SUGAR TRANSPORTER OLIGOMERIC STRUCTURE AND FUNCTION

该提案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机制 膜。 该提案的主要目标是了解糖如何 转运蛋白寡聚化发生以及转运蛋白如何发生 寡聚影响运输功能。 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 糖运输的监管，最终可能具有价值 管理糖尿病等紊乱状态。 我们的研究表明人类红细胞糖转运系统存在 有两种催化活性形式：四聚体（主要种类）和 GLUT1 蛋白的二聚体（次要种类）。 的寡聚状态 糖转运蛋白决定了转运的催化特性。 具体目标 1 询问是否纯化了四聚体 GLUT1，例如 红细胞驻留 GLUT1 是一种多位点变构转运蛋白。 我们的 研究表明底物结合位点的排列 四聚体和二聚体GLUT1是不同的。 四聚体 GLUT1 似乎 表现为 2 个“双位点载体”，同时暴露两种糖 底物的流入和糖流出位点。 二聚体 GLUT1 的行为如 2 独立的“简单载体”，交替暴露糖的流入和 糖流出位点至底物。 我们通过询问来检验这个假设 无论是纯化的、四聚体还是二聚体 GLUT1 都可以结合底物 糖流入和糖流出位点同时进行。 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。 具体目标 2 询问四聚体 GLUT1 中的神秘半胱氨酸是否是 以二硫化物形式存在。 四聚体 GLUT1 仅包含 2 个游离半胱氨酸 每个 GLUT1 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 GLUT1 每个含有 6 个游离半胱氨酸 GLUT1单体。 我们使用差异化羧甲基化程序和 肽图谱鉴定四聚体的隐秘半胱氨酸并 确定隐性半胱氨酸是否是内部二硫键，混合 二硫化物或被脂肪酰化。具体目标 3 询问什么角色 四聚体 GLUT1 的隐性半胱氨酸在 GLUT1 四聚化中发挥作用。 如果特定半胱氨酸的修饰导致 GLUT I 四聚化， 半胱氨酸的缺失可以防止四聚体的形成。 每个 6 GLUT1 半胱氨酸将发生突变。 这些突变的影响 GLUT1寡聚结构、配体结合和糖转运将被 评价。 如果成功的话，这些研究将极大地帮助我们长期 理解糖的序列特异性决定因素的术语目标 转运蛋白的高阶结构和功能。

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.