SUGAR TRANSPORTER OLIGOMERIC STRUCTURE AND FUNCTION

糖转运蛋白寡聚结构和功能

• 批准号: 2430197
• 负责人: ANTHONY CARRUTHERS
• 金额: $ 13.46万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2430197
• 关键词: CHO cells; allosteric site; binding proteins; blood brain barrier; chemical association; chimeric proteins; complementary DNA; computer assisted sequence analysis; conformation; dimer; disulfide bond; erythrocytes; glucose transport; high performance liquid chromatography; human genetic material tag; immunoprecipitation; intermolecular interaction; ligands; membrane transport proteins; protein purification; protein structure function; transfection; western blottings

Cellular nutrient and electrolyte transport is a fundamental but incompletely characterized biological process mediated by two broad classes of transport mechanism - channels and carriers. All mammalian cells transport sugars by a carrier-mediated transport mechanism called glucose uniport or glucose transport. This crucial cellular process provides sugars for ATP synthesis, for maintenance of reducing potential and for the biosynthesis of sugar-containing macromolecules such as glycoproteins, glycolipids and nucleic acids. Sugar transport in muscle, liver and the blood brain barrier are also critically important to organismal energy homeostasis. Impaired transport by these tissues is manifest in diseases that include diabetes, glycogen storage diseases and blood brain barrier disorders. Glucose transport involves catalytic steps common to all carrier-mediated transport mechanisms. Because it is abundantly expressed, the red blood cell and blood brain barrier glucose transport system is amenable to biophysical, biochemical and molecular analysis. This system has thus become a prototype not only for studies of the biologically important sugar transport process but also for understanding the wider family of carrier mechanisms. Despite extensive analysis, the structural basis of protein mediated sugar transport process is unknown. Our long term goal is to understand the molecular mechanism of sugar transport. We propose the following aims in our continuing efforts towards this goal: 1) We have demonstrated that the erythrocyte glucose transporter is an allosteric complex of four identical subunits - the GLUT1 protein. This structure is present in all GLUT1-expressing mammalian cells examined to date. Tetrameric GLUT1 is stabilized by non-covalent interactions between subunits which require a subunit-fold promoted by an intra-subunit disulfide bridge. Disulfide disruption causes transporter dissociation into dimers. Subunits of dimeric and tetrameric GLUT1 appear to interact through N-terminal domains (GLUT1 residues 1-199). Precisely what subsequences mediate these interactions and how these interactions lead to oligomerization are unknown. We outline a co-immunoprecipitation strategy using chimeric transporters to broadly map these domains and to begin to understand their role in GLUT1 oligomerization. 2) Tetrameric GLUT1 transports sugars 15-fold more rapidly than does dimeric GLUT1 and, unlike dimeric GLUT1, is characterized by heterotropic, cooperative interactions between sugar import and export sites. Our recent studies show that cooperative ligand binding is not always required for rapid sugar transport and that other, uncharacterized subunit interactions promote rapid substrate translocation by tetrameric GLUT1. We describe sugar transport and ligand binding experiments that exploit chimeric transporters from Aim 1 to map GLUT1 domains required for rapid transport function and/or cooperative ligand binding. We thereby determine whether these domains are separate from or identical to oligomerization domains.

细胞的营养和电解质运输是基本的，但 不完全刻画的生物过程由两个广泛的 运输机制的类别--渠道和载体。所有哺乳动物 细胞通过载体介导的糖类转运机制 葡萄糖单口或葡萄糖转运。这一关键的细胞过程 为ATP合成提供糖，以维持还原电位 以及含糖大分子的生物合成，如 糖蛋白、糖脂和核酸。糖在肌肉中的运输， 肝脏和血脑屏障对 生物能量动态平衡。这些组织造成的运输障碍是 表现为糖尿病、糖原贮积症和 血脑屏障障碍。 葡萄糖转运涉及所有载体介导的共同的催化步骤 传输机制。因为它被丰富地表达，红色的血液 细胞和血脑屏障葡萄糖转运系统易受 生物物理、生化和分子分析。这一系统因此 成为一个原型不仅对研究生物具有重要意义 糖的运输过程也是为了了解更广泛的家族 载体机构。尽管进行了广泛的分析，但 蛋白质介导的糖转运过程尚不清楚。我们的长期目标 是为了了解糖运输的分子机制。我们建议 以下是我们为实现这一目标而继续努力的目标： 1)我们已经证明了红细胞葡萄糖转运蛋白是一种 四个相同亚基的变构复合体--GLUT1蛋白。这 结构存在于所有表达GLUT1的哺乳动物细胞中 约会。四聚体GLUT1通过非共价相互作用稳定 需要亚基折叠的亚基-由亚基内的亚基启动 二硫化桥。硫化物破坏导致转运体解离 变成二聚体。二聚体和四聚体GLUT1的亚基似乎相互作用 通过N-末端结构域(GLUT1残基1-199)。到底是什么 子序列调节这些交互以及这些交互如何导致 对齐聚反应的影响是未知的。我们概述了一种免疫共沉淀 使用嵌合转运蛋白广泛映射这些结构域的策略 开始了解它们在GLUT1齐聚中的作用。 2)四聚体GLUT1转运糖分的速度是 二聚体GLUT1，与二聚体GLUT1不同，其特征是 食糖进出口之间的异质性、协作性互动 网站。我们最近的研究表明，合作配体结合不是 总是需要快速的糖运输，而另一个，没有特征的 亚基相互作用通过四聚体促进底物快速转运 Glut1.我们描述了糖转运和配体结合实验 利用来自Aim 1的嵌合转运蛋白来映射所需的GLUT1结构域 用于快速转运功能和/或协同配基结合。我们 从而确定这些域是独立的还是相同的 到齐聚结构域。