Arg188 in rice sucrose transporter OsSUT1 is crucial for substrate transport
© Sun and Ward; licensee BioMed Central Ltd. 2012
Received: 15 June 2012
Accepted: 5 October 2012
Published: 21 November 2012
Plant sucrose uptake transporters (SUTs) are H+/sucrose symporters related to the major facilitator superfamily (MFS). SUTs are essential for plant growth but little is known about their transport mechanism. Recent work identified several conserved, charged amino acids within transmembrane spans (TMS) in SUTs that are essential for transport activity. Here we further evaluated the role of one of these positions, R188 in the fourth TMS of OsSUT1, a type II SUT.
The OsSUT1(R188K) mutant, studied by expression in plants, yeast, and Xenopus oocytes, did not transport sucrose but showed a H+ leak that was blocked by sucrose. The H+ leak was also blocked by β-phenyl glucoside which is not translocated by OsSUT1. Replacing the corresponding Arg in type I and type III SUTs, AtSUC1(R163K) and LjSUT4(R169K), respectively, also resulted in loss of sucrose transport activity. Fluorination at the glucosyl 3 and 4 positions of α-phenyl glucoside greatly decreased transport by wild type OsSUT1 but did not affect the ability to block H+ leak in the R188K mutant.
OsSUT1 R188 appears to be essential for sucrose translocation but not for substrate interaction that blocks H+ leak. Therefore, we propose that an additional binding site functions in the initial recognition of substrates. The corresponding Arg in type I and III SUTs are equally important. We propose that R188 interacts with glucosyl 3-OH and 4-OH during translocation.
KeywordsSucrose transporter Major facilitator superfamily Substrate binding Mutagenesis
Sucrose is an important product of photosynthesis, and is the main form of carbohydrate transported in the phloem in most higher plants . Sucrose transporters (SUTs or SUCs) are membrane proteins that facilitate the uptake of sucrose into the cytoplasm . Driven by the electrochemical H+ gradient across the membrane, SUT proteins transport both sucrose and H+ into the cytoplasm at a ratio of 1:1 [3–5]. Mutagenesis or antisense inhibition of SUT genes causes severe defects in plant growth [6–9]. For example, T-DNA insertions in the Arabidopsis AtSUC2 gene resulted in an excess of starch in source leaves, a lack of sucrose in sink tissues, and stunted plant growth .
According to phylogenetic analysis, plant SUTs can be grouped into three types [10, 11]. Type I SUTs are only found in eudicots, and are necessary for phloem loading [6, 8]. Type II transporters are present in all plants, and in monocots they are considered to function in phloem loading [9, 12]. Each plant species has at least one Type III SUT, which is localized in the vacuolar membrane of cells [13–15].
Despite the importance of SUTs in plants, the substrate binding sites and transport mechanism remain largely unknown [16, 17]. His65 in AtSUC1 was identified as the site of substrate-protectable modification by the inhibitor DEPC . Although AtSUC1(H65C) lost sucrose transport activity, H65K and H65R exhibited higher transport rates than the wild-type , indicating His at this position is not essential for transport function. Charged amino acids within transmembrane spans (TMS) were identified using a 3D structural model of type II rice sucrose transporter OsSUT1 and five of them were identified as essential for sucrose transport activity . Among the five amino acids, conservative mutations of Asp177, Arg188, or Asp331 resulted in complete loss of transport activity. In addition, alterations of Arg335 or Glu336 led to large decreases of the sucrose transport activity .
Prior to identification of the first SUT cDNA , substrate analogs were used as inhibitors to investigate sucrose transporter-substrate interactions using leaf discs , protoplasts from cotyledons , or plasma membrane vesicles . Hydroxyls of the glucose ring are thought to be directly involved in substrate binding, while the fructosyl region provides a hydrophobic surface that is also important for binding [21, 22]. Replacement of the glucosyl 4-OH or 3-OH with hydrogen or fluorine showed the most dramatic decrease in substrate recognition [21–23], indicating that the two hydroxyls interact with the SUT protein via hydrogen bonding . Hydrogen substitution or fluorine substitution of the 2-OH [21, 24] or 6-OH  also inhibited substrate transport.
Plant SUT proteins belong to the major facilitator superfamily (MFS), several members of which have been well studied [25–31]. MFS transporters share similar 3D structure [25, 26, 29, 31, 32], and operate via a “rocker-switch” mode [33, 34]. The most extensively investigated MFS protein is lactose permease of E. coli (LacY), which transports lactose and H+ into the cell at a ratio of 1:1 . Arg144 is one of the six irreplaceable amino acids of LacY; it is located in the middle of Helix V, facing the central cavity . A substitution of Arg144 for Lys results in complete loss of lactose transport activity . Arg188 of OsSUT1 has been suggested to function similarly; replacement of Arg188 by Lys results in complete loss of sucrose transport activity . In LacY, Arg144 forms a bifurcated hydrogen bond with 3-OH and 4-OH groups of the galactose moiety of lactose [26, 36–39]. Arg144 also interacts with Glu126 when substrates are absent, and with Glu269 during the substrate transport process [26, 39].
In this paper, the role of Arg188 in the function of type II sucrose transporter OsSUT1 was further explored. The effects of additional mutations on Arg188 in OsSUT1 were tested. Since Arg188 is conserved in all SUTs, we tested the effect of mutations at this position in type I and type III SUTs. The ability of OsSUT1 and OsSUT1(R188K) to rescue the dwarf phenotype of Arabidopsis atsuc2 mutants was also tested. Fluorine derivatives of α-phenyl glucoside were used to probe the roles of hydroxyl groups at the glucosyl 3 and 4 positions in substrate-protein interactions. Based on results from these experiments we propose a putative binding interaction between Arg188 of OsSUT1 and hydroxyl groups of sucrose. A role of Arg188 in the substrate transport process is also suggested.
Arg188 in OsSUT1 is required for transport activity
To further evaluate the role of Arg188 in OsSUT1, substitutions were made with His and Met (Figure 1B). Replacement of Arg with His retains the positive charge, while Met was selected since it has a long side chain similar in size to the side chain of Arg. Oocytes expressing R188H or R188M did not show detectable currents when sucrose was applied (Figure 1B). The upward deflection in currents was only observed for OsSUT1(R188K) and not for OsSUT1(R188H) or OsSUT1(R188M) mutants (Figure 1B). OsSUT1(R188K) localizes to the plasma membrane when expressed in oocytes . The localization of R188H and R188M were not determined in oocytes, therefore the lack of transport activity could be due to transporter inactivity but we cannot rule out protein instability, degradation, or lack of targeting to the plasma membrane.
OsSUT1(R188K) does not function in plants
Loss of function mutations in the Arabidopsis sucrose transporter AtSUC2 result in dwarf plants due to defects in carbohydrate transport in the vascular tissue [8, 41]. Heterozygous SUC2/suc2 plants do not have a visible phenotype and were transformed with either wild type OsSUT1 or OsSUT1(R188K). The constructs also contained the AtSUC2 native promoter and its 3’ UTR. Transformants with the suc2/suc2 background were identified by PCR. OsSUT1 reversed the growth defect of the suc2/suc2 plants, showing growth similar to wild-type SUC2/SUC2 plants (Figure 2B). On the contrary, the OsSUT1(R188K) failed to rescue the suc2/suc2 mutant (Figure 2B). The results indicate that Arg188 is necessary for transport activity in plants. Membrane localization of OsSUT1(R188K) in plants was not tested and therefore we cannot rule out the possibility that this mutant does not correctly localize to the plasma membrane.
Arg corresponding to OsSUT1 R188 is important in type I and III SUTs
The type III SUT LjSUT4 could transport sucrose and β-phenyl glucoside, but not glucose  (Figure 3B). Compared with wild-type LjSUT4, oocytes expressing LjSUT4(R169K) showed no sucrose-inducible inward currents, and no significant block of inward current by sucrose (Figure 3B). As the sucrose-induced inward current of LjSUT4 (−0.019 μA) was 26 times smaller than that of OsSUT1 (−0.491 μA), a block of an inward H+ leak in LjSUT4(R169K) may have been too small to measure. However, the results supported the conclusion that the conserved Arg was crucial for the substrate transport in Type I, II, and III SUTs.
Hydroxyls 3 and 4 in the glucose moiety of sucrose are crucial for substrate transport
For oocytes expressing OsSUT1(R188K), an upward deflection of current was observed in the presence of α-phenyl glucoside (Figure 4B). This indicated that α-phenyl glucoside interacted with the mutant transporter and blocked the H+ leak (Figure 4B). This interaction is likely to be an initial recognition step between potential substrates and SUT proteins, because it occurred between β-phenyl glucoside and OsSUT1(R188K) (Figure 1B). When phenyl-3-deoxy-3-fluoro-α-glucoside or phenyl-4-deoxy-4-fluoro-α-glucoside was applied to oocytes expressing OsSUT1(R188K), a decrease of inward current was again observed (Figure 4B). The two deoxy-fluoro analogs were transported at a lower rate than sucrose by wild-type OsSUT1 (Figure 4A). However, they had equal or better H+ blocking effect compared to sucrose in OsSUT1(R188K) mutant (Figure 4B). This indicated that the 3-OH and 4-OH were more important for the substrate transport step than for the initial substrate recognition step.
The conserved Arg188 of OsSUT1 was previously suggested to be an essential amino acid for substrate transport by SUTs . Here we show that substitution of Arg188 for Lys, His, or Met resulted in loss of substrate-inducible inward currents when expressed in oocytes (Figure 1B). Mutations of the corresponding Arg in Type I and Type III SUTs also showed a complete loss of substrate-inducible inward currents (Figure 3). When expressed in Arabidopsis under AtSUC2 native promoter, OsSUT1 reversed the dwarf phenotype of Arabidopsis atsuc2 mutant but OsSUT1(R188K) did not. These results further support the suggestion that Arg188 of OsSUT1 is essential for transport activity.
Putative binding interactions between Arg188 and sucrose
The sucrose-induced upward deflection in currents observed in oocytes expressing OsSUT1(R188K) (Figure 1B, 1C and 4B) is interesting because it shows that the mutant retains the ability to bind sucrose. However, as shown by expression in yeast and 14C-sucrose uptake experiments, OsSUT1(R188K) does not transport sucrose across the membrane . Lack of 14C-sucrose uptake activity at pH 4.0 or 7.0 supports the idea that OsSUT1(R188K) has a H+ leak that is blocked by sucrose rather than sucrose/H+ antiport activity.
There is evidence that substrate binding involved in blocking the H+ leak is different than substrate binding required for glucoside translocation. First, the mutant OsSUT1(R188K) retains substrate binding but does not translocate substrates. Second, β-phenyl glucoside blocks the H+ leak but is not translocated by wild-type OsSUT1. Third, β-paranitrophenyl glucoside inhibits sucrose transport activity of type II SUT from barley, HvSUT1, but is not a translocated substrate . There is also evidence that a H+ leak in the absence of substrates occurs in wild-type sucrose transporters  but at a lower rate. For example, sucralose acts as a competitive inhibitor of a type II SUT from sugarcane, ShSUT1, but sucralose application alone does not cause an upward shift in currents in ShSUT1-expressing oocytes . We hypothesize that substrate binding that blocks H+ leak is preliminary to substrate binding required for translocation.
Electrophysiological assays using deoxyl-fluoro derivatives showed that the 3-OH and 4-OH of substrates were more important for the substrate transport process than the initial substrate recognition step (Figure 4). Similarly, the interaction between potential substrates and OsSUT1(R188K) suggested that Arg188 of OsSUT1 was more important for substrate transport than for initial substrate recognition. Therefore, it is reasonable to hypothesize that Arg188 of OsSUT1 interacts with 3-OH and 4-OH of the substrate during the transport process.
Arg188 in OsSUT1 appears to have a similar function as Arg144 in LacY. Arg144 of LacY is the only positively charged key amino acid in the N-terminal half of the transporter [26, 27] and it is located in the middle of Helix V, one of the helixes facing the central cavity [26, 39]. The LacY(R144K) mutant has no lactose transport activity , demonstrating that both guanidine groups -NH2 are crucial. Similarly, Arg188 of OsSUT1 is the only positively charged key amino acid identified in the N-terminal half of the transporter . In OsSUT1, Arg188 is predicted to be located in the middle of Helix IV that surrounds the central transport pathway . The OsSUT1(R188K) mutant does not transport sucrose  (Figure 1B, 2, 4B), indicating that both -NH2 of this Arginine are essential.
OsSUT1(R188K) in the sucrose transport process
All previously proposed transport mechanisms for SUTs are in agreement that on the cytoplasmic side of the membrane, sucrose leaves the SUT protein before release of the proton [4, 17, 45]. However, one model involves a sequential loading of the transporter on the extracellular side with proton binding followed by sucrose [4, 17]. Other results support a random binding model in which either sucrose or a proton can bind first on the extracellular side . Our results support an ordered binding of protons followed by sucrose (Figure 5B). In this model, the transporter binds a proton on the extracellular side of the membrane and this facilitates sucrose binding (Figure 5A, stages 1–3). The fully loaded SUT transporter has a conformational change, termed the “rocker-switch” , to face the cytoplasmic side (Figure 5B, stage 3–4). Sucrose and proton are then released (Figure 5B, stage 4–6). The empty carrier then flips back, returning its substrate binding sites to the apoplastic side (Figure 5B, stage 6–1). Some protons bound at the apoplast of a SUT could be released directly to cytoplasm (Figure 5, stage 2–5), bypassing the sucrose-binding process. The un-coupled transport of H+ has been observed in wild-type StSUT1, but the rate was lower than the H+-coupled transport of sucrose .
The OsSUT1(R188K) mutant has a larger uncoupled H+ leak, compared to wild-type OsSUT1, that is blocked by substrate (Figure 4B). However R188K does not transport sucrose. Therefore, it is likely that R188K is blocked just prior to the “rocker switch” step in the transport cycle (Figure 5B, stage 3). Substrate binding inhibits uncoupled transport (Figure 5B, stage 2–5), most likely by progressing the transporter to stage 3 (Figure 5B) where the cycle is blocked. This explanation supports the concept that Arg188 of OsSUT1 is essential for the transport of substrates across the membrane at the “rocker-switch” step (Figure 5B, stage 3–4).
R188 in OsSUT1 was identified as a charged amino acid within the fourth TMS that is important for transport activity . The R188K mutation in OsSUT1 results in a lack of sucrose transport but when assayed by voltage clamping in oocytes, an upward deflection in current occurs when sucrose is applied. The previous suggestion that this represents a substrate-induced block of H+ current  appears to be correct. The inward leak though R188K is independent of Na+ (Figure 1C) and R188K does not appear to function as an antiporter (Figure 2A). We also show that while OsSUT1 is functional in the phloem when expressed in Arabidopsis atsuc2-5, the R188K mutant is not. This amino acid position is also conserved in type I and type III SUTs (Figure 1A) and corresponding mutations in Arabidopsis AtSUC1 and Lotus japonicus LjSUT4 resulted in a loss of transport activity (Figure 3). Deoxy-fluoro derivatives of α-phenyl glucoside were used to investigate substrate binding by OsSUT1. Modification at the glucosyl 3-OH and 4-OH positions significantly reduced transport by wild type OsSUT1. However, substrate-induced leak current block observed in the OsSUT1(R188K) mutant was not affected. Based on these results, we propose 1) that in OsSUT1, R188 is involved in substrate translocation and 2) an additional substrate binding site, independent of R188, functions in initial substrate recognition and block of H+ leak through the R188K mutant.
Constructs for oocyte experiments
Mutagenesis of OsSUT1 in pCR8/GW was performed using the QuikChange II site-directed mutagenesis kit (Stratagene). PCR reactions included dimethyl sulfoxide (DMSO) at a final concentration of 8% to inhibit the formation of secondary structure. The pCR8/GW-OsSUT1(R188H) or pCR8/GW-OsSUT1(R188M) were recombined with oocyte vector pOO2/GW. AtSUC1(R163K) and LjSUT4(R169K) mutants were made using AtSUC1 and LjSUT4 constructs in the POO2/GW vector using the QuikChange II site-directed mutagenesis kit without the addition of DMSO. All sequences were confirmed. cRNAs were prepared using the SP6 mMessage mMachine kit (Ambion). Oocyte preparation and two-electrode voltage clamp recordings (TEVC) were the same as previously described . Oocytes were bathed in modified Na ringer solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 1 mM NaHCO3, 10 mM MgCl2, 10 mM MES-Tris, pH 5.6) or K ringer (115 mM KCl, 1.8 mM CaCl2, 1 mM NaHCO3, 10 mM MgCl2, 10 mM MES-Tris, pH 5.6).
Yeast (Saccharomyces cerevisiae) strain SEY6210 (Matα ura3-52 leu2-3, 112 his3-Δ200trp1-Δ901 lys2-801 suc2-Δ9)  transformed with pDR196/GW-OsSUT1, pDR196/GW-OsSUT1(R188K), or empty vector pDR196/GW was used for 14C-sucrose uptake experiments as previously described . Uptake assays were done at pH 4.0 or 7.0, and SEY6210 cells were incubated in 1 mM sucrose for 5 minutes at 30°C. They were then washed three times using ice-cold 10 mM sucrose, and radioactivity was counted.
Arabidopsis atsuc2 complementation
Constructs for plant transformation were made by assembling the AtSUC2 (At1g22710) promoter in pDONR P4-P1R , the ORF of OsSUT1 or OsSUT1(R188K) in pCR8/GW , and the 3’ UTR of AtSUC2 in pDONR P2R-P3  into the pB7m34/GW binary vector . LR Clonase Plus (Invitrogen) was used for this directional three-fragment recombination. Agrobacterium tumefaciens strain C58C1 containing the constructs was used to transform Arabidopsis atsuc2-5 heterozygous plants (SALK_087046) . Basta-resistant transformants were selected on soil, and homozygous atsuc2-5 lines were identified via PCR.
Phenyl-3-deoxy-3-fluoro-α-D-glucoside and phenyl-4-deoxy-4-fluoro-α-D-glucoside were supplied by Carbosynth Limited (Berkshire, UK).
This work was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy grant DE-FG02-10ER15886 and a Doctoral Dissertation Fellowship from Plant Biological Science program at the University of Minnesota (to YS).
- Hayashi H, Chino M: Chemical composition of phloem sap from the uppermost internode of the rice plant. Plant Cell Physiol. 1990, 31: 247-251.Google Scholar
- Ayre BG: Membrane-transport systems for sucrose in relation to whole-plant carbon partitioning. Mol Plant. 2011, 4: 377-394. 10.1093/mp/ssr014.PubMedView ArticleGoogle Scholar
- Bush DR: Electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport. Plant Physiol. 1990, 93: 1590-1596. 10.1104/pp.93.4.1590.PubMedPubMed CentralView ArticleGoogle Scholar
- Boorer KJ, Loo DD, Frommer WB, Wright EM: Transport mechanism of the cloned potato H+/sucrose cotransporter StSUT1. J Biol Chem. 1996, 271: 25139-25144. 10.1074/jbc.271.41.25139.PubMedView ArticleGoogle Scholar
- Carpaneto A, Geiger D, Bamberg E, Sauer N, Fromm J, Hedrich R: Phloem-localized, proton-coupled sucrose carrier ZmSUT1 mediates sucrose efflux under the control of the sucrose gradient and the proton motive force. J Biol Chem. 2005, 280: 21437-21443. 10.1074/jbc.M501785200.PubMedView ArticleGoogle Scholar
- Riesmeier JW, Willmitzer L, Frommer WB: Evidence for an essential role of the sucrose transporter in phloem loading and assimilate partitioning. EMBO J. 1994, 13: 1-7.PubMedPubMed CentralGoogle Scholar
- Burkle L, Hibberd JM, Quick WP, Kuhn C, Hirner B, Frommer WB: The H+−sucrose cotransporter NtSUT1 is essential for sugar export from tobacco leaves. Plant Physiol. 1998, 118: 59-68. 10.1104/pp.118.1.59.PubMedPubMed CentralView ArticleGoogle Scholar
- Gottwald JR, Krysan PJ, Young JC, Evert RF, Sussman MR: Genetic evidence for the in planta role of phloem-specific plasma membrane sucrose transporters. Proc Natl Acad Sci U S A. 2000, 97: 13979-13984. 10.1073/pnas.250473797.PubMedPubMed CentralView ArticleGoogle Scholar
- Slewinski TL, Meeley R, Braun DM: Sucrose transporter1 functions in phloem loading in maize leaves. J Exp Bot. 2009, 60: 881-892. 10.1093/jxb/ern335.PubMedPubMed CentralView ArticleGoogle Scholar
- Aoki N, Hirose T, Scofield GN, Whitfeld PR, Furbank RT: The sucrose transporter gene family in rice. Plant Cell Physiol. 2003, 44: 223-232. 10.1093/pcp/pcg030.PubMedView ArticleGoogle Scholar
- Reinders A, Sivitz AB, Ward JM: Evolution of plant sucrose uptake transporters (SUTs). Front Plant Sci. 2012, 3: 00022-View ArticleGoogle Scholar
- Scofield GN, Hirose T, Aoki N, Furbank RT: Involvement of the sucrose transporter, OsSUT1, in the long-distance pathway for assimilate transport in rice. J Exp Bot. 2007, 58: 3155-3169. 10.1093/jxb/erm153.PubMedView ArticleGoogle Scholar
- Eom JS, Cho JI, Reinders A, Lee SW, Yoo Y, Tuan PQ, Choi SB, Bang G, Park YI, Cho MH: Impaired function of the tonoplast-localized sucrose transporter in rice, OsSUT2, limits the transport of vacuolar reserve sucrose and affects plant growth. Plant Physiol. 2011, 157: 109-119. 10.1104/pp.111.176982.PubMedPubMed CentralView ArticleGoogle Scholar
- Endler A, Meyer S, Schelbert S, Schneider T, Weschke W, Peters SW, Keller F, Baginsky S, Martinoia E, Schmidt UG: Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach. Plant Physiol. 2006, 141: 196-207. 10.1104/pp.106.079533.PubMedPubMed CentralView ArticleGoogle Scholar
- Reinders A, Sivitz AB, Starker CG, Gantt JS, Ward JM: Functional analysis of LjSUT4, a vacuolar sucrose transporter from Lotus japonicus. Plant Mol Biol. 2008, 68: 289-299. 10.1007/s11103-008-9370-0.PubMedView ArticleGoogle Scholar
- Lemoine R: Sucrose transporters in plants: update on function and structure. Biochim Biophys Acta. 2000, 1465: 246-262. 10.1016/S0005-2736(00)00142-5.PubMedView ArticleGoogle Scholar
- Geiger D: Plant sucrose transporters from a biophysical point of view. Mol Plant. 2011, 4: 395-406. 10.1093/mp/ssr029.PubMedView ArticleGoogle Scholar
- Lu JM, Bush DR: His-65 in the proton-sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity. Proc Natl Acad Sci U S A. 1998, 95: 9025-9030. 10.1073/pnas.95.15.9025.PubMedPubMed CentralView ArticleGoogle Scholar
- Sun Y, Lin Z, Reinders A, Ward JM: Functionally important amino acids in rice sucrose transporter OsSUT1. Biochemistry. 2012, 51: 3284-3291. 10.1021/bi201934h.PubMedView ArticleGoogle Scholar
- Riesmeier JW, Willmitzer L, Frommer WB: Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast. EMBO J. 1992, 11: 4705-4713.PubMedPubMed CentralGoogle Scholar
- Delrot S, Roques N, Descotes G, Mentech J: Recognition of some deoxy-derivatives of sucrose by the sucrose transporter of the plasma membrane. Plant Physiol Biochem. 1991, 29: 25-29.Google Scholar
- Hitz WD, Card PJ, Ripp KG: Substrate recognition by a sucrose transporting protein. J Biol Chem. 1986, 261: 11986-11991.PubMedGoogle Scholar
- Hecht R, Slone JH, Buckhout TJ, Hitz WD, Vanderwoude WJ: Substrate specificity of the H+−sucrose symporter on the plasma membrane of sugar beets (Beta vulgaris L.): transport of phenylglucopyranosides. Plant Physiol. 1992, 99: 439-444. 10.1104/pp.99.2.439.PubMedPubMed CentralView ArticleGoogle Scholar
- Griffin SD, Buxton KD, Donaldson IA: The alpha-D-glucosyl C-2 hydroxyl is required for binding to the H(+)-sucrose transporter in phloem. Biochim Biophys Acta. 1993, 1152: 61-68. 10.1016/0005-2736(93)90231-N.PubMedView ArticleGoogle Scholar
- Hirai T, Heymann JA, Shi D, Sarker R, Maloney PC, Subramaniam S: Three-dimensional structure of a bacterial oxalate transporter. Nat Struct Biol. 2002, 9: 597-600.PubMedGoogle Scholar
- Abramson J, Smirnova I, Kasho V, Verner G, Kaback HR, Iwata S: Structure and mechanism of the lactose permease of Escherichia coli. Science. 2003, 301: 610-615. 10.1126/science.1088196.PubMedView ArticleGoogle Scholar
- Guan L, Mirza O, Verner G, Iwata S, Kaback HR: Structural determination of wild-type lactose permease. Proc Natl Acad Sci U S A. 2007, 104: 15294-15298. 10.1073/pnas.0707688104.PubMedPubMed CentralView ArticleGoogle Scholar
- Law CJ, Almqvist J, Bernstein A, Goetz RM, Huang Y, Soudant C, Laaksonen A, Hovmoller S, Wang DN: Salt-bridge dynamics control substrate-induced conformational change in the membrane transporter GlpT. J Mol Biol. 2008, 378: 828-839. 10.1016/j.jmb.2008.03.029.PubMedView ArticleGoogle Scholar
- Dang S, Sun L, Huang Y, Lu F, Liu Y, Gong H, Wang J, Yan N: Structure of a fucose transporter in an outward-open conformation. Nature. 2010, 467: 734-738. 10.1038/nature09406.PubMedView ArticleGoogle Scholar
- Frillingos S, Sahin-Toth M, Wu J, Kaback HR: Cys-scanning mutagenesis: a novel approach to structure function relationships in polytopic membrane proteins. FASEB J. 1998, 12: 1281-1299.PubMedGoogle Scholar
- Huang Y, Lemieux MJ, Song J, Auer M, Wang DN: Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science. 2003, 301: 616-620. 10.1126/science.1087619.PubMedView ArticleGoogle Scholar
- Yin Y, He X, Szewczyk P, Nguyen T, Chang G: Structure of the multidrug transporter EmrD from Escherichia coli. Science. 2006, 312: 741-744. 10.1126/science.1125629.PubMedPubMed CentralView ArticleGoogle Scholar
- Locher KP, Bass RB, Rees DC: Structural biology. Breaching the barrier. Science. 2003, 301: 603-604. 10.1126/science.1088621.PubMedView ArticleGoogle Scholar
- Karpowich NK, Wang DN: Structural biology. Symmetric transporters for asymmetric transport. Science. 2008, 321: 781-782. 10.1126/science.1161495.PubMedPubMed CentralView ArticleGoogle Scholar
- West IC, Mitchell P: Stoicheiometry of lactose-H+ symport across the plasma membrane of Escherichia coli. Biochem J. 1973, 132: 587-592.PubMedPubMed CentralView ArticleGoogle Scholar
- Frillingos S, Gonzalez A, Kaback HR: Cysteine-scanning mutagenesis of helix IV and the adjoining loops in the lactose permease of Escherichia coli: Glu126 and Arg144 are essential. Biochemistry. 1997, 36: 14284-14290. 10.1021/bi972314d.PubMedView ArticleGoogle Scholar
- Sahin-Toth M, Frillingos S, Lawrence MC, Kaback HR: The sucrose permease of Escherichia coli: functional significance of cysteine residues and properties of a cysteine-less transporter. Biochemistry. 2000, 39: 6164-6169. 10.1021/bi000124o.PubMedView ArticleGoogle Scholar
- Sahin-Toth M, Lawrence MC, Nishio T, Kaback HR: The C-4 hydroxyl group of galactopyranosides is the major determinant for ligand recognition by the lactose permease of Escherichia coli. Biochemistry. 2001, 40: 13015-13019. 10.1021/bi011233l.PubMedView ArticleGoogle Scholar
- Mirza O, Guan L, Verner G, Iwata S, Kaback HR: Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY. EMBO J. 2006, 25: 1177-1183. 10.1038/sj.emboj.7601028.PubMedPubMed CentralView ArticleGoogle Scholar
- Sun Y, Reinders A, LaFleur KR, Mori T, Ward JM: Transport activity of rice sucrose transporters OsSUT1 and OsSUT5. Plant Cell Physiol. 2010, 51: 114-122. 10.1093/pcp/pcp172.PubMedPubMed CentralView ArticleGoogle Scholar
- Srivastava AC, Ganesan S, Ismail IO, Ayre BG: Functional characterization of the Arabidopsis AtSUC2 Sucrose/H+ symporter by tissue-specific complementation reveals an essential role in phloem loading but not in long-distance transport. Plant Physiol. 2008, 148: 200-211. 10.1104/pp.108.124776.PubMedPubMed CentralView ArticleGoogle Scholar
- Dunitz JD, Taylor R: Organic fluorine hardly ever accepts hydrogen bonds. Chem Eur J. 1997, 3: 89-98. 10.1002/chem.19970030115.View ArticleGoogle Scholar
- Sivitz AB, Reinders A, Ward JM: Analysis of the transport activity of barley sucrose transporter HvSUT1. Plant Cell Physiol. 2005, 46: 1666-1673. 10.1093/pcp/pci182.PubMedView ArticleGoogle Scholar
- Reinders A, Sivitz AB, Hsi A, Grof CP, Perroux JM, Ward JM: Sugarcane ShSUT1: analysis of sucrose transport activity and inhibition by sucralose. Plant Cell Environ. 2006, 29: 1871-1880. 10.1111/j.1365-3040.2006.01563.x.PubMedView ArticleGoogle Scholar
- Zhou J, Theodoulou F, Sauer N, Sanders D, Miller AJ: A kinetic model with ordered cytoplasmic dissociation for SUC1, an Arabidopsis H+/sucrose cotransporter expressed in Xenopus oocytes. J Membr Biol. 1997, 159: 113-125. 10.1007/s002329900275.PubMedView ArticleGoogle Scholar
- Robinson JS, Klionsky DJ, Banta LM, Emr SD: Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol. 1988, 8: 4936-4948.PubMedPubMed CentralView ArticleGoogle Scholar
- Karimi M, De Meyer B, Hilson P: Modular cloning in plant cells. Trends Plant Sci. 2005, 10: 103-105. 10.1016/j.tplants.2005.01.008.PubMedView ArticleGoogle Scholar
- Wippel K, Sauer N: Arabidopsis SUC1 loads the phloem in suc2 mutants when expressed from the SUC2 promoter. J Exp Bot. 2012, 63: 669-679. 10.1093/jxb/err255.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.