Domain assembly of the GLUT1 glucose transporter.
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.
Cope
D L
DL
Department of Biochemistry, University of Bath, U.K.
Holman
G D
GD
Baldwin
S A
SA
Wolstenholme
A J
AJ
D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D003571:Cytochalasin B / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D006027:Glycosides; D006801:Humans; D008024:Ligands; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D009036:Moths; D011437:Propylamines; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D004187:Disaccharides; D051272:Glucose Transporter Type 1; D006027:Glycosides; D008024:Ligands; D009004:Monosaccharide Transport Proteins; D011437:Propylamines; D011994:Recombinant Proteins; C495677:SLC2A1 protein, human; C065412:2-N-(4-(1-azitrifluoroethyl)benzoyl)-1,3-bis-(mannos-4-yloxy)-2-propylamine; D003571:Cytochalasin B; D005947:Glucose