The Large Cytoplasmic Loop of the Glucose Transporter GLUT1 Is an Essential Structural Element for Function
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Ingrid Monden
Abstract
Alanine scanning mutagenesis and the introduction of deletions and insertions were used to address the role of the large cytoplasmic loop in 2-deoxyDglucose (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 (Δ236241), 49 (Δ231246), 39 (Δ226251), 28 (Δ221257), 18 (Δ216262), or 10 (Δ213267) amino acid residues resulted in a progressive decrease in 2-DOG uptake. Compared with wildtype GLUT1 the uptake rates varied between 33% for the Δ236241 mutant and 4% for the Δ213267 mutant. Insertional mutagenesis using hexaalanine or hexaglycine to fill in the deletion 236D-241L restored 2-DOG uptake to 73% of wildtype 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 (Δ236241 mutant), whereas the plasma membrane fluorescence of the Δ213267 mutant was comparable with that of waterinjected Xenopus oocytes. Computeraided secondary structure prediction of the loop suggested that it consists of a long αhelix bundle interrupted or kinked by the highly conserved glycine-233.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
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- A Microsomal Ecdysone-Binding Cytochrome P450 from the Insect Locusta migratoria Purified by Sequential Use of Type-II and Type-I Ligands
- The Large Cytoplasmic Loop of the Glucose Transporter GLUT1 Is an Essential Structural Element for Function
- Visual Representation by Atomic Force Microscopy (AFM) of Tomato Spotted Wilt Virus Ribonucleoproteins
- Elimination of Hydrocortisone from the Medium Enables Tissue Plasminogen Activator Gene Expression by Normal and Immortalized Nonmalignant Human Epithelial Cells
- Molecular Cloning and Biochemical Characterisation of Proteases from Staphylococcus epidermidis
- Splice Variants of Human Cathepsin L mRNA Show Different Expression Rates
- Osmolytes as Modulators of Conformational Changes in Serpins
- Protective Activity of Aromatic Amines and Imines against Oxidative Nerve Cell Death
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