Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter.
A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.
Inukai
K
K
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Asano
T
T
Katagiri
H
H
Anai
M
M
Funaki
M
M
Ishihara
H
H
Tsukuda
K
K
Kikuchi
M
M
Yazaki
Y
Y
Oka
Y
Y
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003571:Cytochalasin B / Q000378:metabolism* / Q000494:pharmacology; D003847:Deoxyglucose / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000378:metabolism; D051272:Glucose Transporter Type 1; D007930:Leucine / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry*
D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D014364:Tryptophan; D003847:Deoxyglucose; D007930:Leucine; D005947:Glucose