GLUCOSE TRANSPORTER STRUCTURE AND FUNCTION

描述（由申请人提供）：蛋白质介导的促进葡萄糖转运对于细胞代谢、翻译后蛋白质修饰、细胞抗氧化应激和生物体碳水化合物稳态至关重要。尽管有广泛的研究，葡萄糖转运的机制是未知的。我们的目标是了解蛋白质介导的葡萄糖转运的分子机制。为了实现这一目标，我们建议研究人类葡萄糖转运蛋白GluT 1的结构和功能。该转运蛋白独特地适合于研究，可作为来自红细胞的纯化蛋白、作为来自Cos-7细胞的重组蛋白获得，并且适合于通过在葡萄糖转运无效酵母中表达进行功能筛选。具体的目标1和2集中在结构决定因素和功能的结果，GluT 1寡聚化在细胞膜上。具体目标3和4研究转运蛋白动力学和催化作用。特异性目的1检验GluT 1寡聚化需要GluT 1特异性序列的假设。在过去的研究周期中，我们发现细胞膜GluT 1是一种同源四聚体，由GluT 1特异性寡聚化序列稳定，并被一些去污剂保存，但被其他去污剂破坏。我们现在直接挑战这一假设，通过GluT 1结构域交换和诱变实验，询问转运蛋白寡聚体的结构和功能是否被保留。具体目标2测试的假设，GluT 1寡聚化是必要的，但不足以葡萄糖转运协同性。一些（但不是全部）GluT 1配体促进GluT 1亚基之间的协同性。我们已经映射配体立体化学促进协同的配体，但还没有谷氨酸1。我们使用特定目标1的结构来询问GluT 1寡聚化是否是转运体协同性所必需的，并详细检查GluT 1配体结合位点对协同性的要求。具体目标3测试的假设，即特定的GluT 1结构域的构象动态，并通过使用质谱映射GluT 1结构域暴露于溶剂和小的，亲水性分子与GluT 1反应的底物依赖性的方式在配体的存在下发生可及性变化。具体目标4测试的假设，即新发现的“e（S）”-一个短暂的，运输中间体，从水环境中排除底物-结合e1和e2配体同时。这些生物化学和快速猝灭流配体结合的研究严重挑战四聚体和简单的载体模型的运输和进一步调查的性质e（S）和亚基协同作用在基板闭塞。

DESCRIPTION (provided by applicant): Protein-mediated facilitative glucose transport is essential for cellular metabolism, post-translation protein modification, cellular protection against oxidative stress and organismal carbohydrate homeostasis. In spite of extensive study, the mechanism of glucose transport is unknown. Our goal is to understand the molecular mechanism of protein-mediated glucose transport. To achieve this, we propose to investigate the structure and function of the human glucose transport protein GluT1. This transport protein is uniquely amenable to study being available as a purified protein from red cells, as a recombinant protein from Cos-7 cells and being amenable to function-screens by expression in glucose-transport null yeast. Specific aims 1 and 2 focus on structural determinants and functional consequences of GluT1 oligomerization in the cell membrane. Specific Aims 3 and 4 investigate transporter dynamics and catalysis. Specific Aim 1 tests the hypothesis that GluT1-specific sequence is required for GluT1 oligomerization. During the past grant cycle we discovered that cell membrane GluT1 is a homotetramer stabilized by GluT1-specific oligomerization sequence and preserved by some detergents but destabilized by others. We now challenge this hypothesis directly by GluT1 domain swapping and mutagenesis experiments asking whether transporter oligomeric structure and function are preserved. Specific Aim 2 tests the hypothesis that GluT1 oligomerization is necessary but not sufficient for glucose transport cooperativity. Some (but not all) GluT1 ligands promote cooperativity between GluT1 subunits. We have mapped ligand stereochemistry promoting cooperativity to the ligand but not yet to GluT1. We use constructs of specific aim 1 to ask whether GluT1 oligomerization is necessary for transporter cooperativity and examine GluT1 ligand binding site requirements for cooperativity in detail. Specific Aim 3 tests the hypothesis that specific GluT1 domains are conformationally dynamic and undergo accessibility changes in the presence of ligand by using mass spectrometry to map GluT1 domains exposed to solvent and to small, hydrophilic molecules that react with GluT1 in a substrate-dependent manner. Specific Aim 4 tests the hypothesis that the newly discovered "e(S)" - a transient, transport intermediate that excludes substrate from the aqueous environment - binds e1 and e2 ligands simultaneously. These biochemical and rapid quench-flow ligand binding studies critically challenge the tetramer and simple carrier models for transport and further investigate the nature of e(S) and the role of subunit cooperativity in substrate occlusion.