title: Cysteine-scanning mutagenesis and substituted cysteine accessibility analysis of transmembrane segment 4 of the Glut1 glucose transporter.
abstract: A low resolution model has been proposed for the exofacial conformation of the Glut1 glucose transporter in which eight transmembrane segments form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 4, predicted to be an inner helix in this structural model, was investigated by cysteine-scanning mutagenesis in conjunction with the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A functional, cysteine-less, parental Glut1 molecule was used to produce 21 Glut1 point mutants by individually changing each residue along transmembrane helix 4 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. In striking contrast to all of the other seven predicted inner helices, none of the 21 helix 4 single-cysteine mutants was demonstrably inhibited by pCMBS. However, cysteine substitution within helix 4 resulted in an unusually high number of severely transport-defective mutants. The low absolute transport activities of two of these mutants (G130C and G134C) were due to their extremely low levels of expression, presumably a result of structural instability and consequent degradation in oocytes, suggesting that these two residues play an important role in maintaining the native structure of Glut1. The other two transport-defective mutants (Y143C and E146C) exhibited low specific transport activities, implying that these two residues play an important role in the transport cycle. Based on these data, we conclude that the exoplasmic end of helix 4 lies outside the inner helical bundle in the exofacial configuration of Glut1.
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. mike@cellbio.wustl.edu
authors.forename: Mike
authors.initials: M
authors.lastname: Mueckler
authors.forename: Carol
authors.initials: C
authors.lastname: Makepeace
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D003847:Deoxyglucose / Q000378:metabolism; D005260:Female; D051272:Glucose Transporter Type 1 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D051272:Glucose Transporter Type 1; D011994:Recombinant Proteins; C495677:SLC2A1 protein, human; D003847:Deoxyglucose; D003545:Cysteine