SUGAR TRANSPORTER OLIGOMERIC STRUCTURE AND FUNCTION

糖转运蛋白寡聚结构和功能

• 批准号: 2905478
• 负责人: ANTHONY CARRUTHERS
• 金额: $ 14.54万
• 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/2905478
• 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.

细胞营养和电解质的运输是一个基本的， 不完全表征的生物过程由两个广泛的 传输机制的分类--信道和载波。所有哺乳动物 细胞通过载体介导的转运机制转运糖， 葡萄糖单端口或葡萄糖转运。这个至关重要的细胞过程 为ATP合成提供糖，以维持还原电位 以及用于含糖大分子的生物合成， 糖蛋白、糖脂和核酸。糖在肌肉中的运输， 肝脏和血脑屏障也至关重要， 机体能量平衡这些组织的运输受损， 在包括糖尿病、糖原累积病和 血脑屏障障碍 葡萄糖转运涉及所有载体介导的 运输机制。因为它被大量表达，红色的血液 细胞和血脑屏障葡萄糖转运系统 生物物理、生物化学和分子分析。因此，该系统 不仅成为研究生物学重要性的原型， 糖的运输过程，也为了解更广泛的家庭 载体机制尽管进行了广泛的分析， 蛋白质介导的糖转运过程是未知的。我们的长期目标 是了解糖运输的分子机制。我们提出 在我们为实现这一目标而不断努力的过程中，我们的目标如下： 1)我们已经证明红细胞葡萄糖转运蛋白是一种 四个相同亚基的变构复合物-GLUT 1蛋白。这 结构存在于所有检测的表达GLUT 1的哺乳动物细胞中， 约会四聚体GLUT 1通过以下物质之间的非共价相互作用而稳定： 需要由亚基内启动的亚基折叠的亚基 二硫桥二硫键破坏导致转运蛋白解离 变成二聚体二聚体和四聚体GLUT 1的亚基似乎相互作用 通过N-末端结构域（GLUT 1残基1-199）。但正是这种 顺应性介导这些相互作用，以及这些相互作用如何导致 低聚化的可能性是未知的。我们概述了免疫共沉淀 使用嵌合转运蛋白的策略来广泛地定位这些结构域， 开始了解它们在GLUT 1寡聚化中的作用。 2)四聚体GLUT 1转运糖的速度比正常情况快15倍 二聚体GLUT 1，与二聚体GLUT 1不同，其特征在于 糖进口和出口之间的异向合作相互作用 网站.我们最近的研究表明，合作配体结合不是 糖的快速运输总是需要的，另一个， 亚基相互作用促进四聚体快速底物转运 GLUT 1。我们描述了糖转运和配体结合实验， 利用来自Aim 1的嵌合转运蛋白绘制所需的GLUT 1结构域 用于快速转运功能和/或协同配体结合。我们 从而确定这些结构域是独立的还是相同的 到寡聚化结构域。