Transmembrane segment 3 of the Glut1 glucose transporter is an outer helix.
A model has been proposed for the structure of the Glut1 glucose transporter based on the results of mutagenesis studies and homology modeling in which eight transmembrane segments form an inner helical bundle surrounded by four outer helices. The role of transmembrane segment 3 in this structural model was investigated using cysteine-scanning mutagenesis in conjunction with the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). Twenty-one Glut1 mutants were created from a fully functional, cysteine-less, parental Glut1 molecule by successively changing each residue along transmembrane helix 3 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. Cysteine substitution at methionine 96 abolished transport activity, whereas substitutions at the other positions resulted in either modest reductions or no significant effect on transport activity. In striking contrast to all other helices that have been examined to date, only one of the 21 helix 3 single-cysteine mutants was inhibited by pCMBS, suggesting that only a small portion of this helix is exposed to the external solvent. This result is consistent with predictions based on our current structural model, in which helix 3 is one of four outer helices that surround the inner helical bundle that comprises the aqueous substrate-binding cavity. An updated two-dimensional model for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose-binding site of Glut1 is presented that is consistent with existing experimental data.
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. mike@cellbio.wustl.edu
Mike
M
Mueckler
William
W
Roach
Carol
C
Makepeace
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D003847:Deoxyglucose / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000737:chemistry; D051272:Glucose Transporter Type 1; D006801:Humans; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000378:metabolism; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014982:Xenopus laevis
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; C495677:SLC2A1 protein, human; D013439:Sulfhydryl Reagents; D003553:Cystine; D002731:4-Chloromercuribenzenesulfonate; D003847:Deoxyglucose; D005947:Glucose; D003545:Cysteine