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MFS transporter : Analysis of transport mechanism by correlation of gene and structure

MFS转运蛋白：通过基因和结构的相关性分析转运机制

基本信息

批准号：
16055201
负责人：
KASAHARA Michihiro
金额：
$ 15.49万
依托单位：
Teikyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16055201/
关键词：
Membrane transport Transporter Substrate recognition Chimera Sugar transport glucose galactose malabsorption Sorbitol transport glucose transporter MFS 酵母

项目摘要

1) Substrate recognition of MFS transporterTo 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 transportersThree 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 malabsorptionWe 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.

1)MFS转运蛋白的底物识别为了研究MFS转运蛋白中底物识别的分子机制，我们采用了一种综合的方法，使用酵母高亲和力葡萄糖转运蛋白Hxt 2和其低亲和力蛋白Hxt 1之间的嵌合体。首先，通过在Hxt 2和Hxt 1之间改组12个跨膜片段（TM），发现TM 1、5、7和8是高亲和力转运所必需的。作为下一步，这些TM中的20个不同的氨基酸残基同时改组。饱和诱变结果表明，Leu-59，Leu-61，Leu-201，Asn-331，Phe-366是重要的，Phe-108，Ala-363和瓦尔-316或Ala-368支持最大转运。它们位于转运蛋白的胞质边缘，除了Asn-331似乎位于中心孔，这是基于E. coil MFS转运蛋白GlpT.2）苹果山梨醇转运蛋白的鉴定了苹果主要光合产物山梨醇的三种转运蛋白（MdSOT 3、MdSOT 4和MdSOT 5）。当它们在酵母中异源表达时，它们是MFS转运蛋白并且具有转运活性。MdSOT 3和MdSOT 4表现出与木糖醇和肌醇的竞争，它们的表观Km分别为0.71 mM（MdSOT 3）和3.2 mM（MdSOT 5）。质子载体CCCP强烈抑制山梨醇通过MdSOT 3和MdSOT 5的转运，表明1-1+依赖的主动转运。3）Na+/葡萄糖协同转运蛋白SGLT 1的突变导致葡萄糖/半乳糖吸收不良我们已经证实了第一例土耳其葡萄糖/半乳糖吸收不良患者具有纯合的Thr 460 Pro突变。用爪蟾卵母细胞进行的异源表达实验表明，突变的SGLT 1是无活性的，并被携带到质膜上。该突变体转运蛋白为研究转运蛋白功能的分子机制提供了重要依据。

项目成果

期刊论文数量（28）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Identification of sorbitol transporters expressed in the phloem of apple source leaves.

DOI：
10.1093/pcp/pch121
发表时间：
2004-08
期刊：
Plant & cell physiology
影响因子：
4.9
作者：
Junya Watari;Yoshihiro Kobae;S. Yamaki;K. Yamada;K. Toyofuku;T. Tabuchi;K. Shiratake
通讯作者：
Junya Watari;Yoshihiro Kobae;S. Yamaki;K. Yamada;K. Toyofuku;T. Tabuchi;K. Shiratake

Microsatellite polymorphism in Japanese mongrel dogs.

日本杂种狗的微卫星多态性。