Functional analysis of sugar transporter

糖转运蛋白的功能分析

• 批准号: 14580652
• 负责人: KASAHARA Michihiro
• 金额: $ 2.3万
• 依托单位: Teikyo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580652/
• 关键词: Glucose transporter; Chimera; Substrate recognition; Yeast; GLUT; Hxt2

The yeast Saccharomyces cerevisiae possesses 18 hexose transporters (Hxtl-17, Gal2). They contain 12 transmembrane segments (TMs), and belong to the major facilitator superfamily that is the major transport family for organic solutes. Chimeras were constructed by randomly replacing each of the L2TMs of Hxt2, a high affinity glucose transporter with the corresponding segment of Hxt1, a low affinity glucose transporter (TM shuffling). Characterization of these chimeras revealed that at least TMs 1, 5, 7 and 8 of Hxt2 are required for high-affinity glucose transport activity.To determine which amino acid residues in these 4 TMs are important for high-affinity glucose transport, we comprehensively shuffled all the 20 residues in these regions that differ between Hxt2 and Hxt1. Analysis of 60 independent chimera strains identified as expressing high-affinity and high-capacity glucose transport by the selection on glucose-limited agar plates revealed that Leu-201 in TM5 of Hxt2 is most important. Simulation by molecular dynamics indicated that Leu-201 is situated at the wall of aqueous pore constructed by several TMs including This 1, 5, 7, and 8.

酿酒酵母具有18个己糖转运蛋白（Hxtl-17, Gal2）。它们包含12个跨膜片段（TMs），属于主要助剂超家族，是有机溶质的主要运输家族。嵌合体的构建方法是将高亲和力葡萄糖转运蛋白Hxt2的每个L2TMs随机替换为低亲和力葡萄糖转运蛋白Hxt1的相应片段（TM洗牌）。这些嵌合体的表征表明，Hxt2的至少TMs 1、5、7和8是高亲和力葡萄糖转运活性所必需的。为了确定这4个TMs中哪些氨基酸残基对高亲和力葡萄糖转运起重要作用，我们对这些区域中Hxt2和Hxt1之间不同的所有20个残基进行了综合重组。通过对60株具有高亲和力和高容量葡萄糖转运的独立嵌合体菌株在葡萄糖限制琼脂板上的筛选分析发现，Hxt2的TM5中的Leu-201最为重要。分子动力学模拟表明，Leu-201位于由This 1、5、7、8等TMs组成的水孔壁上。

项目成果

Kasahara, T., Ishiguro,.M., Kasahara, M.: "Comprehensive chimeric analysis of amino acid residues critical for high-affinity glucose transport by Hxt2 of Saccharomyces cerevisiae"J.Biol.Chem.. (In press). (2004)

Kasahara, T.、Ishiguro,.M.、Kasahara, M.：“对酿酒酵母 Hxt2 高亲和力葡萄糖转运至关重要的氨基酸残基的综合嵌合分析”J.Biol.Chem..（出版中）。

Kasahara, T., Kasahara, M.: "Substrate recognition site of yeast high-affinity sugar transporter."Cell Struct.Funct.. 28. 357 (2003)

Kasahara, T., Kasahara, M.：“酵母高亲和力糖转运蛋白的底物识别位点。”Cell Struct.Funct.. 28. 357 (2003)

Kasahara, T., Maeda, M., Kasahara M.: "Comprehensive approach for the study of the substrate recognition sites in glucose transporters."生化学. 75. 762 (2003)

Kasahara, T.、Maeda, M.、Kasahara M.：“葡萄糖转运蛋白底物识别位点的综合研究方法”75. 762 (2003)。

Kasahara, T., Ishiguro, M., Kasahara, M.: "Comprehensive chimeric analysis of amino acid residues critical for high-aftinity glucose transport by Hxt2 of Saccharornyces cerevisiae"J. Biol. Chem.. (in press). (2004)

Kasahara, T.、Ishiguro, M.、Kasahara, M.：“对酿酒酵母 Hxt2 的高亲和力葡萄糖转运至关重要的氨基酸残基的综合嵌合分析”J。

Kasahara, T., Kasahara, M.: "Transmembrane segments 1, 5, 7 and 8 are required for high-affinity glucose transport by Saccharomyces cerevisiae Hxt2 transporter"Biochem. J.. 372. 247-252 (2003)

Kasahara, T.、Kasahara, M.：“酿酒酵母 Hxt2 转运蛋白的高亲和力葡萄糖转运需要跨膜片段 1、5、7 和 8”Biochem。