Abstract
The GLUT1 glucose transporter has been proposed to form an aqueous substrate translocation pathway via the clustering of several amphipathic 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 possible role of transmembrane helix 8 in the formation of this permeation pathway was investigated using cysteine-scanning mutagenesis and 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 segment 8 to a cysteine. The mutant proteins were then expressed in Xenopus oocytes, and their membrane concentrations, 2-deoxyglucose uptake activities, and sensitivities to pCMBS were determined. Four positions within helix 8, alanine 309, threonine 310, serine 313, and glycine 314, were accessible to pCMBS as judged by the inhibition of transport activity. All four of these residues are clustered along one face of a putative alpha-helix. These results suggest that transmembrane segment 8 of GLUT1 forms part of the sugar permeation pathway. Updated two-dimensional models for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose binding site of GLUT1 are proposed that are consistent with existing experimental data and homology modeling based on the crystal structures of two bacterial membrane transporters.
Highlights
Passive transport of glucose across the plasma membrane of mammalian cells is mediated by members of the GLUT (SLC2a) family of membrane glycoproteins
Mutant cDNAs were constructed using oligonucleotide-mediated site-directed mutagenesis in which each of the 21 residues within transmembrane segment 8 was individually changed to a cysteine residue producing 21 mutant GLUT1 molecules, each possessing only a single cysteine residue
As we have observed for the analysis of other GLUT1 helices [13,14,15,16], the single-C mutants were expressed at varying concentrations in the oocyte plasma membrane necessitating the normalization of uptake data to expression levels to directly compare the catalytic activities of the mutants with the C-less parent
Summary
Materials—Xenopus laevis imported African frogs were purchased from Xenopus Express (Homosassa, FL). 2-[3H]Deoxyglucose and diguanosine triphosphate (mRNA cap) were purchased from Amersham Biosciences. Materials—Xenopus laevis imported African frogs were purchased from Xenopus Express (Homosassa, FL). 2-[3H]Deoxyglucose and diguanosine triphosphate (mRNA cap) were purchased from Amersham Biosciences. (Austin, TX), and a TransformerTM site-directed mutagenesis kit was obtained from Clontech. A MegascriptTM RNA synthesis kit was purchased from Ambion, Inc. 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 8 was individually changed to cysteine. Residue number refers to the amino acid numbering for human Glut given in Ref. 9. Amino acids are designated by the single-letter code
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