Analysis of Transmembrane Segment 10 of the Glut1 Glucose Transporter by Cysteine-scanning Mutagenesis and Substituted Cysteine Accessibility

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, pchloromercuribenzenesulfonate (pCMBS). A series of 21 mutants was created from a fully functional, cysteineless, 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 ␣-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.

Analysis of Transmembrane Segment 8 of the GLUT1 Glucose Transporter by Cysteine-scanning Mutagenesis and Substituted Cysteine Accessibility

“…1b). 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. The Oocytes were washed four times in Barth's saline and then subjected to 2-deoxyglucose uptake measurements under the conditions described in the legend to Fig.…”

Structure of, and functional insight into the GLUT family of membrane transporters

“…45,62,63 Cysteine scanning mutagenesis was subsequently employed to probe which residues lined this proposed central pore. [64][65][66][67][68][69][70][71][72][73][74] The resulting model indicated that TMs 2, 4, 5, 7, 8, 11, and possibly 1 and 10 formed a central aqueous transport channel for glucose, whereas TMs 3, 6, 9, and 12 formed a structural scaffold on the outside of the protein. 70 A possible substrate binding site was also proposed, involving Q161, Q282, and W412.…”

Functional Studies of Threonine 310 Mutations in Glut1

“…This hydrophilic amino acid is predicted to be near (5) or at (18) the exofacial end of helix 8, which, in turn, is predicted to be involved in the formation of the water-accessible glucose channel (17,18,35). The threonine hydrophilic side chain with its hydroxyl group may play an important role in forming hydrogen bonds with glucose (36).…”

“…Based on experimental observations of substituted cysteine accessibility, Mueckler and Makepeace argued that a glucosetransporting pathway in Glut1 could be lined by helices 5,7,8,10, and 11 (17). Based on the suggested helical bundle arrangement of Lac permease, Zuniga et al recently proposed a threedimensional model for Glut1 that accounts for mutational and biochemical evidence (18).…”

“…11). Also, whole transmembrane domains or other conserved domains have been studied by site-directed mutagenesis (11)(12)(13)(14)(15)(16)(17). These studies have provided additional insights into the structure/function relationships within Glut1.…”