Domain assembly of the GLUT1 glucose transporter

• Published in: Biochemical journal 1994-06, Vol.300 (2), p.291-294
• Main Authors: COPE, D. L, HOLMAN, G. D, BALDWIN, S. A, WOLSHTENHOLME, A. J
• Format: Article
• Language: English
• Subjects: Affinity Labels; Analytical, structural and metabolic biochemistry; Animals; Azides - metabolism; Baculoviridae - genetics; Binding and carrier proteins; Biological and medical sciences; Cells, Cultured; Cytochalasin B - metabolism; Disaccharides - metabolism; Fundamental and applied biological sciences. Psychology; Glucose - metabolism; Glucose Transporter Type 1; Glycosides; Humans; Ligands; Monosaccharide Transport Proteins - biosynthesis; Monosaccharide Transport Proteins - genetics; Monosaccharide Transport Proteins - metabolism; Moths; Propylamines; Proteins; Rabbits; Recombinant Proteins - genetics; Recombinant Proteins - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3000291

A full-length construct of the glucose transporter isoform GLUT1 has been expressed in Sf9 (Spodoptera frugiperida Clone 9) insect cells, and a photolabelling approach has been used to show that the expressed protein binds the bismannose compound 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propylamine (ATB-BMPA) and cytochalasin B at its exofacial and endofacial binding sites respectively. Constructs of GLUT1 which produce either the N-terminal (amino acids 1-272) or C-terminal (amino acids 254-492) halves are expressed at levels in the plasma membrane which are similar to that of the full-length GLUT1 (approximately 200 pmol/mg of membrane protein), but do not bind either ATB-BMPA or cytochalasin B. When Sf9 cells are doubly infected with virus constructs producing both the C- and N-terminal halves of GLUT1, then the ligand labelling is restored. Only the C-terminal half is labelled, and, therefore, the labelling of this domain is dependent on the presence of the N-terminal half of the protein. These results suggest that the two halves of GLUT1 can assemble to form a stable complex and support the concept of a bilobular structure for the intact glucose transporters in which separate C- and N-domain halves pack together to produce a ligand-binding conformation.