Tryptophan 388 in putative transmembrane segment 10 of the rat glucose transporter Glut1 is essential for glucose transport

The molecular mechanism of substrate recognition in membrane transport is not well understood. Two amino acid residues, Tyr(446) and Trp(455) in transmembrane segment 10 (TM10), have been shown to be important for galactose recognition by the yeast Gal2 transporter; Tyr(446) 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, 16121-16724). 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, Phe(379) and Trp(388), in rat Glut1 corresponding to Tyr(446) and Trp(455) Of Gal2 was therefore studied. Phe(379) and Trp(388) 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 Trp(388) mutants,whereas the Phe(379) 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 (Trp(388), corresponding to Trp(455) of Gal2), rather than that in the middle of TM10 (Phe(379), corresponding to Tyr(446) Of Gal2), is essential for transport activity.