| Budget Amount *help |
¥24,200,000 (Direct Cost: ¥24,200,000)
Fiscal Year 2005: ¥11,600,000 (Direct Cost: ¥11,600,000)
Fiscal Year 2004: ¥12,600,000 (Direct Cost: ¥12,600,000)
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| Research Abstract |
1) Substrate recognition of MFS transporter
To study the molecular mechanism of substrate recognition in MFS transporters, we have adopted a comprehensive approach using chimeras between yeast high-affinity glucose transporter Hxt2 and its low-affinity paralog Hxtl. First, by shuffling 12 transmembrane segments (TMs) between Hxt2 and Hxtl, TMs 1, 5, 7 and 8 were found necessary for high-affinity transport. As the next step, 20 different amino acid residues in these TMs were simultaneously shuffled. Saturation mutagenesis revealed eight residues, Leu-59, Leu-61, Leu-201, Asn-331, Phe-366 were found important and Phe-108, Ala-363 and Val-316 or Ala-368 supported a maximal transport. They were located at the cytoplasmic edge of the transporter, except for Asn-331 that seems to located at the central pore as estimated by a homology model based on a E. coil MFS transporter GlpT.
2) Identification of apple sorbitol transporters
Three transporters for sorbitol, a major photosynthetic product of apple was identified (MdSOT3, MdSOT4 and MdSOT5). They were MFS transporters and active in transport when they were heterologously expressed in yeast. MdSOT3 and MdS0T4 showed competition by xylitol and myo-inositol and their apparent Kms were 0.71 mM (MdSOT3) and 3.2 mM (MdSOT5). A protonophore CCCP strongly inhibited sorbitol transport via MdSOT3 and MdSOT5, indicating 1-1+-dependent active transport.
3) Mutation in Na+/glucose cotransporter SGLT1 that caused glucose/galactose malabsorption
We have confirmed the 1st case of Turkish patient with glucose/galactose malabsorption possessed homozygous Thr460Pro mutation. Heterologous expression expreriments using Xenopus oocytes indicated the mutated SGLT1 was inactive and carried to plasma membrane. This mutant transporter is useful for the study for the molecular mechanism of transport function.
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