SUGAR TRANSPORTER OLIGOMERIC STRUCTURE AND FUNCTION

这一建议代表了我们实验室的持续努力， 了解蛋白质介导的糖跨细胞转运的机制 膜。 这项提案的主要目标是了解糖 转运蛋白寡聚化的发生以及转运蛋白如何 寡聚化影响转运功能。 这些研究将有助于 我们的长期目标是了解物理基础和细胞 调节糖的运输，并最终可能在 管理紊乱状态，如糖尿病。 我们的研究表明，人红细胞糖转运系统存在， 以两种催化活性形式：作为四聚体（主要物质）和作为 GLUT1蛋白的二聚体（次要物质）。 的低聚状态， 糖转运蛋白决定转运的催化性质。 特异性目的1询问纯化的四聚体GLUT 1，如 红细胞驻留GLUT 1是一种多位点变构转运蛋白。 我们 研究表明，底物结合位点的排列， 四聚体和二聚体GLUT 1是不同的。 四聚体GLUT 1似乎 表现为2个“双位点载体”，同时暴露两种糖 流入和糖流出位点到底物。 二聚体GLUT 1的行为与2相同 独立的“简单载体”，其交替地暴露糖流入和 糖流出位点到底物。 我们通过询问 无论是纯化的、四聚的还是二聚的GLUT 1， 糖内流和糖外流位点同时进行。 如果我们的假设 是正确的，只有四聚体应该同时暴露两个位点。 特异性目标2询问四聚体GLUT 1中的隐蔽半胱氨酸是否 以二硫化物存在。 四聚体GLUT 1仅含有2个游离半胱氨酸 每个GLUT 1单体，通过还原剂转化为二聚体，但不是两个 二硫键连接的二聚体。 二聚体GLUT 1含有6个游离半胱氨酸， GLUT 1单体。 我们使用不同的羧甲基化程序， 肽作图以鉴定四聚体的隐蔽半胱氨酸， 确定隐蔽半胱氨酸是否为内部二硫化物，混合 二硫化物或脂肪酰化。具体目标3问： 四聚体GLUT 1的隐蔽半胱氨酸在GLUT 1的四聚化中起作用。 如果特定半胱氨酸的修饰导致GLUT I的四聚化， 半胱氨酸的缺失应防止四聚体的形成。 每个 6个GLUT 1半胱氨酸将发生突变。 这些突变对 GLUT1寡聚体结构、配体结合和糖转运将在 评估。 如果成功的话，这些研究将大大有助于我们长期的 理解糖的序列特异性决定因素的长期目标 更高层次的结构和功能。

This proposal represents a continuing effort by our laboratory to understand the mechanism of protein-mediated sugar transport across cell membranes. The broad goal of this proposal is to understand how sugar transport protein oligomerization occurs and how transport protein oligomerization affects transport function. These studies will assist in our long term goals of understanding the physical basis and the cellular regulation of sugar transport and could ultimately be of value in the management of disordered states such as diabetes. Our studies show that the human erythrocyte sugar transport system exists in two catalytically active forms: as a tetramer (major species) and as a dimer (minor species) of GLUT1 proteins. The oligomeric state of the sugar transport protein determines the catalytic properties of transport. Specific Aim 1 asks whether purified tetrameric GLUT1, like erythrocyte-resident GLUT1, is a multisite, allosteric transporter. Our studies suggest that the arrangement of substrate binding sites in tetrameric and dimeric GLUT1 is different. Tetrameric GLUT1 appears to behave as 2 "two-site carriers" that simultaneously expose both sugar influx and sugar efflux sites to substrate. Dimeric GLUT1 behaves as 2 independent "simple carriers" that alternately expose sugar influx and sugar efflux sites to substrate. We test this hypothesis by asking whether purified, tetrameric or dimeric GLUT1 can bind substrates at sugar influx and at sugar efflux sites simultaneously. If our hypothesis is correct, only the tetramer should expose both sites simultaneously. Specific Aim 2 asks if the cryptic cysteines in tetrameric GLUT1 are present as disulfides. Tetrameric GLUT1 contains only 2 free cysteines per GLUT1 monomer, is converted to dimer by reductant but is not two disulfide-linked dimers. Dimeric GLUT1 contains 6 free cysteines per GLUT1 monomer. We use differential carboxymethylation procedures and peptide mapping to identify the cryptic cysteines of the tetramer and to determine whether the cryptic cysteines are internal disulfides, mixed disulfides or are fatty acylated. Specific Aim 3 asks what role the cryptic cysteines of tetrameric GLUT1 play in tetramerization of GLUT1. If modification of a specific cysteine causes tetramerization of GLUT I, deletion of the cysteine should prevent tetramer formation. Each of the 6 GLUT1 cysteines will be mutated. The effects of these mutations on GLUT1 oligomeric structure, ligand binding and sugar transport will be evaluated. If successful, these studies will greatly assist in our long term goal of understanding sequence-specific determinants of sugar transporter higher order structure and function.