Genetic evidence indicating the identity of the cytochalasin B photolabelled components in rat myoblasts

While photolabelling with cytochalasin B (CB) has been widely used in the identification of eukaryotic glucose transporters, there is presently no unequivocal evidence indicating that the CB-labelled components are indeed the glucose transporters. A combination of biochemical, physiological and genetic manipulations was used in the present investigation to demonstrate that the plasma membrane hexose transporters can indeed by photolabelled by CB. In this study, plasma membranes from glucose-grown and glucose-starved hexose transport mutant D23 and its parental L6 cells were photolyzed in the presence of 3H-CB. The amount of CB bound to the 40-60 kDa region (CB50) was found to be differentially inhibited by D-glucose, 2-deoxy-D-glucose (dGlc) and 3-O-methyl-glucose (MeGlc). Mutant D23 exhibited not only reduced hexose transport activity but also significantly lower level of CB50. Glucose-starvation resulted not only in elevated hexose transport activity but also increased level of CB50. It should be noted glucose-starvation did not have much effect on the hexose transport activity and on the level of CB50 in mutant D23. The present study provides the first genetic evidence indicating that the CB-labelled component(s) are indeed associated with the hexose transport systems.