A conserved amino acid motif (R-X-G-R-R) in the GLUT1 glucose transporter is an important determinant of membrane topology

The Glut1 glucose transporter is one of over 300 members of the major facil itator superfamily of membrane transporters. These proteins are extremely d iverse in substrate specificity and differ in their transport mechanisms. T he two most common features shared by many members of this superfamily are the presence of 12 predicted transmembrane segments and an amino acid motif , R-X-G-R-R, present at equivalent positions within the cytoplasmic loops j oining transmembrane segments 2-3 and 8-9. The structural and functional ro les of the arginine residues within these motifs in Glut1 were investigated by expression of site-directed mutant transporters in Xenopus oocytes foll owed by analyses of intrinsic transport activity and the membrane topology of mutant glycosylation-scanning reporter Glut1 molecules. Substitution of lysine residues for the cluster of 3 arginine residues in each of the 2 cyt oplasmic pentameric motifs of Glut1 revealed no absolute requirement for ar ginine side chains at any of the 6 positions for transport of 2-deoxyglucos e. However, removal of the 3 positive charges at either site by substitutio n of glycines for the arginines completely abolished transport activity as the result of a local perturbation in the membrane topology in which the cy toplasmic loop was aberrantly translocated into the exoplasm along with the two flanking transmembrane segments. Substitution of lysines for the argin ines had no affect on membrane topology. We conclude that the positive char ges in the R-X-G-R-R motif form critical local cytoplasmic anchor points in volved in determining the membrane topology of Glut1. These data provide a simple explanation for the presence of this conserved amino acid motif in h undreds of functionally diverse membrane transporters that share a common p redicted membrane topology.