Overexpression of stomatin depresses GLUT-1 glucose transporter activity

We showed previously that GLUT-1 glucose transporter is associated with stomatin (band 7.2b) in human red blood cell membranes and in Clone 9 cells. We show here that in a mixed population of stably transfected cells, overexpression of either murine or human stomatin resulted in 35-50% reduction in the basal rate of glucose transport. Moreover, there was a correlation between increased expression of stomatin and depression in the rate of glucose transport. In two clones chosen for further study, the similar to 10% and similar to 70% reduction in basal rate of glucose transport was associated with increases in stomatin mRNA and protein expression without a detectable change in GLUT-1 content in plasma membranes of either clone. In the clone overexpressing high levels of stomatin, immunoprecipitated GLUT-1 was associated with a large amount of stomatin as a coimmunoprecipitant. Employing extracts of cells overexpressing human stomatin, we found that stomatin bound to the glutathione-S-transferase (GST) fusion protein containing the COOH-terminal 42-amino acid segment of GLUT-1 but not to GST alone or a GST fusion protein containing the 66-amino acid central loop of GLUT-1. Rat stomatin cDNA was cloned by RT-PCR and found to be highly homologous to mouse (97%) and human (86%) stomatins. These results suggest that overexpression of stomatin results in a depression in the basal rate of glucose transport by decreasing the "intrinsic" activity of GLUT-1, probably through protein-protein interaction.