ANALYSIS OF THE STRUCTURAL FEATURES OF THE C-TERMINUS OF GLUT1 THAT ARE REQUIRED FOR TRANSPORT CATALYTIC ACTIVITY

C-terminally truncated and mutated forms of GLUT1 have been constructe d to determine the minimum structure at the C-terminus required for gl ucose transport activity and ligand binding at the outer and inner bin ding sites. Four truncated mutants have been constructed (CTD24 to CTD 27) in which 24 to 27 amino acids are deleted. In addition, point subs titutions of R468 --> L, F467 --> L and G466 --> E have been produced. Chinese hamster ovary clones which were transfected with these mutant GLUT1s were shown, by Western blotting and cell-surface carbohydrate labelling, to have expression levels which were comparable with the wi ld-type clone. Wild-type levels of 2-deoxy-D-glucose transport activit y were retained only in the clone transfected with the construct in wh ich 24 amino acids were deleted (CTD24). The CTD25, CTD26 and CTD27 cl ones showed markedly reduced transport activity. From a kinetic compar ison of the CTD24 and CTD26 clones it was found that the reduced trans port was mainly associated with a reduced V-max. value for 2-deoxy-D-g lucose uptake but with a slight lowering of the K-m. These data establ ish that the 24 amino acids at the C-terminus of GLUT1 are not require d for the transport catalysis. However, the point mutations of F467L a nd G466E (26 and 27 residues from the C-terminus) did not significantl y perturb the kinetics of 2-deoxy-D-glucose transport. The substitutio n of R468L produced a slight, but significant, lowering of the K-m. Th e ability of the truncated GLUT1s to bind the exofacial ligand, )benzo yl-1,3-bis-(D-mannas-4-yl-oxy)-2-propylamine (ATB-BMPA), and the endof acial ligand, cytochalasin B, were assessed by photolabelling procedur es. The ability to bind ATB-BMPA was retained only in the CTD24 trunca ted mutant and was reduced to levels comparable with those of the non- transfected clone in the other mutant clones. Cytochalasin B labelling was unimpaired in all four mutated GLUT1s. These data establish that a minimum structure at the C-terminus of GLUT1, which is required for the conformational change to expose the exofacial site, includes amino acids at positions Phe-467 and Arg-468; however, these amino acids ar e not individually essential.