The large cytoplasmic loop of the glucose transporter GLUT1 is an essential structural element for function

Alanine scanning mutagenesis and the introduction of deletions and insertio ns were used to address the role of the large cytoplasmic loop in 2-deoxy-D -glucose (2-DOG) uptake by GLUT1 expressed in Xenopus oocytes. Alanine scan ning mutagenesis of 29 amino acid residues that are identical or homologous in GLUT1 to GLUT4 demonstrated that the transport activities of only a few variants were affected. Progressive truncation of the loop by six deletion s leaving intact 59 (Delta 236-241), 49 (Delta 231-246), 39 (Delta 226-251) ,28 (Delta 221-257),18 (Delta 216-262), or 10 (Delta 213-267) amino acid re sidues resulted in a progressive decrease in 2-DOG uptake. Compared with wi ld-type GLUT1 the uptake rates varied between 33% for the Delta 236-241 mut ant and 4% for the Delta 213-267 mutant. Insertional mutagenesis using hexa alanine or hexa-glycine to fill in the deletion D-236-L-241 restored 2-DOG uptake to 73% of wild-type GLUT1 in the case of hexaalanine, whereas hexagl ycine insertion was without effect. Confocal laser microscopy demonstrated that a deletion of six amino acid residues did; not influence the expressio n level in the plasma membrane (Delta 236-241 mutant), whereas the plasma m embrane fluorescence of the Delta 213-267 mutant was comparable with that o f water-injected Xenopus oocytes. Computer-aided secondary structure predic tion of the loop suggested that it consists of a long alpha -helix bundle i nterrupted or kinked by the highly conserved glycine-233.