Amino acid residues responsible for galactose recognition in yeast Gal2 transporter

A novel, systematic approach was used to identify amino acid residues responsible for substrate recognition in the transmembrane 10 region of the Gal2 galactose transporter of Saccharomyces cerevisiae, A mixture of approximately 25,000 distinct plasmids that encode all the combinations of 12 amino acids in transmembrane 10 that are different in Gal2 and the homologous glucose transporter Hxt2 was synthesized. Selection of galactose transport-positive clones on galactose limited agar plates yielded 19 clones, all of which contained the Tyr(446) residue found in Gal2. 14 of the 19 clones contained Trp(455) found in Gal2, whereas the other 5 contained Cys(455), a residue not found in either Gal2 or Hxt2. When Tyr(446) Of Gal2 was replaced with any of the other 19 amino acids, no galactose transport activity was observed in the resulting transporters, indicating that Tyr(446) plays an essential role in the transport of this sugar. Replacement of 2 amino acids of Hxt2 with the corresponding Tyr(446) and Trp(455) Of Gal2 allowed the modified Hxt2 to transport galactose, The K-m of galactose transport for the modified transporter was 8- fold higher than that of Gal2. These results and other evidence unequivocally show that Tyr(446) is essential and Trp(455) is important for the discrimination of galactose versus glucose.