Mueckler M , Makepeace C .

R01 DK43695 NIDDK NIH HHS , R01 DK043695 NIDDK NIH HHS

	Figure 1. Expression of dicysteine mutants in Xenopus oocyte membranes.Stage 5 Xenopus oocytes were injected with water (Sham) or with 50 ng of mRNA encoding the parental reporter construct (C-Tev) or the indicated dicysteine mutant. Two days post injection total oocyte membranes were prepared and subjected to immunoblot analysis using a rabbit polyclonal ab raised against a peptide corresponding to the C-terminal 15 residues of human Glut1.
	Figure 2. 2-Deoxyglucose uptake activity of Glut1 mutants.[3H]-2-DOG uptake (50 µM, 30 min. at 22°C) was measured 2 days after injection of oocytes with 50 ng of mRNA. Activities were normalized to the value measured for oocytes expressing the control C-Tev construct (0.18±0.03 pMoles/oocyte/30 min/unit band intensity). Unit band intensity refers to the relative intensity of the protein bands as measured by immunoblot analysis using a Li-Cor imager and normalizes for varying levels of protein expression for the different mutants. Results represent the mean ± SE of 3–6 independent experiments with 15–20 oocytes per experimental group. Values observed in water-injected oocytes were subtracted.
	Figure 3. Chemical Cross-linking of di-C Mutants.Stage 5 Xenopus oocytes were injected with 50 ng of mRNA encoding the parental reporter construct (C-Tev) or the indicated dicysteine mutants (see Table 1). After incubation of oocytes for 2 days, cross-linking analysis was conducted on purified oocyte membranes as described in “Materials and Methods”. The reactions were quenched by the addition of 2 mM cysteine and oocyte membranes were digested with Tev protease then subjected to SDS-PAGE followed by immunoblotting with rabbit polylclonal ab raised against the C-terminal 15 residues of human Glut1 (red bands) and a mouse monoclonal ab that recognizes an epitope in the N-terminal half of the central cytoplasmic loop (green bands). Note that the full-length ∼54 kD bands were recognized by both antibodies and show up as yellow when the intensity of the detector was increased. “C-TEV” is the control cysteine-less parental construct. “Control” lanes were loaded with membranes that were not subjected to either chemical cross-linking or protease cleavage. “DMSO” lanes were loaded with membranes that were not subjected to chemical cross-linking but were digested with TEV protease. The “o-PDM” and “BMH” lanes were loaded with membranes that were subjected to cross-linking by the respective chemical and then were treated with TEV protease. The ratio of the intensities of the full-length bands in the DMSO lanes to those in the Control lanes thus provide the maximum level of protease cleavage for each mutant. The ratio of the intensities of the full-length bands in the “o-PDM” or ‘BMH” lanes to those in the Control lanes indicate the extent of cross-linking by either reagent. This ratio is termed the cross-linking efficiency or “fraction cross-linked” in Table 2.
	Figure 4. Effect of non-transported ligands on chemical cross-linking.Oocyte membranes were incubated in presence of either vehicle alone (water or ethanol) or 50 µM cytochalasin B or 50 mM ethylidene glucose for 5 min prior to the addition of the indicated concentration of either o-PDM or BMH. Cross-linking efficiency was measured by protease cleavage followed by immunoblot analysis as described in “Materials and Methods”. The water lanes represent the controls for the addition of ethylidene glucose and the ethanol lanes controlled for the addition of cytochalasin B. The DMSO lanes represent samples to which DMSO was added but no cross-linker. These lanes indicate the maximum cleavage observed for each mutant with TEV protease.
	Figure 5. Cross-sectional model of the inner transmembrane helices of Glut1 in the exoplasmic conformation as viewed from the exoplasmic face of the membrane based on experimental results.Amino acid residues subjected to cysteine substitution in the dicysteine mutants are given by the single letter code. Red lines connect residues that were cross-linked by o-PDM or BMH within the helical pairs 2/11 and 5/8.
	Figure 6. Side-view of the orientation of helices 2/11 and 5/8 of Glut1 in the endofacial conformation based on homology modeling.The orientation of the helices is derived from an homology-based model of Glut1 that used the structure of the E. coli Glycerol-3-P Antiporter as the template molecule [17]. Side chains of residues that were mutated to cysteines and subjected to chemical cross-linking analysis are identified by their single letter amino acid codes. Residues that exhibited cross-linking are shown in green in ball and stick form and are connected by dotted lines. Distances between cross-linked residues are given in angstroms (Ã ). Residues that did not exhibit cross-linking are shown in red.

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