Identification of a key residue determining substrate affinity in the human glucose transporter GLUT1.
Asn(331) in transmembrane segment 7 of the yeast Saccharomyces cerevisiae transporter Hxt2 has been identified as a single key residue for high-affinity glucose transport by comprehensive chimera approach. The glucose transporter GLUT1 of mammals belongs to the same major facilitator superfamily as Hxt2 and may therefore show a similar mechanism of substrate recognition. The functional role of Ile(287) in human GLUT1, which corresponds to Asn(331) in Hxt2, was studied by its replacement with each of the other 19 amino acids. The mutant transporters were individually expressed in a recently developed yeast expression system for GLUT1. Replacement of Ile(287) generated transporters with various affinities for glucose that correlated well with those of the corresponding mutants of the yeast transporter. Residues exhibiting high affinity for glucose were medium-sized, non-aromatic, uncharged and irrelevant to hydrogen-bond capability, suggesting an important role of van der Waals interaction. Sensitivity to phloretin, a specific inhibitor for the presumed exofacial glucose binding site, was decreased in two mutants, whereas that to cytochalasin B, a specific inhibitor for the presumed endofacial glucose binding site, was unchanged in the mutants. These results suggest that Ile(287) is a key residue for maintaining high glucose affinity in GLUT1 as revealed in Hxt2 and is located at or near the exofacial glucose binding site.
Laboratory of Biophysics, School of Medicine, Hachioji, Tokyo 192-0395, Japan.
Toshiko
T
Kasahara
Mari
M
Maeda
Eckhard
E
Boles
Michihiro
M
Kasahara
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D055592:Biophysical Phenomena; D005947:Glucose / Q000378:metabolism; D051246:Glucose Transport Proteins, Facilitative / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051272:Glucose Transporter Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity
D051246:Glucose Transport Proteins, Facilitative; D051272:Glucose Transporter Type 1; C084498:HXT2 protein, S cerevisiae; D011994:Recombinant Proteins; C495677:SLC2A1 protein, human; D029701:Saccharomyces cerevisiae Proteins; D005947:Glucose