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 insertions 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 scanning 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 deletions leaving intact 59 (delta236-241), 49 (delta231-246), 39 (delta226-251), 28 (delta221-257), 18 (delta216-262), or 10 (delta213-267) amino acid residues resulted in a progressive decrease in 2-DOG uptake. Compared with wild-type GLUT1 the uptake rates varied between 33% for the delta236-241 mutant and 4% for the delta213-267 mutant. Insertional mutagenesis using hexaalanine or hexaglycine to fill in the deletion 236D-241L restored 2-DOG uptake to 73% of wild-type GLUT1 in the case of hexaalanine, whereas hexaglycine insertion was without effect. Confocal laser microscopy demonstrated that a deletion of six amino acid residues did not influence the expression level in the plasma membrane (delta236-241 mutant), whereas the plasma membrane fluorescence of the delta213-267 mutant was comparable with that of water-injected Xenopus oocytes. Computer-aided secondary structure prediction of the loop suggested that it consists of a long alpha-helix bundle interrupted or kinked by the highly conserved glycine-233.
Monden
I
I
Institut für Pharmakologie, Freie Universität Berlin, Germany.
Olsowski
A
A
Krause
G
G
Keller
K
K
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000465:Algorithms; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism* / Q000648:ultrastructure; D017353:Gene Deletion; D005947:Glucose / Q000378:metabolism; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D005998:Glycine / Q000737:chemistry; D006801:Humans; D008845:Microinjections; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D009865:Oocytes / Q000378:metabolism; D014981:Xenopus
D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495677:SLC2A1 protein, human; C495692:SLC2A4 protein, human; D005947:Glucose; D000409:Alanine; D005998:Glycine