GLUCOSE TRANSPORTER STRUCTURE AND FUNCTION

描述（由申请人提供）：蛋白质介导的葡萄糖转运对于细胞代谢、翻译后蛋白质修饰、细胞抗氧化应激和有机碳水化合物稳态至关重要。尽管进行了广泛的研究，但葡萄糖转运的机制尚不清楚。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GluT1的结构和功能。该转运蛋白是唯一适合于研究的，可以作为红细胞的纯化蛋白，作为Cos-7细胞的重组蛋白，并且可以通过在葡萄糖转运酵母中的表达进行功能筛选。具体目标1和2侧重于细胞膜中GluT1寡聚化的结构决定因素和功能后果。具体目标3和4研究转运蛋白动力学和催化作用。特异性目标1验证了GluT1特异性序列是GluT1寡聚化所必需的假设。在过去的资助周期中，我们发现细胞膜GluT1是一种由GluT1特异性寡聚序列稳定的同四聚体，并被一些洗涤剂保存，但被另一些洗涤剂破坏。我们现在通过GluT1结构域交换和诱变实验直接挑战这一假设，询问转运蛋白低聚体的结构和功能是否保留。特异性目的2验证了GluT1寡聚化是葡萄糖运输协同性的必要条件，但不是充分条件的假设。一些（但不是全部）GluT1配体促进GluT1亚基之间的协同作用。我们已经绘制了促进配体协同作用的配体立体化学图谱，但尚未绘制到GluT1。我们使用特异性目的1的结构来询问GluT1寡聚化是否对转运体协同性是必要的，并详细检查了GluT1配体结合位点对协同性的要求。Specific Aim 3通过质谱法对暴露于溶剂和以底物依赖方式与GluT1反应的亲水小分子的GluT1结构域进行映射，验证了特定的GluT1结构域是构象动态的，并且在配体存在下经历可及性变化的假设。Specific Aim 4测试了新发现的“e(S)”——一种将底物排除在水环境之外的瞬时转运中间体——同时结合e1和e2配体的假设。这些生化和快速猝流配体结合研究对四聚体和简单载体的运输模型提出了严峻的挑战，并进一步研究了e(S)的性质和亚基协同性在底物阻断中的作用。

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.