Cysteine-scanning mutagenesis of transmembrane segment 1 of glucose transporter GLUT1: extracellular accessibility of helix positions.
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.
Heinze
Matthias
M
Institute of Pharmacology, Freie Universität Berlin, Thielallee 67-73, D-14195 Berlin, FRG.
Monden
Ingrid
I
Keller
Konrad
K
D002731:4-Chloromercuribenzenesulfonate / Q000737:chemistry; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000235:genetics; D003545:Cysteine / Q000235:genetics*; D005033:Ethylmaleimide / Q000737:chemistry; D045604:Extracellular Fluid / Q000737:chemistry* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D006801:Humans; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D013439:Sulfhydryl Reagents / Q000737:chemistry; D014981:Xenopus
D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; C495677:SLC2A1 protein, human; D013439:Sulfhydryl Reagents; D002731:4-Chloromercuribenzenesulfonate; D005947:Glucose; D003545:Cysteine; D005033:Ethylmaleimide