Abstract
A low resolution model has been proposed for the exofacial conformation of the Glut1 glucose transporter in which eight transmembrane segments form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 4, predicted to be an inner helix in this structural model, was investigated by cysteine-scanning mutagenesis in conjunction with the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A functional, cysteine-less, parental Glut1 molecule was used to produce 21 Glut1 point mutants by individually changing each residue along transmembrane helix 4 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. In striking contrast to all of the other seven predicted inner helices, none of the 21 helix 4 single-cysteine mutants was demonstrably inhibited by pCMBS. However, cysteine substitution within helix 4 resulted in an unusually high number of severely transport-defective mutants. The low absolute transport activities of two of these mutants (G130C and G134C) were due to their extremely low levels of expression, presumably a result of structural instability and consequent degradation in oocytes, suggesting that these two residues play an important role in maintaining the native structure of Glut1. The other two transport-defective mutants (Y143C and E146C) exhibited low specific transport activities, implying that these two residues play an important role in the transport cycle. Based on these data, we conclude that the exoplasmic end of helix 4 lies outside the inner helical bundle in the exofacial configuration of Glut1.
Highlights
Glut1 was predicted to possess 12 transmembrane helices based on hydrophobicity analysis of the amino acid sequence deduced from the sequence of a human cDNA clone [6]
The results of cysteine-scanning mutagenesis and substituted cysteine accessibility studies are generally consistent with this crude model and implicate transmembrane segments 1 [9], 2 [10], 5 [11], 7 [10, 12], 8 [13], [14], and [15] of Glut1 in the formation of an inner helical bundle that comprises a water-accessible cavity within the membrane
Materials—Imported female African Xenopus laevis frogs were purchased from Xenopus Express (Homosassa, FL), [3H]2-deoxyglucose and diguanosine triphosphate were purchased from Amersham Biosciences, MegascriptTM RNA synthesis kits were purchased from Ambion, Inc. (Austin, TX), and TransformerTM site-directed mutagenesis kits were obtained from Clontech (Palo Alto, CA)
Summary
Materials—Imported female African Xenopus laevis frogs were purchased from Xenopus Express (Homosassa, FL), [3H]2-deoxyglucose and diguanosine triphosphate were purchased from Amersham Biosciences, MegascriptTM RNA synthesis kits were purchased from Ambion, Inc. (Austin, TX), and TransformerTM site-directed mutagenesis kits were obtained from Clontech (Palo Alto, CA). (Austin, TX), and TransformerTM site-directed mutagenesis kits were obtained from Clontech (Palo Alto, CA). General Procedures—Procedures for the site-directed mutagenesis and 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-PAGE and immunoblotting with Glut C-termi-.
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