of StSUT1, a based mechanism drives unloading of sucrose under the control ZmSUT1 orthologue expressed in the phloem of developing of both the sucrose and the pH gradients as well as the mem- tubers, is reduced by antisense repression, reduced fresh brane potential of the phloem and the surrounding tissues. weight accumulation during tuber development was observed (4, 5). Furthermore, indirect measurements with the proton- Acknowledgment—We thank Dr. N. Aoki for the generous supply of coupled monosaccharide transporter CkHUP1 from the green ZmSUT1 cDNA. alga Chlorella and the SGLT1 Na /glucose transporter from REFERENCES human and rabbit suggest that these sugar carriers from sin- 1. Mu¨ nch, E. 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C., and Oparka, K. J. (2001) Plant Cell 13, 385–398 e.g. by increasing the cytosolic sucrose concentration. The di- 6. Tegeder, M., Wang, X. D., Frommer, W. B., Offler, C. E., and Patrick, J. W. (1999) Plant J. 18, 151–161 rection of the transport of the ZmSUT1 symporter is therefore 7. Weber, H., Borisjuk, L., Heim, U., Sauer, N., and Wobus, U. (1997) Plant Cell dependent on the sum of the free energies of both the sucrose 9, 895–908 8. Lemoine, R., Burkle, L., Barker, L., Sakr, S., Kuhn, C., Regnacq, M., Gaillard, and the proton gradient across the membrane. In agreement C., Delrot, S., and Frommer, W. B. (1999) FEBS Lett. 454, 325–330 with the above considerations, we could demonstrate that su- 9. Evert, R. F., and Russin, W. A. (1993) Am. J. Bot. 80, 1310–1317 10. Wright J. P., and Fisher D. B. (1981) Plant Physiol. 67, 845–848 crose could drive protons through ZmSUT1. Recently, the re- 11. Becker, D., Dreyer, I., Hoth, S., Reid, J. D., Busch, H., Lehnen, M., Palme, K., versibility of the human and rabbit Na /glucose co-transport- and Hedrich, R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8123–8128 ers has been documented by measuring the reversion of the 12. Hilgemann, D. (1995) in Single-Channel Recording (Sakmann, B., and Neher, E., eds) 2nd Ed., pp. 307–328, Plenum Press, New York glucose-coupled Na current. Like the proton-coupled disac- 13. Aoki, N., Hirose, T., Takahashi, S., Ono, K., Ishimaru, K., and Ohsugi, R. charide carrier ZmSUT1, the sodium-coupled SGLT1 shows (1999) Plant Cell Physiol. 40, 1072–1078 more than one order of magnitude difference between the sugar 14. Hirose, T., Imaizumi, N., Scofield, G. N., Furbank, R. T., and Ohsugi, R. (1997) Plant Cell Physiol. 38, 1389–1396 affinities of the two transport modes, indicating a functional 15. Ku¨ hn, C. (2003) Plant Biol. 5, 215–232 asymmetry of both carrier types. Under physiological condi- 16. Marger, M. D., and Saier, M. H., Jr. (1993) Trends Biochem. Sci. 18, 13–20 17. Saier, M. H., Jr., Beatty, J. T., Goffeau, A., Harley, K. T., Heijne, W. H., Huang, tions the inverse transport mode of SGLT1 is highly improba- S. C., Jack, D. L., Jahn, P. S., Lew, K., Liu, J., Pao, S. S., Paulsen, I. T., ble because of the low affinity of the sugar carrier. In the plant Tseng, T. T., and Virk, P. S. (1999) J. Mol. Microbiol. Biotechnol. 1, 257–279 18. Zeuthen, T., Hamann, S., and la Cour, M. (1996) J. Physiol. (Lond.) 497, 3–17 phloem, however, both transport modes of ZmSUT1 are prob- 19. Boorer, K. J., Loo, D. D. F., Frommer, W. B., and Wright, E. M. (1996) J. Biol. able (see model in Fig. 7). In maize source leaves, extracellular Chem. 271, 25139–25144 sucrose concentrations of 2.6 mM were measured (28). Assum- 20. Deeken, R., Geiger, D., Fromm, J., Koroleva, O., Ache, P., Langenfeld-Heyser, R., Sauer, N., May, S. T., and Hedrich, R. (2002) Planta 216, 334–344 ing a pH gradient of 1.5 units and a phloem membrane 21. Zhou, J.-J., Theodoulou, F., Sauer, N., Sanders, D., and Miller, A. J. (1997) J. potential of 150 mV (29), a perfect proton-coupled ZmSUT1 Membr. Biol. 159, 113–125 would allow a theoretical phloem sucrose accumulation of up to 22. Truernit, E., and Sauer, N. (1995) Planta 196, 564–570 23. Gottwald, J. R., Krysan, P. J., Young, J. C., Evert, R. F., and Sussman, M. R. 26 M (according to Equation 3, with n /n  1). Directly H suc (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13979–13984 measured sucrose concentrations of maize phloem sap revealed 24. Komor, E., and Tanner, W. (1974) J. Gen. Physiol. 64, 568–581 25. Komor, E., and Tanner, W. (1974) Nature 248, 511–512 sucrose contents of 0.85 M (28). In the sink phloem, however, 26. Quick, M., Tomasevic, J., and Wright, E. M. (2003) Biochemistry 42, the conditions are different. The external sucrose concentration 9147–9152 27. Sauer, G. A., Nagel, G., Koepsell, H., Bamberg, E., and Hartung, K. (2000) is reduced to 1 mM or less because of the activity of cell wall- FEBS Lett. 469, 98–100 bound invertases (for example, Ref. 30) and the surrounding 28. Lohaus, G., Hussmann, M., Pennewiss, K., Schneider, H., Zhu, J. J., and sucrose taking up (sucking) sink tissues with hyperpolarized Sattelmacher, B. (2000) J. Exp. Bot. 351, 1721–1732 29. van Bel, A. J. E. (1993) Prog. Bot. 54, 134–150 membrane potentials negative to 180 mV. Symplasmic un- 30. Roitsch, T., Balibrea, M. E., Hofmann, M., Proels, R., and Sinha, A. K. (2003) loading is unlikely in maize because of the lack of plasmodes- J. Exp. Bot. 54, 513–524 mata in the protophloem and metaphloem (9). Furthermore, in 31. van Bel A. J. E., and Ehlers K. (2000) in Cambium: The Biology of Wood Formation (Savidge, R., Barnett, J. R., and Napier, R., eds) pp. 85–99, Bios, the region of the release phloem the proton motive force across Oxford the phloem membrane is less strong because of the reduced size 32. Ache, P., Becker, D., Deeken, R., Dreyer, I., Weber, H., Fromm, J., and Hedrich, R. (2001) Plant J. 27, 571–580 (or even absence) of the energy-supplying companion cells (29, 31). Therefore the phloem membrane potential mainly depends J. B. Hafke, unpublished data, and A. J. E. van Bel, personal P. Ache, J. Fromm, and R. Hedrich, unpublished data. communication. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Phloem-localized, Proton-coupled Sucrose Carrier ZmSUT1 Mediates Sucrose Efflux under the Control of the Sucrose Gradient and the Proton Motive Force *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2005 Elsevier Inc.
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0021-9258
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1083-351X
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10.1074/jbc.m501785200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 22, Issue of June 3, pp. 21437–21443, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Phloem-localized, Proton-coupled Sucrose Carrier ZmSUT1 Mediates Sucrose Efflux under the Control of the Sucrose Gradient and the Proton Motive Force* Received for publication, February 16, 2005, and in revised form, March 31, 2005 Published, JBC Papers in Press, April 1, 2005, DOI 10.1074/jbc.M501785200 Armando Carpaneto‡§ , Dietmar Geiger§ , Ernst Bamberg**, Norbert Sauer‡‡, Jo¨rg Fromm§§, ¶¶ and Rainer Hedrich From the Julius-von-Sachs-Institute for Biosciences, Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Platz 2, D-97082 Wu¨rzburg, Germany, the ‡Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, I-16149 Genova, Italy, the **Max-Plank-Institute for Biophysics, Marie Curie Strasse 15, D-60439 Frankfurt am Main, Germany, the ‡‡University Erlangen-Nu¨rnberg, Molecular Plant Physiology, Staudtstrasse 5, D-91058 Erlangen, Germany, and the §§Technical University Mu¨nchen, Holzforschung, Winzererstrasse 45, D-80797 Mu¨nchen, Germany ing vascular cells. The hydrostatic pressure difference between The phloem network is as essential for plants as the vascular system is for humans. This network, assembled source and sink tissues drives the mass flow of water and by nucleus- and vacuole-free interconnected living cells, nutrients in the phloem vessels (1). In sink tissues, which are represents a long distance transport pathway for nutri- dependent on carbon supply via the phloem, a symplasmic ents and information. According to the Mu¨ nch hypoth- unloading of sucrose along its concentration gradient has been esis, osmolytes such as sucrose generate the hydrostatic shown for many plant species (2). Interestingly, however, su- pressure that drives nutrient and water flow between crose/H symporter transcripts and proteins have also been the source and the sink phloem (Mu¨ nch, E. (1930) Die localized in sink tissues, suggesting a role in sink loading/ Stoffbewegungen in der Pflanze, Gustav Fischer, Jena, retrieval or unloading of sucrose via these transporters (see Germany). Although proton-coupled sucrose carriers Ref. 2 for review). SUT1 from the potato, for example, has been have been localized to the sieve tube and the companion detected in the sieve elements of mature source leaves as well cell plasma membrane of both source and sink tissues, as in developing sink leaves, roots (3), and tubers (4, 5). Using knowledge of the molecular representatives and the a sink-specific antisense inhibition for SUT1 under the control mechanism of the sucrose phloem efflux is still scant. We of a tuber-specific promoter, Ku¨hn et al. (2003) (4) could dem- expressed ZmSUT1, a maize sucrose/proton symporter, onstrate the involvement of SUT1 in early tuber development in Xenopus oocytes and studied the transport character- istics of the carrier by electrophysiological methods. and, thus, phloem unloading. Further evidence for a sucrose Using the patch clamp techniques in the giant inside-out export system was added by the localization of sucrose/H patch mode, we altered the chemical and electrochemi- symporters expressed in symplasmically isolated tissues such cal gradient across the sucrose carrier and analyzed the as developing embryos (6, 7) and growing pollen tubes (8). currents generated by the proton flux. Thereby we could Furthermore Evert and Russin (1993) could show, for example, show that ZmSUT1 is capable of mediating both the that symplasmic unloading in maize is unlikely because of the sucrose uptake into the phloem in mature leaves lack of plasmodesmata in the protophloem and metaphloem of (source) as well as the desorption of sugar from the developing leaves (9). Although proton-coupled sucrose carriers phloem vessels into heterotrophic tissues (sink). As pre- have been localized to the sieve tubes and companion cell dicted from a perfect molecular machine, the ZmSUT1- plasma membranes of both source and sink tissues, the molec- mediated sucrose-coupled proton current was reversi- ular representatives and mechanism of the sucrose phloem ble and depended on the direction of the sucrose and pH efflux is still scant. gradient as well as the membrane potential across the In the present study we tested the biophysical properties and transporter. thermodynamics of ZmSUT1, a maize sucrose carrier ex- pressed at a high level in Xenopus laevis oocytes. Specific To ensure adequate partitioning of sucrose throughout the sucrose transport inhibitors are not available, but Xenopus plant body, sucrose has to be translocated from the mesophyll oocytes, like all other creatures apart from plants, do not trans- cells to the sieve element-companion cell complex. Because of port sucrose. Therefore oocytes are well suited for sucrose the energy-dependent sucrose/H symporter in apoplasmic transport studies. Using this sucrose-insensitive system we loading plant species, the transport sugar accumulates at con- could demonstrate that this sugar carrier mediates both su- centrations of several hundred mM to 1 molar in the conduct- crose uptake and release. Upon a drop in membrane potential and/or pH gradient, ZmSUT1 would release sucrose from, for example, sink phloem and thus seem to represent the molecu- * This work was funded in part by Deutsche Forschungsgemeinschaft grants (to R. H.). The costs of publication of this article were defrayed in lar equivalent for the sucrose efflux carrier. Based on our part by the payment of page charges. This article must therefore be biophysical characterization of ZmSUT1, the “source and sink hereby marked “advertisement” in accordance with 18 U.S.C. Section mode” of this transporter is discussed in respect to in planta 1734 solely to indicate this fact. § These authors contributed equally to this work. phloem loading and unloading conditions. In principle, each This author’s stay in Wu¨ rzburg, Germany was funded by an SFB individual transporter should be reversible. But, in contrast to 487 guest scientist stipend and a research fellowship of the Von Hum- the reversible transporters from the animal field and from boldt Foundation. bacteria that have been described to date, ZmSUT1 is the first ¶¶ To whom correspondence should be addressed. Tel.: 49-931-8886101; Fax: 49-931-8886157; E-mail: hedrich@botanik.uni-wuerzburg.de. that works in both directions under physiological conditions. This paper is available on line at http://www.jbc.org 21437 This is an Open Access article under the CC BY license. 21438 Reversibility of the Sucrose Transporter ZmSUT1 MATERIALS AND METHODS Patch Clamp Measurements—Giant patch recording (12) was per- formed in inside-out configuration on ZmSUT1 expressing Xenopus Aphid Breeding—Aphids of the species Rhopalosiphum padi were oocytes. Borosilicate glass pipettes were pulled and fire-polished to bred on barley and maize grown in a climate chamber under a 14-h have a final tip with a diameter between 25 and 30 m. Oocytes were photoperiod. bathed in an external solution consisting of 30 mM KCl, 1 mM CaCl , 1.5 Experimental Setup—Plant aphid cages were applied to the mature mM MgCl ,1mM GdCl , 145 mM sorbitol, and 10 mM MES/Tris, pH 5.6. 2 3 leaves of a 4-week-old potted maize. Aphids feeding on a leaf were After the seal was obtained, the external solution was changed (30 mM dissected from their stylets using a laser as described previously (10). KCl, 1 mM EGTA, 2 mM MgCl , 145 mM (or 500 mM) sorbitol, and 10 mM The recording electrodes were brought in contact with the phloem Tris/MES, pH 7.5), and the patch was excised. The recording pipette exudate appearing at the cut end of the stylet. The leaf was cut 15 cm was then placed in front of a polyethylene tube in connection with the proximal to the tip, and the cut end was incubated with artificial pond desired ionic solutions that were driven by gravity. The standard cyto- water containing the reference electrode (silver/silver chloride) and 1 1 solic solution contained 30 mM KCl, 1 mM EGTA, and 2 mM MgCl . The mM NaCl, 0.1 mM KCl, 0.1 mM CaCl , 100 mM sorbitol, and 1 mM MES, cytosolic sucrose concentration ranged from 0 to 500 mM, as indicated in adjusted to pH 6.0 with Tris. Sucrose pulses were applied by perfusion the remaining text where noted; sorbitol was appropriately added to of artificial pond water solution. Phloem potential measurements were each cytosolic solution to have a total sugar concentration of 500 mM. recorded according to (10). Cytosolic pH was 7.5 or 5.6 (with 10 mM Tris/MES or MES/Tris). The Two-electrode Voltage Clamp (TEVC) Analysis in Xenopus Oocytes— TM standard pipette solution was 30 mM KCl, 1 mM CaCl , 1.5 mM MgCl , 2 2 ZmSUT1 cRNA was prepared using the mMESSAGE mMACHINE 145 mM sorbitol, and 10 mM MES/Tris, pH 5.6; sucrose was added at RNA transcription kit (Ambion Inc., Austin, TX). Oocyte preparation concentrations of 0.5, 5, and 50 mM as indicated in the text where noted. and cRNA injection have been described elsewhere (11). In TEVC stud- Currents, filtered at 10 or 100 Hz and sampled at 200 or 400 Hz, were ies, oocytes were perfused with a standard solution containing 30 mM recorded with an EPC9 amplifier using Pulse 8.3 software (Heka Ele- KCl, 1 mM CaCl , and 1.5 mM MgCl based on Tris/MES buffers for pH 2 2 ktronic GmbH, Lembrecht, Germany). Data were analyzed by custom- values from 5.6 to 8.0 or based on citrate/Tris buffers for the pH values made programs using Igor (Wavemetrics; Lake Oswego, OR). 4.5 and 5.0. The sucrose concentrations and pH values are indicated in Figs. 2–4 and 6 (and the corresponding legends) and throughout the RESULTS text where noted. All solutions were adjusted to 220 mosmol kg using D-sorbitol. Steady state currents were obtained by stepping the mem- ZmSUT1 was isolated from maize and expressed in source brane potential from the holding potential of 0 mV to a series of 500-ms and sink tissues such as mature leave blades and sheaths as test pulses from 60 to 130 mV in 10-mV decrements. Difference well as pedicels and seeds (13). High sequence homologies to currents were calculated by subtracting the currents in the absence of the rice sucrose transporter OsSUT1 (14) and to known sucrose sucrose from the currents in its presence. The sucrose-induced steady state currents were measured in respect to ligand concentrations and transporters from the dicot species group ZmSUT1 into the membrane potential. At each test potential the currents were fitted to SUT2 subfamily of sucrose transporters with high sucrose af- the Michaelis-Menten equation shown in Equation 1, finity (for review, see Ref. 15). ZmSUT1 is a member of a large S S family of membrane proteins mediating the transport of sug- I  I [S]/([S]  K ) (Eq. 1) max m ars, amino acids, and osmolytes across membranes. These car- where the substrate (S) is either [sucrose] or [H ]. These fits yielded in riers share the typical 12-transmembrane-spanning -helix S H the maximal currents I for sucrose and I for H and the max max structure (16, 17). In most eukaryotic cells these transporters H  S half-maximal ligand concentrations K for H and K for sucrose. m m couple the uptake of their substrates to electrochemical ion Intracellular pH Measurements—PH-sensitive microelectrodes were gradients generated by the H -orNa /K -ATPase. pulled from borosilicate capillary (TW100F-3; WPI, Sarasota, FL) using a laser puller (P2000; Sutter Instruments, Novato, CA) and silanized Using the aphid stylet technique on maize leaf blades, it could with dimethyldichlorosilane (Fulka, Steinheim, Germany) at 200 °C for be shown that the addition of sucrose reversibly depolarized the 15 min. The tips of the pH microelectrodes were filled with hydrogen phloem potential (Fig. 1). To elucidate the transport characteris- ionophore I mixture B (Fulka) and then back-filled with a buffer con- tics of the underlying sucrose/H transporter activity with re- taining 40 mM KH PO ,23mM NaOH, and 150 mM NaCl (pH 6.8). Only 2 4 spect to sucrose affinity gradients and proton motive force, we electrodes with a linear slope of 55–60 mV/pH unit over the calibration heterologously expressed ZmSUT1 in Xenopus laevis oocytes. range before and after measurement were used. Signals were recorded with an electrometer (Model FD 223; WPI) in parallel to the currents in Functional analysis was performed using both the TEVC tech- the voltage clamp mode of a TEVC amplifier (Turbo TEC 10CD; npi nique and the patch clamp technique. Oocytes expressing Zm- electronic GmbH, Tamm, Germany). On the basis of the calibration SUT1 efficiently imported radio-labeled sucrose with uptake curve for the pH microelectrodes, the internal pH (pH ) of the oocytes rates of 6 nmol per hour and oocyte, whereas non-injected oocytes was calculated in consideration of the membrane potential. 14 did not accumulate sucrose in detectable amounts (Fig. 2A). To C Sucrose Uptake Experiments—In each experiment, 10 ZmSUT1- monitor the movement of protons accompanying the sucrose injected oocytes or 10 control oocytes were incubated in 0.05 Ci/ml C sucrose with a final sucrose concentration of 5 mM in the standard transport, we simultaneously recorded sucrose-induced ionic cur- solution at pH 5.6. At defined time points the oocytes were rapidly rents and changes in cytoplasmic pH by TEVC and proton- washed three times in ice-cold standard solution and transferred to selective microelectrodes (18). Upon the addition of sucrose to the liquid scintillation vials containing scintillation mixture (Emulsifier- external solution, large inward currents were elicited (Fig. 2B, TM 14 Safe ; Packard, Meriden, CT). The C radioactivity was counted in a upper trace). Inward currents were accompanied by a decrease in liquid scintillation analyzer (Model 1900CA; Packard), and the sucrose pH by up to 0.5 units within 10 min (Fig. 2B, lower trace). After uptake per oocyte was calculated from three independent experiments for each time point. the removal of sucrose from the bath medium, the inward cur- [ C]Sucrose Release Experiments—Control oocytes and ZmSUT1- rents returned to the pre-sucrose level again, whereas the recov- injected oocytes were loaded with 0.5 Ci of radiolabeled sucrose with a ery of pH was delayed. Control oocytes showed neither sucrose- TM final sucrose concentration of 50 mM by injection (Picospritzer II; induced currents nor sucrose-dependent changes in pH . General Valve Co., Fairfield, NJ). After a 10-min washing period in Stepwise increases in sucrose concentrations resulted in a grad- ice-cold ND96, each single oocyte was transferred into 200 lofthe ual rise in ZmSUT1-mediated currents (Fig. 2C). In the current standard solution at pH 5.6 or pH 5.6 in the presence of 10 mM acetate. After2hthe C radioactivity of the incubation-solution was measured clamp mode, membrane depolarization in response to different in a scintillation counter. The oocytes were rapidly washed in ice-cold sucrose concentrations could be recorded as well (Fig. 2D). Like standard solution and transferred to the scintillation mixture for count- the current response in Fig. 2C, the degree of membrane depo- ing the C radioactivity in the liquid scintillation analyzer. The per- larization depended on the sucrose concentration applied (up to centage of sucrose release was calculated. 50 mV with 10 mM sucrose). When the steady-state currents recorded in presence of extracellular sucrose concentrations be- tween 0.5 and 50 mM were plotted against the membrane poten- The abbreviations used are: MES, 4-morpholineethanesulfonic acid; pH , internal pH; TEVC, two-electrode voltage clamp. tial, ZmSUT1 currents increased upon hyperpolarization and i Reversibility of the Sucrose Transporter ZmSUT1 21439 FIG.1. Phloem potential measure- ments. Top, side-view of R. padi in feed- ing position on the upper side of a maize leaf (32). Bottom left, front view of R. padi sucking on maize with its stylet inserted into a sieve element of a vascular bundle. Bottom right, after the aphid is separated from its stylet by a laser pulse, the stylet stump exuded sieve tube sap to which the tip of a microelectrode was at- tached (400). Application of sucrose via the apoplast depolarizes phloem poten- tial, pointing to a proton-coupled cotrans- porter. Upon removal of sucrose, the membrane potential repolarized. FIG.2. ZmSUT1 is a sucrose/H symporter. A, uptake of C sucrose (5 mM final concentration) into ZmSUT1-in- jected and non-injected Xenopus oocytes over a time scale of 60 min at pH 5.6. B, parallel measurements of sucrose- dependent inward currents (upper trace) and the cytosolic pH (lower trace)ofa ZmSUT1-injected oocyte in response to 5 mM sucrose at an external pH of 5.6 and a holding potential (V )of 60 mV. Su- crose-induced currents are accompanied byadecreaseincytosolicpH.C,thesucrose- dependent inward currents were moni- tored in response to a stepwise increase in sucrose concentrations. V 60 mV. D, sucrose concentration-dependent mem- brane depolarization caused by a series of different sucrose concentrations at pH 5.6. 21440 Reversibility of the Sucrose Transporter ZmSUT1 pH 6.5 the sucrose affinity was reduced. Both K voltage curves could be fitted with a single exponential function, allowing us to extrapolate K to measured phloem potentials of up to 180 S S mV (20). A K of 3.7 mM at pH 5.6 and a K of 12.4 mM at pH m m 6.5 were calculated. The maximal carrier current I values max were found to be voltage-dependent also (not shown), decreasing linearly with negative-going membrane potentials. To study the proton coupling of ZmSUT1-mediated sucrose transport, the steady-state currents were measured as a func- tion of voltage and pH in the presence of 5 mM sucrose (not shown). As predicted for a proton-coupled transport process, in the pH range between 6.5 and 4.5 ZmSUT1 currents increased with increasing proton concentration and hyperpolarization. At pH values 7.0 no significant inward currents could be de- tected. The currents at selected voltages were plotted against the H concentration (not shown) and fitted by a single Michae- H H lis-Menten equation to calculate K and I (not shown). m max The proton affinity K of ZmSUT1 exponentially increased with hyperpolarizing membrane potentials (Fig. 3C). This be- havior is in line with the results for the sucrose affinities K (compare Fig. 3B). Thus, both the apparent affinity constants and the I values for sucrose as well as for protons decrease max upon hyperpolarization. To study the inverse transport mode of ZmSUT1 and its affinity toward cytosolic sucrose, we applied the giant patch clamp technique to ZmSUT1-expressing oocytes. In the inside- out configuration we varied the “cytosolic” sucrose concentra- tion in the presence of either 0.5, 5, or 50 mM extracellular (pipette) sucrose. Upon a stepwise increase in cytosolic sucrose from 0 to 50, 100, 200, and 500 mM in the presence of 50 mM in the pipette, a progressive decrease in inward current was measured (Fig. 4A). This effect was completely reversible; in- ward currents reached their pre-stimulus levels after the re- moval of cytosolic sucrose. Non-injected oocytes, however, did not respond to variations in the cytosolic sucrose concentration. When plotting the average currents shown in Fig. 4A as a function of the cytosolic sucrose concentration, data could be fitted by a Michaelis-Menten equation (Fig. 4A, continuous line) characterized by an apparent K of 160 mM (Fig. 4D). The inset of Fig. 4D depicts the extrapolation of the sucrose-induced currents from 2 to 3 M, a concentration range in which ZmSUT1 FIG.3. Voltage-, sucrose-, and pH-dependence of ZmSUT1. A, currents would reverse direction (I  0 at 2.38 M sucrose). steady-state, sugar-dependent, inward currents (mean  S.D.; n  4) at When the extracellular sucrose concentration was decreased to different potentials at pH 5.6 were plotted as a function of the external 5mM or even 0.5 mM, the ZmSUT1-mediated currents reversed sucrose concentration. Steady-state currents (currents in the absence of sucrose were subtracted) were normalized to the current induced by 10 direction at physiological cytosolic sucrose levels (Fig. 4, B and mM sucrose and a membrane potential of 100 mV. Curves were fitted C). In the presence of 5 mM external sucrose, a K of 278 mM with a Michaelis-Menten function. B, apparent affinity constants of was calculated (Fig. 4E). A rise in cytosolic sucrose concentra- ZmSUT1 K (deduced from panel A) as a function of the membrane tion above 314 mM even inverted the current direction. Upon a potential. K decreases exponentially upon hyperpolarization. Data were fitted with a single exponential function ([S]  [S ] exp (V/ ), further decrease in extracellular sucrose concentration to 0.5 0 0 where S is substrate) and extrapolated to more positive and more mM and the absence of cytosolic sucrose, only very small inward negative voltages. The fitting parameters at pH 5.6 were S  16.1 currents remained (Fig. 4C). Under these conditions, however, mM 0.7 mM and   122 mV  8 mV, and at pH 6.5 S  67 mM 3 0 0 a rise in cytosolic sucrose concentration to just 50 mM inverted mM and   108 mV  10 mV. C, the half-maximal proton concentra- the ZmSUT1 current already. From the Michaelis-Menten fit a tion K , was determined from the Michaelis-Menten fit (not shown) S H and plotted against the membrane potential. Like K , K was voltage- m m K of 362 mM and a zero current value at 31 mM was obtained dependent and could be fitted with a single exponential function as in (Fig. 4F). When plotting the K values versus the external panel B, with S  15.4 M 0.3 M and   98 mV  5 mV. 0 0 sucrose concentration, a decrease in K with the rise in exter- nal sucrose concentration became evident (not shown). were saturated at 30 mM sucrose (not shown). Plotting the cur- Likewise, the cytosolic sucrose concentration causing the rents as a function of the sucrose concentration a single Michae- ZmSUT1 current to change direction was plotted as a function lis-Menten function could be fitted to the individual, voltage-de- of external sucrose (Fig. 5). Under the equilibrium conditions pendent sucrose saturation curves (Fig. 3A). These current- depicted in Equation 2, concentration curves are hyperbolic in shape, suggesting that just one sucrose molecule binds to the transporter. The apparent n   n   0(Eq.2) suc suc H H affinity constant of ZmSUT1, K , exhibited pronounced voltage- and pH-dependence (Fig. 3B; compare also Ref. 19). Hyperpolar- where n and n are the number of moles of sucrose and suc H cyt ext izing voltages increased the apparent affinity to sucrose from 16 protons transported through the membrane,      is suc suc mM at0mVto7.2mM at 100 mV and pH 5.6. Upon a change to the difference between the cytosolic and external chemical poten- Reversibility of the Sucrose Transporter ZmSUT1 21441 FIG.4. Changes in cytosolic sucrose feedback on the magnitude and direction of ZmSUT1 currents. A–C, ZmSUT1 currents recorded in inside-out giant patches in the presence of 50 mM (A),5mM (B), and 0.5 mM external sucrose (C). Schematic representations above each graph depict the proton and sucrose concentrations; cytosolic and external pH was 7.5 and 5.6, respectively, and cytosolic sucrose concentrations were elevated from 0 to 50, 100, 200, and 500 mM as indicated. The membrane potential was clamped to 0 mV. D–F, averaged currents gained from experiments shown in panels A–C were plotted versus the corresponding cytosolic sucrose concentrations. Data were fitted by the Michaelis-Menten equation I  I ([Suc] /[Suc]  K )  I , where I  11.0 pA, K  161 mM, and I  10.3 pA (E); I  4.83 pA, K  278 mM, and I  2.56 1 cyt cyt m 0 1 m 0 1 m 0 pA (F); and I  536 fA, K  362 mM, and I  42.7 fA (G). The inset of panel D shows the current extrapolation for cytosolic sucrose concentrations 1 m 0 ranging from 2 to 3 M. FIG.5. Stoichiometry between H and sucrose of ZmSUT1. The cytosolic sucrose concentration that induces zero current, obtained by experiments as shown in Fig. 3, is plotted against the external sucrose concentration. The con- tinuous lines were obtained by the equi- librium equation (Equation 3) with V 0, pH  pH  1.9, and different val- cyt ext ues for n and n (the thicker line corre- suc H sponds to a 1:1 stoichiometry of the ZmSUT1 transporter). The same experi- mental conditions as those in Fig. 3 were used. cyt ext tial (or molar free energy) of sucrose, and      is revealed that the ZmSUT1 transporter has a 1 Suc/1 H stoi- H H H the difference between the cytosolic and external electro-chemi- chiometry (compare Refs. 19 and 21). cal potential of protons. Therefore, Equation 3, shown here, In agreement with a perfectly coupled thermodynamic ma- chine, the positive current in Fig. 4 represents the sucrose n FV H m (pHcyt  pHext  ) n 2.303RT [Suc]  [Suc] 10 suc (Eq. 3) cyt ext gradient-driven efflux of protons against the proton gradient. To study the two transport modes of ZmSUT1 in the absence of is another way in which Equation 2 can be written. The con- the proton motive force, in Fig. 6A we stepped the cytosolic tinuous lines in Fig. 5 are obtained by Equation 2 with V  0 mV and using different values for n and n . This analysis sucrose concentration from 0 to 500 mM (5 mM sucrose in the suc H 21442 Reversibility of the Sucrose Transporter ZmSUT1 FIG.7. Model for apoplasmic sucrose loading and unloading by sucrose/H symporters, modified after Ache et al. (32). The source site of the sieve element (SE)-companion cell (CC) complex is characterized by an outward-directed sucrose and an inward-directed H gradient. The membrane potential is hyperpolarized because of the FIG.6. Inward and inverse transport mode of ZmSUT1. A, activity of the H -ATPases localized in the companion cells. Under change in direction of ZmSUT1 currents upon changes in cytosolic these conditions, sucrose is accumulated in the phloem cells by su- sucrose concentration from 0 to 500 mM recorded in inside-out giant crose/H symporters like ZmSUT1. In the sink phloem the apoplastic patches. Note that the pH was a symmetrical 5.6 on both sides of the concentrations of sucrose is reduced, and the membrane potential is membrane. External sucrose was 5 mM, and the membrane voltage was depolarized to values around 60 mV. In this region the membrane 0 mV. B, similar experiment as in panel A, but performed in the potential mainly depends on the potassium conductance because of the presence of 50 mM external sucrose. This recording was chosen because reduced size (or even absence) of the energy-supplying companion cells. a fast rundown of the current was apparent. The decay of both positive Thus, the proton motive force is decreased. This regime directs Zm- and negative currents could be fitted by single exponentials with the SUT1 into the inverse transport mode, and sucrose is released. F, same time constant (  40 s). C, percentage of sucrose release from fructose; G, glucose; S, sucrose. [ C]sucrose-injected, ZmSUT1-expressing, and control oocytes. The percentage of sucrose release was measured after2hof oocyte incuba- tion ina1mM sucrose solution at pH 5.6 or pH 5.6 plus 5 mM acetate. in Fig. 4B, the ZmSUT1 currents were subject of a fast “run- down,” most likely due to the loss of regulatory cytosolic factors. pipette) in the absence of a pH gradient. With [Suc]  0mM cyt Interestingly, in Fig. 6B the decay of both inward and outward and the absence of a membrane potential, we recorded an currents could be fitted by single exponential functions (dashed inward current as expected from the steep inward-directed lines) with the same time constant. This indicates that both sucrose gradient. Inverting the sucrose gradient by increasing transport modes of ZmSUT1 are perfectly coupled via the su- [Suc] to 500 mM, the carrier current reversed direction. In the cyt crose gradient and proton motive force. presence of an inward-directed pH gradient, however, the mag- Under the conditions of the sink phloem, the sucrose gradient nitude of outward currents was smaller (compare Fig. 4). In- drives the efflux of protons and sucrose. To mimic this situation ward currents could be re-established again upon removal of in the oocyte system in Fig. 6C, ZmSUT1-expressing oocytes were the disaccharide. Following a rise in the extracellular sucrose injected with [ C]sucrose (final concentration of 50 mM), and the concentration from 5 to 50 mM and the absence of cytosolic release of the radioactively labeled sucrose was measured. In sucrose, carrier currents remained inward (Fig. 6B). During ZmSUT1-oocytes, but not in water-injected control-oocytes, pro- bath perfusion to [Suc]  500 mM, currents changed direc- cyt nounced sucrose release was measured. As expected from our tion. These experiments indicate that the sucrose gradient can thermodynamic assumptions, the sucrose-release was enhanced drive the proton flux and vice versa. In the experiment depicted when the cytosol was acidified by acetate treatment. Reversibility of the Sucrose Transporter ZmSUT1 21443 DISCUSSION on the potassium conductance mediated by K channels (32). Direct measurements in these sink phloem cells revealed mem- Because of the localization of a sucrose/H transporter in brane potentials of only 60 mV. At an apoplasmic sucrose sink tissues, it has previously been speculated that phloem concentration of 1 mM, a phloem sap sucrose concentration of unloading may be mediated by the same sucrose-H symport- 0.85 M, and a pH gradient of 1 unit of sucrose release would ers that are responsible for phloem loading (for example, Ref. occur at membrane potentials positive from 115 mV (accord- 22). The direct demonstration that ZmSUT1, a member of the ing to Equation 3 with n /n  1). This regime directs Zm- phloem sucrose carrier family, acts either in the source mode or H suc SUT1 into the inverse transport mode, and sucrose is released. sink mode for the life-maintaining uptake, and the adsorption The present work revealed the functional asymmetry of the of sucrose is underpinned by genetic evidence. Arabidopsis phloem sucrose carrier ZmSUT1. Our data, for the first time, mutants, which lack the ZmSUT1 homologue AtSUC2, are demonstrate the “sink mode” of this pivotal carrier type, pro- strongly impaired in phloem loading and unloading of sucrose, vide for the molecular mechanism of phloem sucrose release, which results in stunted growth, retarded development, and and explain the severe phenotype of phloem H /sucrose carrier sterility (23). Phloem unloading of sucrose is required for loss-of-function mutants and antisense-repression plants. In starch formation in storage tissues, such as the grains of cere- contrast to symplasmic unloading, this sucrose/H symporter- als or potato tubers. When the copy number of StSUT1, a based mechanism drives unloading of sucrose under the control ZmSUT1 orthologue expressed in the phloem of developing of both the sucrose and the pH gradients as well as the mem- tubers, is reduced by antisense repression, reduced fresh brane potential of the phloem and the surrounding tissues. weight accumulation during tuber development was observed (4, 5). Furthermore, indirect measurements with the proton- Acknowledgment—We thank Dr. N. Aoki for the generous supply of coupled monosaccharide transporter CkHUP1 from the green ZmSUT1 cDNA. alga Chlorella and the SGLT1 Na /glucose transporter from REFERENCES human and rabbit suggest that these sugar carriers from sin- 1. Mu¨ nch, E. 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Journal of Biological ChemistryAmerican Society for Biochemistry and Molecular Biology

Published: Jun 3, 2005

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