Substitution of tyrosine 293 of GLUT1 locks the transporter into an outward facing conformation.
Tyrosines 292 and 293 in the mammalian glucose transporter GLUT1 have been substituted by either isoleucine or phenylalanine. Chinese hamster ovary clones that were transfected with Tyr-292-->Ile, Tyr-292-->Phe, Tyr-293-->Ile, and Tyr-293-->Phe constructs of GLUT1 were shown, by Western blotting and cell surface carbohydrate labeling, to have expression levels that were comparable with the wild type. The Vmax for 2-deoxy-D-glucose transport was markedly reduced only as a result of the Tyr-293-->Ile mutation. The ability of the Tyr-293-->Ile mutated GLUT1 to bind the exofacial ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine (ATB-BMPA) and the endofacial ligand cytochalasin B were assessed by photolabeling procedures. The ability to bind the bis-mannose compound was unimpaired, whereas the ability to bind cytochalasin B was totally abolished, and the level of labeling was lower than in the nontransfected clone. Affinities of the wild-type and Tyr-293-->Ile GLUT1 for D-glucose, the exofacial ligands (ATB-BMPA and 4,6-O-ethylidene-D-glucose), and the endofacial ligand (cytochalasin B) were assessed by the ability of these agents to displace the radioactive ATB-BMPA photolabel. These data indicated that the Tyr-293-->Ile substitution produced no change in the affinity for D-glucose, a relatively small enhancement in the affinity for exofacial ligands, but a large approximately 300-fold reduction in affinity for cytochalasin B, suggesting that the mutated GLUT1 is locked in an outward facing conformation. The observation that the Tyr-293-->Ile mutant transporter can bind nontransported C4 and C6 substituted hexose analogues but cannot catalyze transport is interpreted as indicating that Tyr-293 is involved in closing the exofacial site around C4 and C6 of D-glucose in the transport catalysis process.
Mori
H
H
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Hashiramoto
M
M
Clark
A E
AE
Yang
J
J
Muraoka
A
A
Tamori
Y
Y
Kasuga
M
M
Holman
G D
GD
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003571:Cytochalasin B / Q000378:metabolism; D017931:DNA Primers; D003847:Deoxyglucose / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D051272:Glucose Transporter Type 1; D006027:Glycosides; D006801:Humans; D007532:Isoleucine; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017354:Point Mutation; D011437:Propylamines; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection; D014443:Tyrosine
D000345:Affinity Labels; D001386:Azides; D017931:DNA Primers; D004187:Disaccharides; D051272:Glucose Transporter Type 1; D006027:Glycosides; D009004:Monosaccharide Transport Proteins; D011437:Propylamines; D011994:Recombinant Proteins; C495677:SLC2A1 protein, human; D007532:Isoleucine; C065412:2-N-(4-(1-azitrifluoroethyl)benzoyl)-1,3-bis-(mannos-4-yloxy)-2-propylamine; D003571:Cytochalasin B; D014443:Tyrosine; D010649:Phenylalanine; D003847:Deoxyglucose