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Biochemistry
2009 Jun 30;4825:5934-42. doi: 10.1021/bi900521n.
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Model of the exofacial substrate-binding site and helical folding of the human Glut1 glucose transporter based on scanning mutagenesis.
Mueckler M
,
Makepeace C
.
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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.
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
Abramson,
Structural comparison of lactose permease and the glycerol-3-phosphate antiporter: members of the major facilitator superfamily.
2004,
Pubmed
Alisio,
Relative proximity and orientation of helices 4 and 8 of the GLUT1 glucose transporter.
2004,
Pubmed
,
Xenbase
Barnett,
Structural requirements for binding to the sugar-transport system of the human erythrocyte.
1973,
Pubmed
Birnbaum,
Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein.
1986,
Pubmed
Blodgett,
Analysis of glucose transporter topology and structural dynamics.
2008,
Pubmed
Brahm,
Kinetics of glucose transport in human erythrocytes.
1983,
Pubmed
Carruthers,
Facilitated diffusion of glucose.
1990,
Pubmed
Cloherty,
Human erythrocyte sugar transport is incompatible with available carrier models.
1996,
Pubmed
Cloherty,
Sugar transporter regulation by ATP and quaternary structure.
2001,
Pubmed
De Vivo,
Glucose transporter protein syndromes.
2002,
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
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
Hresko,
Topology of the Glut 1 glucose transporter deduced from glycosylation scanning mutagenesis.
1994,
Pubmed
,
Xenbase
Hresko,
Discrete structural domains determine differential endoplasmic reticulum to Golgi transit times for glucose transporter isoforms.
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
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
Joost,
Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators.
2002,
Pubmed
Kaback,
Site-directed alkylation and the alternating access model for LacY.
2007,
Pubmed
Kahlenberg,
Structural requirements of D-glucose for its binding to isolated human erythrocyte membranes.
1972,
Pubmed
Kahn,
Lilly lecture 1995. Glucose transport: pivotal step in insulin action.
1996,
Pubmed
Kasahara,
Reconstitution and purification of the D-glucose transporter from human erythrocytes.
1977,
Pubmed
Keller,
Functional expression of the human HepG2 and rat adipocyte glucose transporters in Xenopus oocytes. Comparison of kinetic parameters.
1989,
Pubmed
,
Xenbase
Leitch,
ATP-dependent sugar transport complexity in human erythrocytes.
2007,
Pubmed
Lemieux,
Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure.
2007,
Pubmed
Levine,
Properties of the human erythrocyte glucose transport protein are determined by cellular context.
2005,
Pubmed
Liu,
The predicted ATP-binding domains in the hexose transporter GLUT1 critically affect transporter activity.
2001,
Pubmed
,
Xenbase
Marger,
A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport.
1993,
Pubmed
Marshall,
Domains that confer intracellular sequestration of the Glut4 glucose transporter in Xenopus oocytes.
1993,
Pubmed
,
Xenbase
Montel-Hagen,
Erythrocyte Glut1 triggers dehydroascorbic acid uptake in mammals unable to synthesize vitamin C.
2008,
Pubmed
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,
Analysis of transmembrane segment 10 of the Glut1 glucose transporter by cysteine-scanning mutagenesis and substituted cysteine accessibility.
2002,
Pubmed
,
Xenbase
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,
Facilitative glucose transporters.
1994,
Pubmed
Olsowski,
Cysteine-scanning mutagenesis of flanking regions at the boundary between external loop I or IV and transmembrane segment II or VII in the GLUT1 glucose transporter.
1998,
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
Pessino,
Evidence that functional erythrocyte-type glucose transporters are oligomers.
1991,
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
Santer,
Fanconi-Bickel syndrome--a congenital defect of the liver-type facilitative glucose transporter. SSIEM Award. Society for the Study of Inborn Errors of Metabolism.
1998,
Pubmed
Scheepers,
The glucose transporter families SGLT and GLUT: molecular basis of normal and aberrant function.
2004,
Pubmed
Schürmann,
Role of conserved arginine and glutamate residues on the cytosolic surface of glucose transporters for transporter function.
1997,
Pubmed
Seatter,
QLS motif in transmembrane helix VII of the glucose transporter family interacts with the C-1 position of D-glucose and is involved in substrate selection at the exofacial binding site.
1998,
Pubmed
,
Xenbase
Tamori,
Substitution at Pro385 of GLUT1 perturbs the glucose transport function by reducing conformational flexibility.
1994,
Pubmed
Taylor,
Symmetrical kinetic parameters for 3-O-methyl-D-glucose transport in adipocytes in the presence and in the absence of insulin.
1981,
Pubmed
Wellner,
The role of cysteine residues in glucose-transporter-GLUT1-mediated transport and transport inhibition.
1994,
Pubmed
,
Xenbase
Wellner,
From triple cysteine mutants to the cysteine-less glucose transporter GLUT1: a functional analysis.
1995,
Pubmed
,
Xenbase
Wheeler,
Infinite-cis kinetics support the carrier model for erythrocyte glucose transport.
1988,
Pubmed
Wheeler,
The glucose transporter of mammalian cells.
1985,
Pubmed
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
Zottola,
Glucose transporter function is controlled by transporter oligomeric structure. A single, intramolecular disulfide promotes GLUT1 tetramerization.
1995,
Pubmed
van Iwaarden,
What we can learn from the effects of thiol reagents on transport proteins.
1992,
Pubmed
