Contribution to substrate recognition of two aromatic amino acid residues in putative transmembrane segment 10 of the yeast sugar transporters Gal2 and Hxt2.
The comprehensive study of chimeras between the Gal2 galactose transporter and the Hxt2 glucose transporter of Saccharomyces cerevisiae has shown that Tyr446 is essential and Trp455 is important for galactose recognition by Gal2. Consistent with this finding, replacement of the corresponding Phe431 and Tyr440 residues of Hxt2 with Tyr and Trp, respectively, allowed Hxt2 to transport galactose, suggesting that the two amino acid residues in putative transmembrane segment 10 play a definite role in galactose recognition (Kasahara, M., Shimoda, E., and Maeda, M. (1997) J. Biol. Chem. 272, 16721-16724). Replacement of Trp455 of Gal2 with any of the other 19 amino acids was shown to reduce galactose transport activity to between 0 and <20% of that of wild-type Gal2. The role of Phe431 in Hxt2 was similarly studied. Other than Phe, only Tyr at position 431 was able to support glucose transport activity, at the reduced level of <20%. In contrast, replacement of Tyr440 of Hxt2 with other amino acids revealed that most replacements, with the exception of Pro and charged amino acids, supported glucose transport activity. The importance of residue 431 in sugar recognition was more pronounced in a modified Hxt2 in which Tyr440 was replaced with Trp. Glucose transport was supported only by the aromatic amino acids Phe, Tyr, and Trp at position 431, and galactose transport was supported only by Tyr. These results suggest that an aromatic amino acid located in the middle of transmembrane segment 10 (Tyr446 in Gal2 and Phe431 in Hxt2) plays a critical role in substrate recognition in the yeast sugar transporter family to which Gal2 and Hxt2 belong.
Kasahara
M
M
Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo 192-0395, Japan. kasahara@main.teikyo-u.ac.jp
Maeda
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D051246:Glucose Transport Proteins, Facilitative; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism*; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine / Q000378:metabolism*
D017931:DNA Primers; C106901:GAL2 protein, S cerevisiae; D051246:Glucose Transport Proteins, Facilitative; C084498:HXT2 protein, S cerevisiae; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; D010649:Phenylalanine; D005947:Glucose; D005690:Galactose