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 facilitator superfamily of membrane transporters. These proteins are extremely diverse in substrate specificity and differ in their transport mechanisms. The 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 joining transmembrane segments 2-3 and 8-9. The structural and functional roles of the arginine residues within these motifs in Glut1 were investigated by expression of site-directed mutant transporters in Xenopus oocytes followed 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 cytoplasmic pentameric motifs of Glut1 revealed no absolute requirement for arginine side chains at any of the 6 positions for transport of 2-deoxyglucose. However, removal of the 3 positive charges at either site by substitution of glycines for the arginines completely abolished transport activity as the result of a local perturbation in the membrane topology in which the cytoplasmic loop was aberrantly translocated into the exoplasm along with the two flanking transmembrane segments. Substitution of lysines for the arginines had no affect on membrane topology. We conclude that the positive charges in the R-X-G-R-R motif form critical local cytoplasmic anchor points involved in determining the membrane topology of Glut1. These data provide a simple explanation for the presence of this conserved amino acid motif in hundreds of functionally diverse membrane transporters that share a common predicted membrane topology.
Sato
M
M
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Mueckler
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D017124:Conserved Sequence / Q000235:genetics*; D005786:Gene Expression Regulation; D051272:Glucose Transporter Type 1; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D008845:Microinjections; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D014981:Xenopus
D051272:Glucose Transporter Type 1; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human