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Transmembrane segment 6 of the Glut1 glucose transporter is an outer helix and contains amino acid side chains essential for transport activity.
Mueckler M
,
Makepeace C
.
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Experimental data and homology modeling suggest a structure for the exofacial configuration of the Glut1 glucose transporter in which 8 transmembrane helices form an aqueous cavity in the bilayer that is stabilized by four outer helices. The role of transmembrane segment 6, predicted to be an outer helix in this model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzene-sulfonate (pCMBS). A fully functional Glut1 molecule lacking all 6 native cysteine residues was used as a template to produce a series of 21 Glut1 point mutants in which each residue along helix 6 was individually changed to cysteine. These mutants were expressed in Xenopus oocytes, and their expression levels, functional activities, and sensitivities to inhibition by pCMBS were determined. Cysteine substitutions at Leu(204) and Pro(205) abolished transport activity, whereas substitutions at Ile(192), Pro(196), Gln(200), and Gly(201) resulted in inhibition of activity that ranged from approximately 35 to approximately 80%. Cysteine substitutions at Leu(188), Ser(191), and Leu(199) moderately augmented specific transport activity relative to the control. These results were dramatically different from those previously reported for helix 12, the structural cognate of helix 6 in the pseudo-symmetrical structural model, for which none of the 21 single-cysteine mutants exhibited reduced activity. Only the substitution at Leu(188) conferred inhibition by pCMBS, suggesting that most of helix 6 is not exposed to the external solvent, consistent with its proposed role as an outer helix. These data suggest that helix 6 contains amino acid side chains that are critical for transport activity and that structurally analogous outer helices may play distinct roles in the function of membrane transporters.
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
Baldwin,
Purification and reconstitution of glucose transporter from human erythrocytes.
1989,
Pubmed
Baldwin,
Mammalian passive glucose transporters: members of an ubiquitous family of active and passive transport proteins.
1993,
Pubmed
Barnett,
Structural requirements for binding to the sugar-transport system of the human erythrocyte.
1973,
Pubmed
Carruthers,
Facilitated diffusion of glucose.
1990,
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
Heinze,
Cysteine-scanning mutagenesis of transmembrane segment 1 of glucose transporter GLUT1: extracellular accessibility of helix positions.
2004,
Pubmed
,
Xenbase
Hresko,
Discrete structural domains determine differential endoplasmic reticulum to Golgi transit times for glucose transporter isoforms.
1994,
Pubmed
,
Xenbase
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
Huang,
Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli.
2003,
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
Marshall,
Domains that confer intracellular sequestration of the Glut4 glucose transporter in Xenopus oocytes.
1993,
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,
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,
Structure, function and biosynthesis of GLUT1.
1997,
Pubmed
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,
Facilitative glucose transporters.
1994,
Pubmed
Mueckler,
Glutamine 161 of Glut1 glucose transporter is critical for transport activity and exofacial ligand binding.
1994,
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
Pessin,
Mammalian facilitative glucose transporter family: structure and molecular regulation.
1992,
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
Wellner,
From triple cysteine mutants to the cysteine-less glucose transporter GLUT1: a functional analysis.
1995,
Pubmed
,
Xenbase
