Glucose transporter function is controlled by transporter oligomeric structure. A single, intramolecular disulfide promotes GLUT1 tetramerization.
The human erythrocyte glucose transporter is an allosteric complex of four GLUT1 proteins whose structure and substrate binding properties are stabilized by reductant-sensitive, noncovalent subunit interactions [Hebert, D. N., & Carruthers, A. (1992) J. Biol. Chem. 267, 23829-23838]. In the present study, we use biochemical and molecular approaches to isolate specific determinants of transporter oligomeric structure and transport function. When unfolded in denaturant, each subunit (GLUT1 protein) of the transporter complex exposes two sulfhydryl groups. Four additional thiol groups are accessible following subunit exposure to reductant. Assays of subunit disulfide bridge content suggest that two inaccessible sulfhydryl groups form an internal disulfide bridge. Differential alkylation/peptide mapping/N-terminal sequence analyses show that a GLUT1 carboxyl-terminal peptide (residues 232-492) contains three inaccessible sulfhydryl groups and that an N-terminal GLUT1 peptide (residues 147-261/299) contains two accessible thiols. The carboxyl-terminal peptide most likely contains the intramolecular disulfide bridge since neither its yield nor its electrophoretic mobility is altered by addition of reductant. Each GLUT1 cysteine was changed to serine by oligonucleotide-directed, in vitro mutagenesis. The resulting transport proteins were expressed in CHO cells and screened by immunofluorescence microscopy for their ability to expose tetrameric GLUT1-specific epitopes. Serine substitution at cysteine residues 133, 201, 207, and 429 does not inhibit exposure of tetrameric GLUT1-specific epitopes. Serine substitution at cysteines 347 or 421 prevents exposure of tetrameric GLUT1-specific epitopes. Hydrodynamic analysis of GLUT1/GLUT4 chimeras expressed in and subsequently solubilized from CHO cells indicates that GLUT1 residues 1-199 promote chimera dimerization and permit GLUT1/chimera heterotetramerization. This GLUT1 N-terminal domain is insufficient for chimera tetramerization which additionally requires GLUT1 residues 200-463. Extracellular reductants (dithiothreitol, beta-mercaptoethanol, or glutathione) reduce erythrocyte 3-O-methylglucose uptake by up to 15-fold. This noncompetitive inhibition of sugar uptake is reversed by the cell-impermeant, oxidized glutathione. Reductant is without effect on sugar exit from erythrocytes. Dithiothreitol doubles the cytochalasin B binding capacity of erythrocyte-resident glucose transporter, abolishes allosteric interactions between substrate binding sites on adjacent subunits, and occludes tetrameric GLUT1-specific GLUT1 epitopes in situ. CHO cell-resident GLUT1 structure and transport function are similarly affected by extracellular reductant. We conclude that each subunit of the glucose transporter contains an extracellular disulfide bridge (Cys347 and Cys421) that stabilizes transporter oligomeric structure and thereby accelerates transport function.
Zottola
R J
RJ
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01605, USA.
Cloherty
E K
EK
Coderre
P E
PE
Hansen
A
A
Hebert
D N
DN
Carruthers
A
A
D019325:3-O-Methylglucose; D000478:Alkylation; D000595:Amino Acid Sequence; D001483:Base Sequence; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D051272:Glucose Transporter Type 1; D006801:Humans; D046911:Macromolecular Substances; D008757:Methylglucosides / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D017510:Protein Folding; D017421:Sequence Analysis; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004220:Disulfides; D051272:Glucose Transporter Type 1; D046911:Macromolecular Substances; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human; D013438:Sulfhydryl Compounds; D019325:3-O-Methylglucose; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D004229:Dithiothreitol