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BMC Biochem
2012 Nov 21;13:26. doi: 10.1186/1471-2091-13-26.
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Arg188 in rice sucrose transporter OsSUT1 is crucial for substrate transport.
Sun Y
,
Ward JM
.
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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.
Figure 1. A conserved Arg in OsSUT1 is crucial for sucrose-induced currents. (A) Arg188 in OsSUT1 is conserved in all three types of SUTs. (B) Substrate-induced currents in oocytes expressing OsSUT1 wild type or Arg188 mutants at pH 5.6. The currents were induced by 30 mM sucrose, β-phenyl-glucoside, or glucose, at −118 mV. Error bars indicate SE (n=3). (C) Oocyte expressing OsSUT1(R188K) shows block of inward leak currents by 30 mM sucrose. Currents were measured in Na ringer or K ringer (Na-free) at pH 5.6, and a holding potential of −40 mV. Results in C are from one oocyte that is representative of four experiments. Pipette potential was zeroed when bath solutions were changed.
Figure 2. Analysis of the function of OsSUT1 (R188K) in yeast and plants. (A) 14C-sucrose uptake by yeast SEY6210 expressing OsSUT1 wild type, OsSUT1(R188K) mutant, and the empty vector pDR196. Assays were performed at 30°C with 1 mM sucrose in Na phosphate buffer at pH 4.0 or pH 7.0 for 5 minutes. Data are presented as mean ± SE (n=3). (B) 30-day Arabidopsis plants, including the wild type control (SUC2/SUC2), suc2-5 homozygous (suc2/suc2), suc2 homozygous transformed with OsSUT1, and suc2 homozygous transformed with OsSUT1(R188K).
Figure 3. Analysis of corresponding mutations in type I and type III SUTs. (A) Substrate-induced currents in oocytes expressing type I sucrose transporter AtSUC1 wild type and AtSUC1(R163K). (B) LjSUT4 wild type, and LjSUT4(R169K). In A and B, currents were measured at −118 mV, pH 5.6, with a substrate concentration of 30 mM. Error bars indicate SE (n=3).
Figure 4. Currents induced by deoxy-fluoro derivatives of α-phenyl glucoside in oocytes expressing OsSUT1 wild type or OsSUT1(R188K). (A) Xenopus oocytes expressing OsSUT1 wild type were voltage clamped at -40mV in Na ringer solution at pH 5.6. Currents were recorded in response to application of the following substrates at 30 mM: α-phenyl glucoside (phenyl-glu), phenyl-3-deoxy-3-fluoro-α-glucoside (3deoxy-3F), phenyl-4-deoxy-4-fluoro-α-glucoside (4deoxy-4F), and sucrose (suc). Zero current is indicated (−0). (B) Currents recorded in oocytes expressing OsSUT1(R188K) in response to the same substrates and using the same conditions as in A.
Figure 5. Model for the function of Arg188 in OsSUT1. (A) Upper: known interaction between Arg144 of LacY and the galactose ring of lactose [38]. Lower: Suggested interaction between Arg188 of OsSUT1 and the glucose ring of sucrose. (B) Kinetic model for a SUT protein. C represents the SUT protein, S represents sucrose, and H is the proton. The dashed arrows between the two [CH] statuses indicate H+ flows across through SUTs without transporting sucrose. In the OsSUT1(R188K) mutant, sucrose binds to the transporter from the apoplastic side, but cannot be released to the cytoplastic side, keeping the mutated protein in the [CHS] apoplastic status (step 3).
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