SUBSTITUTION AT PRO(385) OF GLUT1 PERTURBS THE GLUCOSE-TRANSPORT FUNCTION BY REDUCING CONFORMATIONAL FLEXIBILITY

The mammalian glucose transporter, GLUT1, is capable of alternating be tween two conformations which expose either an outward- or inward-faci ng ligand binding site. The possibility that these conformational chan ges are related to the presence of prolines and glycines in transmembr ane region 10 was investigated by site-directed mutagenesis. Chinese h amster ovary clones which were transfected with Pro385 --> Ile and Pro 385 --> glycine mutations of GLUT1 were shown, by Western blotting and cell surface carbohydrate labeling, to have expression levels which w ere comparable with the wild type. The transport activity was markedly reduced as a result of the Pro385 --> isoleucine but not in the Pro38 5 --> glycine mutation. The loss of transport activity in the Pro385 - -> isoleucine clone was associated with loss of labeling by the exofac ial photoaffinity ligand, yl)benzoyl-1,3-bis(D-mannos-4-yloxy)-2-propy lamine (ATB-BMPA), but there was no loss in labeling by the inside sit e-directed ligand cytochalasin B. These results suggest that the trans porter cannot adopt the outward-directed conformation in the Pro385 -- > isoleucine clone. By contrast, the glycine substitution for proline at this position resulted in a retention of the ligand binding propert ies at both inside and outside sites. We suggest a putative mode of op eration of the transporter which involves conformational flexibility a bout the prolines in transmembrane segment 10 such that helices 11 and 12 can alternately either pack against the outside (ATB-BMPA binding) site in helices 7, 8, and 9 or against the inner (cytochalasin B bind ing) site at the base of transmembrane segment 10.