Modulation of GLUT1 intrinsic activity in clone 9 cells by inhibition of oxidative phosphorylation

Brief (1-2 h) exposure of Clone 9 cells to inhibitors of oxidative phosphorylation such as azide is known to markedly increase glucose uptake. Clone 9 cells express GLUT1 but not GLUT2, -3, and -4, and the azide effect was not accompanied by any increase in cellular or plasma membrane GLUT1 level. To identify the molecular event underlying this apparent increase in GLUT1 intrinsic activity, we studied the acute effects of azide on the substrate binding activity of GLUT1 in Clone 9 cells by measuring glucose-sensitive cytochalasin B binding. The glucose-displaceable, cytochalasin B binding activity was barely detectable in membranes isolated from Clone 9 cells under control conditions but was readily detectable after a 60-min incubation of cells in the presence of 5 mM azide showing a 3-fold increase in binding capacity with no change in binding affinity. Furthermore, the cytochalasin B binding activity of purified human erythrocyte GLUT1 reconstituted in liposomes was significantly reduced in the presence of cytosol derived from azide-treated Clone 9 cells but not in the presence of cytosol from control cells; this effect was heat-labile and abolished by the presence of the peptide corresponding to the GLUT1 COOH-terminal sequence. These results suggest that a cytosolic protein in Clone 9 cells binds to GLUT1 at its COOH-terminal domain and inhibits its substrate binding and that azide-induced metabolic alteration releases GLUT1 from this inhibitory interaction. Studying the binding of cytosolic proteins derived from 35S-labeled Clone 9 cells to glutathione S-transferase fusion protein containing glucose transporter COOH-terminal sequences, we identified 28- and 70-kDa proteins that bind specifically to the cytoplasmic domain of GLUT1 and GLUT4 in vitro. We also found a 32P-labeled, 85-kDa protein that binds to GLUT4 but not to GLUT1 and only in cytosol derived from azide-treated cells. The roles, if any, of these glucose transporter-binding proteins in the azide-sensitive modulation of GLUT1 substrate binding activity in Clone 9 cells are yet to be determined.