REPLACEMENT OF BATH TRYPTOPHAN RESIDUES AT 388 AND 412 COMPLETELY ABOLISHED CYTOCHALASIN-B PHOTOLABELING OF THE GLUT1 GLUCOSE-TRANSPORTER

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose trypt ophans 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-3 88, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mut ant and the Trp-412 mutant which had leucine at 388 and 412 respective ly. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously fo r the Trp-412 mutant. Cytochalasin B labelling was finally abolished c ompletely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT 1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the label ling efficiency of these transporters is similar. These findings sugge st that cytochalasin B binds to the transmembrane domain of the glucos e transporter in the vicinity of helix 10-11, and is inserted covalent ly by photoactivation at either the 388 or the 412 site.