Abstract
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.
Highlights
Facilitated diffusion of glucose across the membranes of animal cells is mediated by members of the Glut (SLC2a) family of membrane glycoproteins
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
Hydroxyl- and amide-containing amino acid side chains within these helices were predicted to form the sugar-binding site(s) of Glut1 via hydrogen bond formation with glucose hydroxyl groups [9]. Experimental support for this structural model has come from cysteine-scanning mutagenesis and substituted cysteine accessibility studies, which implicate transmembrane segments 1 [10], 2 [11], 5 [12], 7 [11, 13], 8 [14], [15], and [16] of Glut1 in the formation of an inner helical bundle that comprises a water-accessible cavity within the membrane
Summary
Materials—Xenopus laevis imported African frogs were purchased from Xenopus Express (Homosassa, FL), 3H-2-deoxyglucose and diguanosine triphosphate (mRNA cap) were purchased from Amersham Biosciences, the MegascriptTM RNA synthesis kit was purchased from Ambion Inc. (Austin, TX), and the TransformerTM sitedirected mutagenesis kit was obtained from Clontech (Palo Alto, CA). (Austin, TX), and the TransformerTM sitedirected mutagenesis kit was obtained from Clontech (Palo Alto, CA). Sequencing of human Glut cDNA and the in vitro transcription and purification of Glut mRNAs [19], isolation, microinjection, and incubation of Xenopus oocytes [20], preparation of purified oocyte plasma membranes and indirect immunofluorescence laser confocal microscopy [21], SDS-polyacrylamide gel electrophoresis and TABLE I Cysteine scanning mutagenesis of helix 3. 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 3 was individually changed to cysteine. Refers to the amino acid numbering for human Glut given by Mueckler et al [7].
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