SERINE-294 AND THREONINE-295 IN THE EXOFACIAL LOOP DOMAIN BETWEEN HELICE-7 AND HELICE-8 OF GLUCOSE TRANSPORTERS (GLUT) ARE INVOLVED IN THE CONFORMATIONAL ALTERATIONS DURING THE TRANSPORT PROCESS

The role of a conserved polar motif (STS) in the exofacial loop betwee n helices 7 and 8 of GLUT4 for transporter function was investigated b y site-directed mutagenesis and expression of the constructs in COS-7 cells. Reconstituted glucose-transport activity, cytochalasin B bindin g and photolabelling with the exofacial label thyl)benzoyl-1,3-bis-(D- mannosyloxy)-2-propylamine (ATB-BMPA) were assayed in membranes from t ransfected cells and corrected for immunoreactivity of expressed trans porters. Replacement of Ser-294 with Ala or Thr suppressed transport a ctivity and cytochalasin B binding. ATB-BMPA photolabelling was normal in S294A mutants, and even increased in S294T mutants. Replacement of Thr-295 with Ala suppressed transport activity and cytochalasin B bin ding, whereas ATB-BMPA photolabelling was normal; substitution of Ser failed to alter the investigated parameters. Similarly, exchanging Ser -296 for Ala generated a normally functioning protein. The data sugges t that Ser-294 and Thr-295 are involved in the conformational change i n GLUT during the transport process, and that their substitution may a rrest the transporter in an outward-facing conformation.