Hruz P W, Mueckler M M
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1999 Dec 17;274(51):36176-80. doi: 10.1074/jbc.274.51.36176.
The human erythrocyte facilitative glucose transporter (Glut1) is predicted to contain 12 transmembrane spanning alpha-helices based upon hydropathy plot analysis of the primary sequence. Five of these helices (3, 5, 7, 8, and 11) are capable of forming amphipathic structures. A model of GLUT1 tertiary structure has therefore been proposed in which the hydrophilic faces of several amphipathic helices are arranged to form a central aqueous channel through which glucose traverses the hydrophobic lipid bilayer. In order to test this model, we individually mutated each of the amino acid residues in transmembrane segment 7 to cysteine in an engineered GLUT1 molecule devoid of all native cysteines (C-less). Measurement of 2-deoxyglucose uptake in a Xenopus oocyte expression system revealed that nearly all of these mutants retain measurable transport activity. Over one-half of the cysteine mutants had significantly reduced specific activity relative to the C-less protein. The solvent accessibility and relative orientation of the residues within the helix was investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl directed reagent p-chloromercuribenzene sulfonate (pCMBS). Cysteine replacement at six positions (Gln(282), Gln(283), Ile(287), Ala(289), Val(290), and Phe(291)), all near the exofacial side of the cell membrane, produced transporters that were inhibited by incubation with extracellular pCMBS. Residues predicted to be near the cytoplasmic side of the cell membrane were minimally affected by pCMBS. These data demonstrate that the exofacial portion of transmembrane segment 7 is accessible to the external solvent and provide evidence for the positioning of this alpha-helix within the glucose permeation pathway.
基于对一级序列的亲水性图谱分析，预计人类红细胞易化葡萄糖转运体（Glut1）含有12个跨膜的α螺旋。其中五个螺旋（3、5、7、8和11）能够形成两亲性结构。因此，有人提出了GLUT1三级结构模型，其中几个两亲性螺旋的亲水面排列形成一个中央水通道，葡萄糖通过该通道穿过疏水脂质双层。为了验证该模型，我们在一个不含所有天然半胱氨酸（无C）的工程化GLUT1分子中，将跨膜片段7中的每个氨基酸残基单独突变为半胱氨酸。在非洲爪蟾卵母细胞表达系统中测量2-脱氧葡萄糖摄取量，结果显示几乎所有这些突变体都保留了可测量的转运活性。超过一半的半胱氨酸突变体相对于无C蛋白具有显著降低的比活性。通过测定突变转运体对巯基导向试剂对氯汞苯磺酸盐（pCMBS）抑制的敏感性，研究了螺旋内残基的溶剂可及性和相对取向。在六个位置（Gln(282)、Gln(283)、Ile(287)、Ala(289)、Val(290) 和Phe(291)）进行半胱氨酸替换，所有这些位置都靠近细胞膜的外表面，产生的转运体在与细胞外pCMBS孵育时受到抑制。预计靠近细胞膜胞质侧的残基受pCMBS的影响最小。这些数据表明跨膜片段7的外表面部分可被外部溶剂接触，并为该α螺旋在葡萄糖渗透途径中的定位提供了证据。
