Mueckler Mike, Makepeace Carol
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2006 Dec 1;281(48):36993-8. doi: 10.1074/jbc.M608158200. Epub 2006 Oct 3.
A model has been proposed for the exofacial configuration of the Glut1 glucose transporter in which eight transmembrane domains form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 12, predicted to be an outer helix in this hypothetical model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A previously characterized functional cysteine-less Glut1 molecule was used to produce 21 Glut1 point mutants by changing each residue along helix 12 to a cysteine residue. These mutants were then expressed in Xenopus oocytes, and their protein levels, functional activities, and sensitivities to pCMBS were determined. Strikingly, in contrast to all nine other predicted Glut1 transmembrane helices that have been previously examined by this method, none of the 21 helix 12 single-cysteine mutants exhibited significant inhibition of specific transport activity. Also unlike most other Glut1 transmembrane domains in which solvent-accessible residues lie along a single face of the helix, mutations in five consecutive residues predicted to lie close to the exofacial face of the membrane resulted in sensitivity to pCMBS-induced transport inhibition. These results suggest that helix 12 plays a passive stabilizing role in the structure of Glut1 and is not directly involved in the transport mechanism. Additionally, the pCMBS data indicate that the predicted exoplasmic end of helix 12 is completely exposed to the external solvent when the transporter is in its exofacial configuration.
有人提出了一种关于Glut1葡萄糖转运蛋白胞外构象的模型，其中八个跨膜结构域形成一个内部螺旋束，由四个外部螺旋稳定。在这个假设模型中，跨膜片段12被预测为一个外部螺旋，通过半胱氨酸扫描诱变和使用膜不透性、巯基特异性试剂对氯汞苯磺酸盐（pCMBS）的取代半胱氨酸可及性方法来研究其作用。使用先前表征的无半胱氨酸的功能性Glut1分子，通过将螺旋12上的每个残基替换为半胱氨酸残基来产生21个Glut1点突变体。然后将这些突变体在非洲爪蟾卵母细胞中表达，并测定它们的蛋白质水平、功能活性和对pCMBS的敏感性。令人惊讶的是，与先前用这种方法研究的所有其他九个预测的Glut1跨膜螺旋不同，21个螺旋12单半胱氨酸突变体中没有一个表现出对特异性转运活性的显著抑制。也与大多数其他Glut1跨膜结构域不同，在这些结构域中，溶剂可及残基位于螺旋的单个面上，预测靠近膜胞外表面的五个连续残基中的突变导致对pCMBS诱导的转运抑制敏感。这些结果表明，螺旋12在Glut1的结构中起被动稳定作用，不直接参与转运机制。此外，pCMBS数据表明，当转运蛋白处于其胞外构象时，螺旋12的预测胞外末端完全暴露于外部溶剂。
