ABSTRACT
Transmembrane segment 1 of the cysteine-less GLUT1 glucose transporter was subjected to cysteine-scanning mutagenesis. The majority of single-cysteine mutants were functional transporters, as assessed by 2-deoxy-d-glucose uptake or 3-O-methyl-d-glucose transport. Substitution of cysteine for Leu-21, Gly-22, Ser-23, Gln-25, and Gly-27, however, led to uptake rates that were less than 10% of that of the nonmutated cysteine-less GLUT1. NEM, a membrane-permeable agent, was used to identify positions that are sensitive to transport alteration by sulfhydryl reagents, whereas uptake modification by the membrane-impermeant pCMBS indicated accessibility to water-soluble solutes from the external cell environment. Twelve of the 21 single-cysteine mutants were significantly (p < 0.01) affected by NEM, and on the basis of this sensitivity, four positions were identified by pCMBS to form a water-accessible surface within helix 1. The pCMBS-sensitive positions are localized at the exofacial C-terminal end along a circumference of the helix.
Subject(s)
Cysteine/genetics , Extracellular Fluid/chemistry , Glucose/metabolism , Monosaccharide Transport Proteins/chemistry , Mutagenesis, Site-Directed , Peptide Fragments/chemistry , 4-Chloromercuribenzenesulfonate/chemistry , Amino Acid Substitution/genetics , Animals , Cell Membrane/chemistry , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Membrane Permeability/genetics , Ethylmaleimide/chemistry , Extracellular Fluid/metabolism , Glucose Transporter Type 1 , Humans , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Oocytes/chemistry , Oocytes/metabolism , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Structure, Secondary/genetics , Protein Structure, Tertiary/genetics , Protein Transport/genetics , Sulfhydryl Reagents/chemistry , XenopusABSTRACT
The functional consequences of an in vivo heterozygous insertion mutation in the human facilitated glucose transporter isoform 1 (GLUT1) gene were investigated. The resulting frameshift in exon 10 changed the primary structure of the C-terminus from 42 in native GLUT1 to 61 amino acid residues in the mutant. Kinetic studies on a patient's erythrocytes were substantiated by expressing the mutant cDNA in Xenopus laevis oocytes. K(m) and V(max) values were clearly decreased explaining pathogenicity. Targeting to the plasma membrane was comparable between mutant and wild-type GLUT1. Transport inhibition by cytochalasin B was more effective in the mutant than in the wild-type transporter. The substrate specificity of GLUT1 remained unchanged.
