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Comparative Study
, 48 (25), 5934-42

Model of the Exofacial Substrate-Binding Site and Helical Folding of the Human Glut1 Glucose Transporter Based on Scanning Mutagenesis

Affiliations
Comparative Study

Model of the Exofacial Substrate-Binding Site and Helical Folding of the Human Glut1 Glucose Transporter Based on Scanning Mutagenesis

Mike Mueckler et al. Biochemistry.

Abstract

Transmembrane helix 9 of the Glut1 glucose transporter was analyzed by cysteine-scanning mutagenesis and the substituted cysteine accessibility method (SCAM). A cysteine-less (C-less) template transporter containing amino acid substitutions for the six native cysteine residues present in human Glut1 was used to generate a series of 21 mutant transporters by substituting each successive residue in predicted transmembrane segment 9 with a cysteine residue. The mutant proteins were expressed in Xenopus oocytes, and their specific transport activities were directly compared to that of the parental C-less molecule whose function has been shown to be indistinguishable from that of native Glut1. Only a single mutant (G340C) had activity that was reduced (by 75%) relative to that of the C-less parent. These data suggest that none of the amino acid side chains in helix 9 is absolutely required for transport function and that this helix is not likely to be directly involved in substrate binding or translocation. Transport activity of the cysteine mutants was also tested after incubation of oocytes in the presence of the impermeant sulfhydryl-specific reagent, p-chloromercuribenzene sulfonate (pCMBS). Only a single mutant (T352C) exhibited transport inhibition in the presence of pCMBS, and the extent of inhibition was minimal (11%), indicating that only a very small portion of helix 9 is accessible to the external solvent. These results are consistent with the conclusion that helix 9 plays an outer stabilizing role for the inner helical bundle predicted to form the exofacial substrate-binding site. All 12 of the predicted transmembrane segments of Glut1 encompassing 252 amino acid residues and more than 50% of the complete polypeptide sequence have now been analyzed by scanning mutagenesis and SCAM. An updated model is presented for the outward-facing substrate-binding site and relative orientation of the 12 transmembrane helices of Glut1.

Figures

Figure 1
Figure 1
Helical wheel representation of helix 9. Transmembrane helix 9 of Glut1 viewed from the exoplasmic surface of the membrane. Amino acids are represented by the single-letter code. The red arrow points to the residue where cysteine substitution resulted in an inhibition of transport activity, and the black arrow points to the single residue that is exposed to the external solvent according to pCMBS reactivity.
Figure 2
Figure 2
Expression of helix 9 single-C mutant transporters in Xenopus oocytes. Stage 5 Xenopus oocytes were injected with 50 ng of wild-type, C-less, or mutant C-less mRNAs; 2 days later, frozen sections were prepared and analyzed by indirect immunofluorescence laser confocal microscopy, or oocytes were used to prepare purified membrane fractions for immunoblot analysis. (A) Confocal micrographs of oocytes expressing each of the 21 single-C mutants. (B) Immunoblots of 10 μg of total oocyte membrane protein loaded per lane. Rabbit antiserum A674 raised against the 15 C-terminal residues of human Glut1 was used at a 1:500 dilution. Numbers above the lanes at the right represent the amounts in nanograms of human erythrocyte Glut1 loaded in each lane as a quantitative standard.
Figure 3
Figure 3
2-Deoxyglucose uptake activity of helix 9 single-C mutants. [3H]-2-Deoxyglucose uptake (50 μM, 30 min at 22 °C) and the plasma membrane content of each single-C mutant were quantitated 2 days after injection of mRNAs. Results represent the mean ± standard error of 5–10 independent experiments, each experiment employing 15–20 oocytes per experimental group. The star denotes a p of <0.001 for the single-C mutant compared to parental C-less Glut1. Background values observed in sham-injected oocytes were subtracted.
Figure 4
Figure 4
Effect of pCMBS on the transport activity of helix 9 single-C mutants. Three days after injection of mRNAs, groups of 15–20 oocytes were incubated in the presence or absence of 0.5 mM pCMBS in Barth's saline at 22 °C for 15 min. Oocytes were washed four times in Barth's saline and then subjected to 2-deoxyglucose uptake measurements under the conditions described in the legend of Figure 3. Results represent the mean ± standard error of 5–11 independent experiments, each experiment employing 15–20 oocytes per experimental group. Data are expressed as relative uptake activity, i.e., uptake observed in the presence of pCMBS divided by the uptake observed in the absence of pCMBS. C-less represents the parental cysteine-less Glut1 construct. V165C is a well-characterized positive control whose activity is inhibited by pCMBS (37). A star denotes a p of <0.05, activity with vs without prior incubation in the presence of pCMBS.
Figure 5
Figure 5
Summary of mutagenesis data and SCAM analysis of Glut1. The membrane is represented by a black rectangle, and the 12 transmembrane segments are numbered consecutively from the N-terminus to the C-terminus. The termini of the transmembrane segments are assigned as proposed in ref (17). Amino acid residues are identified by the single-letter code. Amino acid residues that have been subjected to mutagenesis are colored green. Residues that represent sites of pCMBS sensitivity are colored purple. Residues corresponding to mutations that inhibit transport activity by >90% are colored yellow. Putative substrate-binding residues are colored red. See the text for a detailed discussion.
Figure 6
Figure 6
Low-resolution cartoon for the arrangement of the 12 transmembrane helices and of the exofacial binding site of Glut1. Proposed model of the exofacial glucose-binding site as viewed from the outside of the cell. For the sake of simplicity, all transmembrane segments are drawn as perfect helices perpendicular to the plane of the membrane. Glucose is not drawn to scale. The dotted lines represent possible hydrogen bonds formed between glucose hydroxyl groups and various side chains on Glut1. Numbered residues are accessible to pCMBS from the external solvent. The arrows on the right represent the probable displacement of helices 1 and 4 in the exofacial configuration of the transporter as compared to the cytoplasmic configuration. Residues in helices 4 (G145) and 8 (L325) that are within ~6 Å of each other as determined by chemical cross-linking experiments are indicated. Most helices are positioned such that their pCMBS-accessible residues face a proposed central cavity open to the extracellular fluid. Helices 1 and 12 exhibit no periodicity in their pCMBS reactivity. They are oriented such that only hydrophobic side chains face the lipid bilayer. The orientation of helices 4 and 8 is consistent with the results of chemical cross-linking experiments (55).

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