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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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4-Chloromercuribenzenesulfonate / chemistry
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Amino Acid Substitution / genetics
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Animals
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Cell Membrane / chemistry
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Cell Membrane / genetics
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Cell Membrane / metabolism
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Cell Membrane Permeability / genetics
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Cysteine / genetics*
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Ethylmaleimide / chemistry
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Extracellular Fluid / chemistry*
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Extracellular Fluid / metabolism
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Glucose / metabolism*
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Glucose Transporter Type 1
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Humans
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Monosaccharide Transport Proteins / chemistry*
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Monosaccharide Transport Proteins / genetics
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Monosaccharide Transport Proteins / metabolism
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Mutagenesis, Site-Directed*
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Oocytes / chemistry
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Oocytes / metabolism
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Peptide Fragments / chemistry*
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Peptide Fragments / genetics
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Peptide Fragments / metabolism
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Protein Structure, Secondary / genetics
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Protein Structure, Tertiary / genetics
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Protein Transport / genetics
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Sulfhydryl Reagents / chemistry
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Xenopus
Substances
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Glucose Transporter Type 1
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Monosaccharide Transport Proteins
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Peptide Fragments
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SLC2A1 protein, human
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Sulfhydryl Reagents
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4-Chloromercuribenzenesulfonate
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Glucose
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Cysteine
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Ethylmaleimide