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 Tyr446 residue found in Gal2. 14 of the 19 clones contained Trp455 found in Gal2, whereas the other 5 contained Cys455, a residue not found in either Gal2 or Hxt2. When Tyr446 of Gal2 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 Trp455 of Gal2 allowed the modified Hxt2 to transport galactose. The Km of galactose transport for the modified transporter was 8-fold higher than that of Gal2. These results and other evidence unequivocally show that Tyr446 is essential and Trp455 is important for the discrimination of galactose versus glucose.
Kasahara
M
M
Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo 192-03, Japan. kasahara@main.teikyo-u.ac.jp
Shimoda
E
E
Maeda
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000378:metabolism; D005690:Galactose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D051246:Glucose Transport Proteins, Facilitative; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; C106901:GAL2 protein, S cerevisiae; D051246:Glucose Transport Proteins, Facilitative; C084498:HXT2 protein, S cerevisiae; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C023550:galactose-binding protein; D014443:Tyrosine; D014364:Tryptophan; D005947:Glucose; D005690:Galactose