Amino acid residues responsible for galactose recognition in yeast Gal2 transporter

Abstract

A novel, systematic approach was used to identify amino acid residues responsible for substrate recognition in the transmembrane 10 region of the Ga12 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 Ga12 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 Tyr446 residue found in Ga12. 14 of the 19 clones contained Trp445 found in Ga12, whereas the other 5 contained Cys445, a residue not found in either Ga12 or Hxt2. When Tyr446 of Ga12 was replaced with any of the other 19 amino acids, no galactose transport activity was observed in the resulting transporters, indicating that Tyr446 plays an essential role in the transport of this sugar. Replacement of 2 amino acids of Hxt2 with the corresponding Tyr446 and Trp445 of Ga12 allowed the modified Hxt2 to transport galactose. The K(m) of galactose transport for the modified transporter was 8-fold higher than that of Ga12. These results and other evidence unequivocally show that Tyr446 is essential and Trp445 is important for the discrimination of galactose versus glucose.