FUNCTIONAL CONSEQUENCES OF PROLINE MUTATIONS IN THE PUTATIVE TRANSMEMBRANE SEGMENT-6 AND SEGMENT-10 OF THE GLUCOSE-TRANSPORTER GLUT1

Proline residues are thought to play a characteristic structural and/o r dynamic role in various membrane proteins [Williams, K. A. and Deber , C. M. (1991) Biochemistry 30, 8919-8923]. By use of site-directed mu tagenesis and functional expression of mutant glucose transporters in Xenopus oocytes, we investigated the effects of single proline substit utions in the putative helices 6 and 10 on GLUT1-mediated glucose tran sport. Proline residues of helix 6, that are conserved in all human gl ucose-transporter isoforms except for the human GLUT2, were mutated ei ther to alanine or to the corresponding residues of GLUT2, i.e. to his tidine (P187H), arginine (P196R) or phenylalanine (P205F). In addition , the three proline amino acids within the domain G382-P-G-P-I-P of he lix 10 were individually replaced with either alanine or glutamine res idues. In all cases, transport function was retained when each individ ual 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 mu tant transporter protein in plasma membrane and total membrane prepara tions was found to be decreased, as detected by immunoblotting, no sin gle 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 singl e proline position of helix 10 almost completely abolished 2-deoxy-D-g lucose uptake. Thus, the specific chemical structure of the side chain of the substituted amino acid rather than the unique property of prol ine residues for cis-trans isomerization seemed to determine the conse quences on glucose transport.