Topology of the Glut 1 glucose transporter deduced from glycosylation scanning mutagenesis.
The erythrocyte glucose transporter (Glut 1) is predicted to contain 12 membrane-spanning domains based on the hydropathy plot of its deduced 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 transporter mutants were expressed both in vitro using a rabbit reticulocyte lysate translation system and in vivo in Xenopus oocytes. The cytoplasmic or exofacial orientation of each soluble domain was inferred from the glycosylation state of the corresponding insertion mutant. The results from the cell-free system were aberrant in that two topological orientations were observed when the epitope was inserted into any of the short cytoplasmic loops or the NH2 terminus. The in vivo data, however, were in complete agreement with the proposed 12-helix model. Therefore, the multiple topologies observed in vitro probably resulted from the inability of the cell-free system to facilitate the proper folding of the insertion mutants into the membrane. 2-Deoxyglucose uptake data on the glycosylation mutants indicated that epitope insertion into the NH2 terminus, the large central loop, or the second, third, or fifth exofacial loop had no dramatic effect on the activity of the transporter. However, insertion into the other soluble domains either completely abolished or significantly reduced transport activity.
Hresko
R C
RC
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Kruse
M
M
Strube
M
M
Mueckler
M
M
D000595:Amino Acid Sequence; D000818:Animals; D051272:Glucose Transporter Type 1; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics; D016254:Mutagenesis, Insertional; D009865:Oocytes; D014176:Protein Biosynthesis; D011487:Protein Conformation; D014981:Xenopus
D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human