Unity and diversity in active transport in cell membrans

细胞膜主动运输的统一性和多样性

• 批准号: 09044310
• 负责人: TSUCHIYA Tomofusa
• 金额: $ 4.1万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044310/
• 关键词: Active transport; Structure and function; Unity and diversity; Symporter; Antiporter

It is important to analyze many transport systems for the investigation of unity and diversity in structure, function and mechanism in active transport proteins. Fortunately, we have found and investigated many active transport systems in bacterial cells so far. In the past two years, we have obtained the following results.1) We analyzed many NaィイD1+ィエD1-coupled transport systems, mainly NaィイD1+ィエD1/melibiose symporter and NaィイD1+ィエD1/serine symporter of Escherichia coli and some other bacteria. We characterized the symporters from biochemical view point and genetic view point. We established methods for over production, large-scale purification and reconstitution of the NaィイD1+ィエD1/melibiose symporter protein and NaィイD1+ィエD1/serine symporter protein. This opened up a way for further analysis in structure, function and mechanism an active transport proteins.2) We identified regions and amino acid residues important or necessary for ion-coupling or substrate recognition in the NaィイD1+ィエD1/melibiose symporter protein through mutant analysis and site-directed mutagenesis. Homology search and motif analysis were also useful for such analysis.3) We found new transport systems and cloned their genes in some bacteria. Biochemical and genetic analyses further revealed unity and diversity in transport proteins.Collaborations with Prof. Wilson and Prof. Konings were very valuable foe this project.

分析许多转运系统对于研究活性转运蛋白结构、功能和机制的统一性和多样性非常重要。幸运的是，迄今为止我们已经发现并研究了细菌细胞中的许多主动转运系统。近两年来，我们得到了以下成果：1)我们分析了许多NaィイD1+ィエD1耦合转运系统，主要是大肠杆菌和其他一些细菌的NaィイD1+ィエD1/蜜二糖同向转运蛋白和NaィイD1+ィエD1/丝氨酸同向转运蛋白。我们从生化角度和遗传学角度对同向转运蛋白进行了表征。我们建立了NaiiD1+iiD1/蜜二糖同向转运蛋白和NaiiD1+iiD1/丝氨酸同向转运蛋白的过量生产、大规模纯化和重建方法。这为进一步分析主动转运蛋白的结构、功能和机制开辟了道路。2)通过突变体分析和定点诱变，我们鉴定了NaiiD1+ィエD1/蜜二糖同向转运蛋白中离子偶联或底物识别重要或必需的区域和氨基酸残基。同源搜索和基序分析对于此类分析也很有用。3）我们发现了新的转运系统并在一些细菌中克隆了它们的基因。生化和遗传分析进一步揭示了转运蛋白的统一性和多样性。与Wilson教授和Konings教授的合作对于这个项目非常有价值。

项目成果

Noriko Okazaki: "Mutants of Citorobacter freundii that transport and utilize melibiose"Journal of Bacteriology. 180. 3480-3482 (1998)

Noriko Okazaki：“运输和利用蜜二糖的弗氏柠檬酸杆菌突变体”细菌学杂志。

Wakano Ogawa: "Cloning and expression of the gene for the na+-coupled serine transport from Escherichia coli and characteristics of the transport"Journal of Bacteriology. 180. 6749-6752 (1998)

若野小川：“大肠杆菌 na 偶联丝氨酸转运基因的克隆和表达以及转运的特征”细菌学杂志。

Hiroki Inoue: "pH-dependent growth retardation of Escherichia coli caused by overproduction of Na+/H+ antiporter"Biol. Pharm. Bull.. 21. 1128-1133 (1998)

Hiroki Inoue：“Na /H 逆向转运蛋白过量产生导致大肠杆菌 pH 依赖性生长迟缓”Biol。

Wakano Ogawa: "Isolation and characterization of an Escherichia coli mutant lacking the major serine transporter,and cloning of a serine transporter gene." J.Biochem.122. 1241-1245 (1997)

Wakano Okawa：“缺乏主要丝氨酸转运蛋白的大肠杆菌突变体的分离和表征，以及丝氨酸转运蛋白基因的克隆。”

Noriko Okazaki: "Sequence of a melibiose transporter gene of Enterobacter cloacae"Biochim. Biopys. Acta. 1354. 7-12 (1997)

Noriko Okazaki：“阴沟肠杆菌蜜二糖转运蛋白基因的序列”Biochim。