Kasahara T, Kasahara M
Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo 192-0395, Japan.
J Biol Chem. 1998 Oct 30;273(44):29113-7. doi: 10.1074/jbc.273.44.29113.
The molecular mechanism of substrate recognition in membrane transport is not well understood. Two amino acid residues, Tyr446 and Trp455 in transmembrane segment 10 (TM10), have been shown to be important for galactose recognition by the yeast Gal2 transporter; Tyr446 was found to be essential in that its replacement by any of the other 19 amino acids abolished transport activity (Kasahara, M., Shimoda, E., and Maeda, M. (1997) J. Biol. Chem. 272, 16721-16724). The Glut1 glucose transporter of animal cells belongs to the same Glut transporter family as does Gal2 and thus might be expected to show a similar mechanism of substrate recognition. The role of the two amino acids, Phe379 and Trp388, in rat Glut1 corresponding to Tyr446 and Trp455 of Gal2 was therefore studied. Phe379 and Trp388 were individually replaced with each of the other 19 amino acids, and the mutant Glut1 transporters were expressed in yeast. The expression level of most mutants was similar to that of the wild-type Glut1, as revealed by immunoblot analysis. Glucose transport activity was assessed by reconstituting a crude membrane fraction of the yeast cells in liposomes. No significant glucose transport activity was observed with any of Trp388 mutants, whereas the Phe379 mutants showed reduced or no activity. These results indicate that the two aromatic amino acids in TM10 of Glut1 are important for glucose transport. However, unlike Gal2, the residue at the cytoplasmic end of TM10 (Trp388, corresponding to Trp455 of Gal2), rather than that in the middle of TM10 (Phe379, corresponding to Tyr446 of Gal2), is essential for transport activity.
膜转运中底物识别的分子机制尚未完全明确。跨膜片段10（TM10）中的两个氨基酸残基Tyr446和Trp455，已被证明对酵母Gal2转运蛋白识别半乳糖很重要；发现Tyr446至关重要，因为用其他19种氨基酸中的任何一种取代它都会消除转运活性（Kasahara, M., Shimoda, E., and Maeda, M. (1997) J. Biol. Chem. 272, 16721 - 16724）。动物细胞的Glut1葡萄糖转运蛋白与Gal2属于同一Glut转运蛋白家族，因此可能具有类似的底物识别机制。因此，研究了大鼠Glut1中与Gal2的Tyr446和Trp455相对应的两个氨基酸Phe379和Trp388的作用。将Phe379和Trp388分别用其他19种氨基酸取代，并在酵母中表达突变型Glut1转运蛋白。免疫印迹分析显示，大多数突变体的表达水平与野生型Glut1相似。通过将酵母细胞的粗膜部分重组到脂质体中来评估葡萄糖转运活性。任何Trp388突变体均未观察到明显的葡萄糖转运活性，而Phe379突变体的活性则降低或无活性。这些结果表明，Glut1的TM10中的两个芳香族氨基酸对葡萄糖转运很重要。然而，与Gal2不同，TM10胞质端的残基（Trp388，对应于Gal2的Trp455）而非TM10中间的残基（Phe379，对应于Gal2的Tyr446）对转运活性至关重要。
