Kasahara M, Shimoda E, Maeda M
Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo, Japan.
FEBS Lett. 1996 Jul 1;389(2):174-8. doi: 10.1016/0014-5793(96)00567-4.
A systematic series of chimeras between Ga12 galactose transporter and Hxt2 glucose transporter in yeast was produced to delineate the essential domain for substrate recognition. A domain of 101 amino acids close to the COOH-terminus that has been previously identified as the critical substrate recognition region was further divided into four subdomains, by introducing five restriction enzyme sites at exactly corresponding locations of both genes without changing coding amino acids. When each of all possible 16 modified genes was expressed, all the galactose transport-active chimeras were found to possess Ga12-derived transmembrane segment (TM) 10. Of the 35 amino acids in the TM1O region, only 12 differ between Ga12 and Hxt2, indicating that these 12 amino acids include the critical residue(s) responsible for the differential recognition of galactose and glucose in these transporters.
在酵母中构建了一系列由Ga12半乳糖转运蛋白和Hxt2葡萄糖转运蛋白组成的嵌合体,以确定底物识别的关键结构域。先前已确定靠近COOH末端的101个氨基酸结构域为关键底物识别区域,通过在两个基因的精确对应位置引入五个限制性酶切位点而不改变编码氨基酸,将该结构域进一步划分为四个亚结构域。当所有可能的16个修饰基因分别表达时,发现所有具有半乳糖转运活性的嵌合体都含有源自Ga12的跨膜片段(TM)10。在TM10区域的35个氨基酸中,Ga12和Hxt2之间只有12个不同,这表明这12个氨基酸包含负责这些转运蛋白对半乳糖和葡萄糖进行差异识别的关键残基。