Mueckler Mike, Makepeace Carol
Department of Cell Biology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA.
Biochemistry. 2009 Jun 30;48(25):5934-42. doi: 10.1021/bi900521n.
Transmembrane helix 9 of the Glut1 glucose transporter was analyzed by cysteine-scanning mutagenesis and the substituted cysteine accessibility method (SCAM). A cysteine-less (C-less) template transporter containing amino acid substitutions for the six native cysteine residues present in human Glut1 was used to generate a series of 21 mutant transporters by substituting each successive residue in predicted transmembrane segment 9 with a cysteine residue. The mutant proteins were expressed in Xenopus oocytes, and their specific transport activities were directly compared to that of the parental C-less molecule whose function has been shown to be indistinguishable from that of native Glut1. Only a single mutant (G340C) had activity that was reduced (by 75%) relative to that of the C-less parent. These data suggest that none of the amino acid side chains in helix 9 is absolutely required for transport function and that this helix is not likely to be directly involved in substrate binding or translocation. Transport activity of the cysteine mutants was also tested after incubation of oocytes in the presence of the impermeant sulfhydryl-specific reagent, p-chloromercuribenzene sulfonate (pCMBS). Only a single mutant (T352C) exhibited transport inhibition in the presence of pCMBS, and the extent of inhibition was minimal (11%), indicating that only a very small portion of helix 9 is accessible to the external solvent. These results are consistent with the conclusion that helix 9 plays an outer stabilizing role for the inner helical bundle predicted to form the exofacial substrate-binding site. All 12 of the predicted transmembrane segments of Glut1 encompassing 252 amino acid residues and more than 50% of the complete polypeptide sequence have now been analyzed by scanning mutagenesis and SCAM. An updated model is presented for the outward-facing substrate-binding site and relative orientation of the 12 transmembrane helices of Glut1.
通过半胱氨酸扫描诱变和取代半胱氨酸可及性方法（SCAM）对葡萄糖转运蛋白Glut1的跨膜螺旋9进行了分析。使用一种不含半胱氨酸（C-less）的模板转运蛋白，该蛋白对人Glut1中存在的六个天然半胱氨酸残基进行了氨基酸替换，通过用半胱氨酸残基替换预测跨膜片段9中的每个连续残基，生成了一系列21种突变转运蛋白。突变蛋白在非洲爪蟾卵母细胞中表达，并将其特异性转运活性与亲本C-less分子的活性直接进行比较，已证明该亲本分子的功能与天然Glut1的功能无明显差异。只有一个突变体（G340C）的活性相对于C-less亲本有所降低（降低了75%）。这些数据表明，螺旋9中的氨基酸侧链对于转运功能并非绝对必需，并且该螺旋不太可能直接参与底物结合或转运。在不渗透的巯基特异性试剂对氯汞苯磺酸盐（pCMBS）存在的情况下孵育卵母细胞后，也测试了半胱氨酸突变体的转运活性。只有一个突变体（T352C）在pCMBS存在时表现出转运抑制，并且抑制程度最小（11%），表明螺旋9只有非常小的一部分可被外部溶剂接触。这些结果与以下结论一致，即螺旋9对预计形成外表面底物结合位点的内部螺旋束起到外部稳定作用。现在已经通过扫描诱变和SCAM分析了Glut1的所有12个预测跨膜片段，这些片段包含252个氨基酸残基以及超过50%的完整多肽序列。给出了一个关于Glut1向外的底物结合位点和12个跨膜螺旋相对取向的更新模型。
