Abstract
The Glut1 glucose transporter has been proposed to form an aqueous sugar translocation pathway through the lipid bilayer via the clustering of several transmembrane helices (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). The participation of transmembrane helix 10 in the formation of this putative aqueous tunnel 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 10 to cysteine. Each mutant was then expressed in Xenopus oocytes, and its plasma membrane content, 2-deoxyglucose uptake activity, and sensitivity to pCMBS were measured. Helix 10 exhibited a highly distinctive reaction profile to scanning mutagenesis whereby cysteine substitution at residues within the cytoplasmic N-terminal half of the helix tended to increase specific transport activity, whereas substitution at residues within the exoplasmic C-terminal half of the helix tended to decrease specific transport activity. Four residues within helix 10 were clearly accessible to pCMBS as judged by inhibition or stimulation of transport activity. All four of these residues were clustered along one face of a putative alpha-helix. These results combined with previously published data suggest that transmembrane segment 10 of Glut1 forms part of the sugar permeation pathway. Two-dimensional models for the conformation of the 12 transmembrane helices and the exofacial glucose-binding site of Glut1 are proposed that are consistent with existing experimental data.
Highlights
Facilitative transport of glucose into mammalian cells is mediated by members of the Glut (SLC2a) family of membrane glycoproteins
To determine which transmembrane residues are accessible to the external aqueous solvent and may comprise part of the sugar permeation pathway, transport activity was measured for each of the 21 mutants after incubation in the presence of the membrane-impermeant sulfhydryl-specific reagent, pCMBS (Fig. 3B), and compared with the activities measured in the presence of vehicle alone (Fig. 3A)
Among the five transmembrane segments of Glut1 that have been analyzed far by cysteine-scanning mutagenesis, helix 10 exhibits a unique pattern of sensitivity wherein residues within the exoplasmic half of the helix tend to be inhibited by cysteine substitution, and residues within the cytoplasmic half of the helix are stimulated by substitution
Summary
Materials—Xenopus laevis imported African frogs were purchased from Xenopus Express (Homosassa, FL), [2-3H]deoxyglucose and. CDNA encoding cysteine-less human Glut was subjected to oligonucleotide-mediated, site-directed mutagenesis, creating a series of 21 mutant cDNAs in which each of the 21 residues within transmembrane helix 10 was individually changed to cysteine. Residue No refers to the amino acid numbering for human Glut given in Muecker et al [8]
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