Transmembrane segment 5 of the Glut1 glucose transporter is an amphipathic helix that forms part of the sugar permeation pathway.
Transmembrane segment 5 of the Glut1 glucose transporter has been proposed to form an amphipathic transmembrane helix that lines the substrate translocation pathway (Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229, 941-945). This hypothesis was tested using cysteine-scanning mutagenesis in conjunction with the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A series of 21 mutants was created from a fully functional, cysteine-less, parental Glut1 molecule by changing each residue within putative transmembrane segment 5 to cysteine. Each mutant was then expressed in Xenopus oocytes and its steady-state protein level, 2-deoxyglucose uptake activity, and sensitivity to pCMBS were measured. All 21 mutants exhibited measurable transport activity, although several of the mutants exhibited reduced activity due to a corresponding reduction in steady-state protein. Six of the amino acid side chains within transmembrane segment 5 were clearly accessible to pCMBS in the external medium, as determined by inhibition of transport activity, and a 7th residue showed inhibition that lacked statistical significance because of the extremely low transport activity of the corresponding mutant. All 7 of these residues were clustered along one face of a putative alpha-helix, proximal to the exoplasmic surface of the plasma membrane. These results comprise the first experimental evidence for the existence of an amphipathic transmembrane alpha-helix in a glucose transporter molecule and strongly suggest that transmembrane segment 5 of Glut1 forms part of the sugar permeation pathway.
Mueckler
M
M
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. mike@cellbio.wustl.edu
Makepeace
C
C
D002731:4-Chloromercuribenzenesulfonate; D000818:Animals; D001692:Biological Transport; D050260:Carbohydrate Metabolism; D018076:DNA, Complementary; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000235:genetics; D014981:Xenopus
D018076:DNA, Complementary; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D002731:4-Chloromercuribenzenesulfonate