The predicted ATP-binding domains in the hexose transporter GLUT1 critically affect transporter activity

The glucose transporter GLUT1 has three short amino acid sequences (domains I-III) with homology to typical ATP-binding domains. GLUT1 is a facilitati ve transporter, however, and transports its substrates down a concentration gradient without a specific requirement For energy or hydrolysis of ATP. T herefore, we assessed the functional role of the predicted ATP-binding doma ins in GLUT1 by site-directed mutagenesis and expression in Xenopus oocytes , For each mutant, we determined the level of protein expression and the ki netics of transport under zero-trans influx, zero-trans efflux, and equilib rium exchange conditions. Although all five mutants were expressed at level s similar to that of the wild-type GLUT1, each single amino acid chance in domains I or III profoundly affected GLUT1 function. The mutants Cly(116)-- > Ala in domain L and Gly(332)--> Ala in domain III exhibited only 10-20% o f the transport activity of the wild-type GLUT1. The mutants Gly(111)--> Al a in domain I and Leu(336)--> Ala in domain III showed altered kinetic prop erties: neither the apparent K-m nor the V-max for 3-methylglucose transpor t were increased under equilibrium exchange conditions, and they did not sh ow the expected level of countertransport acceleration. The mutant Lys(117) --> Arg in domain I showed a marked increase in the apparent K-m for 3-meth ylglucose transport under zero-trans efflux and equilibrium exchange condit ions while maintaining countertransport acceleration. These results indicat e that the predicted ATP-binding domains I and III in GLUT1 are important c omponents of the region in GLUT1 involved in transport of the substrate and that their integrity is: critical for maintaining the activity and kinetic properties of the transporter.