Glutamine 161 of Glut1 glucose transporter is critical for transport activity and exofacial ligand binding.
The possible role of 5 transmembrane amino acid residues in the function of the Glut1 glucose transporter was investigated by site-directed mutagenesis. The residues were chosen based on their containing hydroxyl or amide side chains capable of hydrogen bonding to glucose and their complete conservation in Glut1 through Glut5. Asn100, Gln161, Gln200, Tyr292, and Tyr293 were individually replaced by hydrophobic and/or polar residues, and the mutants were analyzed by expression in Xenopus oocytes. Substitution of leucine or asparagine for Gln161 reduced the relative transport activity of Glut1 by 50- and 10-fold, respectively, as measured by uptake of 2-deoxyglucose normalized to the plasma membrane content of the mutant transporters. Substitutions at the other residues had either a modest (approximately 2-fold) effect or no effect on the relative transport activity. The Ki for binding of the exofacial ligand 4,6-O-ethylidene-alpha-D-glucose to Glut1 was increased by 18-fold upon substitution of asparagine for Gln161. The Km for zero-trans influx of 2-deoxyglucose was not affected by the substitution of asparagine for Gln161, but the catalytic turnover was decreased by 7.5-fold. These data, combined with the high degree of conservation of Gln161 among the specific members of the 12-helix membrane transporter superfamily that transport hexoses, support the contention that Gln161 forms part of the exofacial substrate-binding site of these molecules. However, the decreased transport activity resulting from mutations at Gln161 is likely due to an additional effect on the rate of a conformational change involved in net substrate uptake. This constitutes the first evidence that a residue within the NH2 terminal half of Glut1 is critical for transport function.
Mueckler
M
M
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Weng
W
W
Kruse
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D005260:Female; D005947:Glucose / Q000031:analogs & derivatives* / Q000378:metabolism; D051272:Glucose Transporter Type 1; D005973:Glutamine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D014982:Xenopus laevis
D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D005973:Glutamine; C013984:ethylidene glucose; D005947:Glucose