How glucose transporter structure affects its function

葡萄糖运输蛋白(GLUT)催化平衡的细胞糖运输，是 人体内的碳水化合物稳态，包含12个跨膜的α螺旋(TM)，它们形成一个 由支架(疏水性TMS)稳定的易位途径(两亲性TMS 1、2、4、5、7、8、10和11 3、6、9和12)，采用向内、遮挡和向外3种构象状态。差距 我们对谷氨酸的理解是：1)底物如何结合；2)底物如何转运；3)如何 谷氨酸寡聚。我们解决了这些差距，并询问在人类疾病-GLUT1中每个过程是如何改变的 虚证(GLUT1-DS)。特定目标1检验了底物与供过于求结合的假设 易位途径。Glut1转运葡萄糖但不转运果糖，而GLUT5则不转运果糖 葡萄糖。突变研究表明，TM 2、7和11有助于GLUT5果糖结合和TM 5， 7、8、10和11与GLUT1葡萄糖结合。使用同源扫描突变，我们询问是否替换 特定的GLUT5易位到GLUT1将GLUT1从葡萄糖转换为果糖转运体，并 反之亦然，我们问哪些残基对结合至关重要。纯化的人GLUT1 Will的质谱学研究 揭示哪些GLUT1残基被光亲和底物/拮抗剂修饰，哪些是底物- 防止氢-氚交换(H/DX)？然后我们询问关键的底物结合残基是否 在GLUT1-DS中更改。《特定目标2》验证了运输涉及易位途径的假设 构象变化由骨架TM6途径TM1相互作用控制。谷氨酸1氢 (80%)与溶剂氚或氚进行构象敏感交换，但哪些氨基酸 交易所未知。我们将通过H/DX-MS对捕获的纯化的人GLUT1进行鉴定 向内、遮挡和向外方向。Glut1-GLUT4同源扫描突变显示TM6 通过以下方式控制GLUT1和GLUT4向内和向外的构象变化速率 与TM1相互作用。我们问这是否是所有谷氨酸的标志，我们调查TM6残基与哪些残基相互作用 TM1通过色氨酸扫描和二半胱氨酸交联突变，并询问这些残基是否在 Glut1-DS。特定目标3检验过剩二聚化和四聚化决定的假说 运输效率，并由特定的转位和支架TM介导。Glut1形成Ho- 二聚体和同源四聚体，而谷氨酸2-4形成同源二聚体。谷氨酸不会异位齐聚。 将GLUT1支架TM9替换到GLUT3中，使GLUT3四聚化并与 Glut1.GLUT3TM9使GLUT1解离成二聚体。途径TM 5、8、2和11可能负责 用于异构体特定二聚化。我们建议直接检验这一假设，并概述实验以 研究寡聚体状态如何影响GLUT活性，以及是否存在导致GLUT1-DS的突变 也影响GLUT1四元结构和/或四元结构对功能的影响。

Glucose transport proteins (GLUTs) catalyze equilibrative, cellular sugar transport, are essential for carbohydrate homeostasis in humans, contain 12 membrane-spanning alpha helices (TMs) which form a translocation pathway (amphipathic TMs 1, 2, 4, 5, 7, 8, 10 and 11) stabilized by a scaffold (hydrophobic TMs 3, 6, 9 and 12) and adopt 3 conformational states: the inward, the occluded and the outward orientations. Gaps in our understanding of the GLUTs are: 1) How substrates bind; 2) How substrates are translocated; 3) How GLUTs oligomerize. We address these gaps and ask how each process is altered in a human disease - GLUT1 deficiency syndrome (GLUT1-DS). Specific Aim 1 tests the hypothesis that substrates bind in the GLUT translocation pathway. GLUT1 transports glucose but not fructose while GLUT5 transports fructose not glucose. Mutagenesis studies suggest that TMs 2, 7 and 11 contribute to GLUT5 fructose binding and TMs 5, 7, 8, 10 and 11 to GLUT1 glucose binding. Using homology scanning mutagenesis, we ask if substitution of specific GLUT5 translocation TMs into GLUT1 converts GLUT1 from a glucose to a fructose transporter and vice versa and we ask which residues are critical for binding. Mass spectrometry of purified human GLUT1 will reveal which GLUT1 residues are modified by photo-affinity substrates/antagonists and which are substrate- protected against hydrogen-deuterium exchange (H/DX)? We then ask if critical substrate binding residues are altered in GLUT1-DS. Specific Aim 2 tests the hypothesis that transport involves translocation pathway conformational changes controlled by scaffold TM6 - pathway TM1 interactions. GLUT1 hydrogens (80%) undergo conformation-sensitive exchange with solvent tritium or deuterium but which amino acids exchange is unknown. We will identify them by H/DX-mass spectrometry of purified human GLUT1 trapped in inward, occluded and outward orientations. GLUT1-GLUT4 homology scanning mutagenesis shows that TM6 controls the rate of conformational change between inward and outward GLUT1 and GLUT4 orientations by interacting with TM1. We ask if this is a hallmark of all GLUTs, we investigate which TM6 residues interact with TM1 by tryptophan scanning and dicysteine cross-linking mutagenesis and ask if these residues are altered in GLUT1-DS. Specific Aim 3 tests the hypothesis that GLUT dimerization and tetramerization determine transport efficiency and are mediated by specific translocation and scaffold TMs. GLUT1 forms homo- dimers and homo-tetramers whereas GLUTs 2-4 form homo-dimers. The GLUTs do not hetero-oligomerize. Substituting GLUT1 scaffold TM9 into GLUT3 allows GLUT3 to tetramerize and to form heterocomplexes with GLUT1. GLUT3 TM9 causes GLUT1 to dissociate into dimers. Pathway TMs 5, 8, 2 and 11 may be responsible for isoform specific dimerization. We propose to test this hypothesis directly and outline experiments to investigate how GLUT activity is affected by oligomeric state and whether mutations that cause GLUT1-DS also affect GLUT1 quaternary structure and/or the effects of quaternary structure on function.