Functional consequences of proline mutations in the putative transmembrane segments 6 and 10 of the glucose transporter GLUT1.
Proline residues are thought to play a characteristic structural and/or dynamic role in various membrane proteins [Williams, K.A. & Deber, C.M. (1991) Biochemistry 30, 8919-8923]. By use of site-directed mutagenesis and functional expression of mutant glucose transporters in Xenopus oocytes, we investigated the effects of single proline substitutions in the putative helices 6 and 10 on GLUT1-mediated glucose transport. Proline residues of helix 6, that are conserved in all human glucose-transporter isoforms except for the human GLUT2, were mutated either to alanine or to the corresponding residues of GLUT2, i.e. to histidine (P187H), arginine (P196R) or phenylalanine (P205F). In addition, the three proline amino acids within the domain G382-P-G-P-I-P of helix 10 were individually replaced with either alanine or glutamine residues. In all cases, transport function was retained when each individual proline residue was replaced with alanine. Substitution of proline 196 by arginine (P196R), however, resulted in reduction of 2-deoxy-D-glucose uptake rates by approximately 70%. Since the amount of this mutant transporter protein in plasma membrane and total membrane preparations was found to be decreased, as detected by immunoblotting, no single proline residue seemed to play a critical role in maintaining the catalytic activity of GLUT1. However, structural changes introduced by incorporation of the neutral polar amino acid glutamine at each single proline position of helix 10 almost completely abolished 2-deoxy-D-glucose uptake. Thus, the specific chemical structure of the side chain of the substituted amino acid rather than the unique property of proline residues for cis-trans isomerization seemed to determine the consequences on glucose transport.
Institut für Pharmakologie der Freien Universität Berlin, Germany.
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Wellner
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Monden
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Mueckler
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Keller
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary; D051272:Glucose Transporter Type 1; D006801:Humans; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D018076:DNA, Complementary; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human; D011392:Proline