TOPOLOGY OF THE GLUT-1 GLUCOSE-TRANSPORTER DEDUCED FROM GLYCOSYLATIONSCANNING MUTAGENESIS

The erythrocyte glucose transporter (Glut 1) is predicted to contain 1 2 membrane-spanning domains based on the hydropathy plot of its deduce d amino acid sequence, The membrane topology of Glut 1 was analyzed by a scanning mutagenesis procedure in which the glycosylated exofacial domain of Glut 4 was inserted independently into each of the putative hydrophilic soluble domains of an aglyco-Glut 1 construct. The transpo rter mutants were expressed both in vitro using a rabbit reticulocyte lysate translation system and in vivo in Xenopus oocytes. The cytoplas mic or exofacial orientation of each soluble domain was inferred from the glycosylation state of the corresponding insertion mutant. The res ults from the cell-free system were aberrant in that two topological o rientations were observed when the epitope was inserted into any of th e short cytoplasmic loops or the NH2 terminus. The in vivo data, howev er, were in complete agreement with the proposed 12-helix model. There fore, the multiple topologies observed in vitro probably resulted from the inability of the cell-free system to facilitate the proper foldin g of the insertion mutants into the membrane. 2-Deoxyglucose uptake da ta on the glycosylation mutants indicated that epitope insertion into the NH2 terminus, the large central loop, or the second, third, or fif th exofacial loop had no dramatic effect on the activity of the transp orter. However, insertion into the other soluble domains either comple tely abolished or significantly reduced transport activity.