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 (Å). Residues that did not exhibit cross-linking are shown in red.

Abramson, Structure and mechanism of the lactose permease of Escherichia coli. 2003, Pubmed

Abramson, Structure and mechanism of the lactose permease of Escherichia coli. 2003, Pubmed
Alisio, Purification and characterization of mammalian glucose transporters expressed in Pichia pastoris. 2010, Pubmed
Appleman, Rapid kinetics of the glucose transporter from human erythrocytes. Detection and measurement of a half-turnover of the purified transporter. 1985, Pubmed
Appleman, Kinetics of the purified glucose transporter. Direct measurement of the rates of interconversion of transporter conformers. 1989, Pubmed
Baker, Asymmetry of the hexose transfer system in human erythrocytes. Experiments with non-transportable inhibitors. 1978, Pubmed
Barnett, Evidence for two asymmetric conformational states in the human erythrocyte sugar-transport system. 1975, Pubmed
Bass, Use of site-directed cysteine and disulfide chemistry to probe protein structure and dynamics: applications to soluble and transmembrane receptors of bacterial chemotaxis. 2007, Pubmed
Birnbaum, Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein. 1986, Pubmed
Bloch, Inhibition of glucose transport in the human erythrocyte by cytochalasin B. 1973, Pubmed
Carruthers, Facilitated diffusion of glucose. 1990, Pubmed
Carruthers, Inhibitions of sugar transport produced by ligands binding at opposite sides of the membrane. Evidence for simultaneous occupation of the carrier by maltose and cytochalasin B. 1991, Pubmed
Carruthers, Will the original glucose transporter isoform please stand up! 2009, Pubmed
Cloherty, Human erythrocyte sugar transport is incompatible with available carrier models. 1996, Pubmed
Dang, Structure of a fucose transporter in an outward-open conformation. 2010, Pubmed
Devés, Cytochalasin B and the kinetics of inhibition of biological transport: a case of asymmetric binding to the glucose carrier. 1978, Pubmed
Forrest, On the accuracy of homology modeling and sequence alignment methods applied to membrane proteins. 2006, Pubmed
Garcia, Amino acid substitutions at tryptophan 388 and tryptophan 412 of the HepG2 (Glut1) glucose transporter inhibit transport activity and targeting to the plasma membrane in Xenopus oocytes. 1992, Pubmed , Xenbase
Gibbs, Proteolytic dissection as a probe of conformational changes in the human erythrocyte glucose transport protein. 1988, Pubmed
Gorga, Equilibria and kinetics of ligand binding to the human erythrocyte glucose transporter. Evidence for an alternating conformation model for transport. 1981, Pubmed
Green, Quantitative evaluation of the lengths of homobifunctional protein cross-linking reagents used as molecular rulers. 2001, Pubmed
Griffin, Inhibition of glucose transport in human erythrocytes by cytochalasins: A model based on diffraction studies. 1982, Pubmed
Hashiramoto, Site-directed mutagenesis of GLUT1 in helix 7 residue 282 results in perturbation of exofacial ligand binding. 1992, Pubmed
Heinze, Cysteine-scanning mutagenesis of transmembrane segment 1 of glucose transporter GLUT1: extracellular accessibility of helix positions. 2004, Pubmed , Xenbase
Holman, Photolabelling of the hexose transporter at external and internal sites: fragmentation patterns and evidence for a conformational change. 1987, Pubmed
Hresko, Topology of the Glut 1 glucose transporter deduced from glycosylation scanning mutagenesis. 1994, Pubmed , Xenbase
Hruz, Cysteine-scanning mutagenesis of transmembrane segment 7 of the GLUT1 glucose transporter. 1999, Pubmed , Xenbase
Hruz, Cysteine-scanning mutagenesis of transmembrane segment 11 of the GLUT1 facilitative glucose transporter. 2000, Pubmed , Xenbase
Hruz, Structural analysis of the GLUT1 facilitative glucose transporter (review). 2001, Pubmed
Huang, Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. 2003, Pubmed
Inukai, Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter. 1994, Pubmed
Jung, Structural basis of human erythrocyte glucose transporter function in reconstituted system. Hydrogen exchange. 1986, Pubmed
Karim, Binding of cytochalasin B to trypsin and thermolysin fragments of the human erythrocyte hexose transporter. 1987, Pubmed
Lowe, The kinetics and thermodynamics of glucose transport in human erythrocytes: indications for the molecular mechanism of transport. 1989, Pubmed
Manolescu, Facilitated hexose transporters: new perspectives on form and function. 2007, Pubmed
Mueckler, Analysis of transmembrane segment 8 of the GLUT1 glucose transporter by cysteine-scanning mutagenesis and substituted cysteine accessibility. 2004, Pubmed , Xenbase
Mueckler, Sequence and structure of a human glucose transporter. 1985, Pubmed
Mueckler, Analysis of transmembrane segment 10 of the Glut1 glucose transporter by cysteine-scanning mutagenesis and substituted cysteine accessibility. 2002, Pubmed , Xenbase
Mueckler, Glutamine 161 of Glut1 glucose transporter is critical for transport activity and exofacial ligand binding. 1994, Pubmed , Xenbase
Mueckler, Identification of an amino acid residue that lies between the exofacial vestibule and exofacial substrate-binding site of the Glut1 sugar permeation pathway. 1997, Pubmed , Xenbase
Mueckler, Transmembrane segment 5 of the Glut1 glucose transporter is an amphipathic helix that forms part of the sugar permeation pathway. 1999, Pubmed , Xenbase
Mueckler, Transmembrane segment 3 of the Glut1 glucose transporter is an outer helix. 2004, Pubmed , Xenbase
Mueckler, Cysteine-scanning mutagenesis and substituted cysteine accessibility analysis of transmembrane segment 4 of the Glut1 glucose transporter. 2005, Pubmed , Xenbase
Mueckler, Transmembrane segment 12 of the Glut1 glucose transporter is an outer helix and is not directly involved in the transport mechanism. 2006, Pubmed , Xenbase
Mueckler, Transmembrane segment 6 of the Glut1 glucose transporter is an outer helix and contains amino acid side chains essential for transport activity. 2008, Pubmed , Xenbase
Mueckler, Model of the exofacial substrate-binding site and helical folding of the human Glut1 glucose transporter based on scanning mutagenesis. 2009, Pubmed , Xenbase
Olsowski, Cysteine scanning mutagenesis of helices 2 and 7 in GLUT1 identifies an exofacial cleft in both transmembrane segments. 2000, Pubmed , Xenbase
Pao, Major facilitator superfamily. 1998, Pubmed
Robichaud, Determinants of ligand binding affinity and cooperativity at the GLUT1 endofacial site. 2011, Pubmed
Saier, Families of transmembrane sugar transport proteins. 2000, Pubmed
Salas-Burgos, Predicting the three-dimensional structure of the human facilitative glucose transporter glut1 by a novel evolutionary homology strategy: insights on the molecular mechanism of substrate migration, and binding sites for glucose and inhibitory molecules. 2004, Pubmed
Thorens, Glucose transporters in the 21st Century. 2010, Pubmed
Vidaver, Inhibition of parallel flux and augmentation of counter flux shown by transport models not involving a mobile carrier. 1966, Pubmed
Wellner, From triple cysteine mutants to the cysteine-less glucose transporter GLUT1: a functional analysis. 1995, Pubmed , Xenbase
Wood, Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins. 2003, Pubmed
Yin, Structure of the multidrug transporter EmrD from Escherichia coli. 2006, Pubmed