GLUCOSE TRANSPORTER STRUCTURE AND FUNCTION

描述：蛋白质介导的促进葡萄糖转运对于 细胞代谢和有机体碳水化合物稳态。尽管 广泛的研究，葡萄糖转运的机制是未知的。远程 该实验室的目标是了解 蛋白质介导的葡萄糖转运。为达致这个目标，我们建议 研究人葡萄糖转运蛋白的结构和功能 Glut1。这种哺乳动物葡萄糖转运蛋白是唯一适合 分析可作为来自红细胞的纯化蛋白质和作为 在Cos-7细胞中表达并从Cos-7细胞亲和纯化的重组蛋白。 具体目的1、4和5检验Glut 1作为一种蛋白质发挥功能的假设。 在细胞膜中的协同同源四聚体。具体目标1问，如果 四聚体Glut 1是通过研究 不同去污剂和脂质对转运蛋白寡聚体大小的影响。具体目标4 通过配体结合的停流分析来测量Glut 1协同性， 如果通过半胱氨酸诱变和通过 异源复合物形成也影响Glut 1协同性。具体目标5 利用了最近发现的一个意想不到的Glut 1协同效应的结果， （通过竞争性抑制剂的亚饱和水平的运输刺激） 询问Glut 1寡聚体大小的改变是否影响这种合作行为。 碳水化合物库也将筛选潜在的“动力学激活剂” 交通工具。 具体目标2和3涉及Glut 1结构。具体目标2建议 色氨酸诱变，以鉴定介导Glut 1色氨酸残基 当配体特异性结合糖时，Glut 1色氨酸荧光猝灭 或糖出口地。具体目标3映射Glut 1糖摄取和输出 通过使用位点特异性光配体、肽图谱和肽 测序将对确定的位点进行丙氨酸扫描诱变 重组Glut 1将被重新定位并测试活性以确认 或者反驳功能丧失

DESCRIPTION: Protein-mediated facilitative glucose transport is vital to cellular metabolism and organismal carbohydrate homeostasis. In spite of extensive study, the mechanism of glucose transport is unknown. The long-range goal of this laboratory is to understand the molecular mechanism of protein-mediated glucose transport. To achieve this goal, we propose to investigate the structure and function of the human glucose transport protein Glut1. This mammalian glucose transport protein is uniquely amenable to analysis being available as a purified protein from red blood cells and as a recombinant protein expressed in, and affinity-purified from Cos-7 cells. Specific Aims 1, 4, and 5 test the hypothesis that Glut1 functions as a cooperative homo tetramer in the cell membrane. Specific Aim 1 asks if tetrameric Glut1 is an artifact of isolation by investigating the effects of different detergents and lipids on transporter oligomeric size. Specific Aim 4 measures Glut1 cooperativity by stopped flow analysis of ligand binding to ask if perturbations of Glut1 oligomeric size by cysteine mutagenesis and by heterocomplex formation also affect Glut1 cooperativity. Specific Aim 5 exploits an unanticipated and recently discovered result of Glut1 cooperativity (transport stimulation by subsaturating levels of competitive inhibitors) to ask if altered Glut1 oligomeric size affects this cooperative behavior. Carbohydrate libraries will also be screened for potential "kinetic activators" of transport. Specific Aims 2 and 3 address Glut1 structure. Specific Aim 2 proposes tryptophan mutagenesis to identify Glut1 tryptophan residue(s) that mediate Glut1 tryptophan fluorescence quenching when ligands bind specifically at sugar uptake or sugar export sites. Specific Aim 3 maps Glut1 sugar uptake and export domains by use of site-specific photo-ligands, peptide mapping and peptide sequencing. Identified sites will be subjected to alanine scanning mutagenesis and the recombinant Glut1 will be remapped and tested for activity to confirm or refute loss of function.