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 a re stabilized by reductant-sensitive, noncovalent subunit interactions [Hebert, D. N., & Carruthers, A. (1992) J. Biol, Chem. 267, 23829-238 383]. In the present study, we use biochemical and molecular approache s to isolate specific determinants of transporter oligomeric structure and transport function. When unfolded in denaturant, each subunit (GL UT1 protein) of the transporter complex exposes two sulfhydryl groups. Four additional thiol groups are accessible following subunit exposur e to reductant. Assays of subunit disulfide bridge content suggest tha t 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) contai ns three inaccessible sulfhydryl groups and that an N-terminal GLUT1 p eptide (residues 147-261/299) contains two accessible thiols. The carb oxyl-terminal peptide most likely contains the intramolecular disulfid e bridge since neither its yield nor its electrophoretic mobility is a ltered by addition of reductant. Each GLUT1 cysteine was changed to se rine by oligonucleotide-directed, in vitro mutagenesis. The resulting transport proteins were expressed in CHO cells and screened by immunof luorescence microscopy for their ability to expose tetrameric GLUT1-sp ecific epitopes. Serine substitution at cysteine residues 133, 201, 20 7, and 429 does not inhibit exposure of tetrameric GLUT1-specific epit opes. Serine substitution at cysteines 347 or 421 prevents exposure of tetrameric GLUT1-specific epitopes. Hydrodynamic analysis of GLUT1/GL UT4 chimeras expressed in and subsequently solubilized from CHO cells indicates that GLUT1 residues 1-199 promote chimera dimerization and p ermit GLUT1/chimera heterotetramerization. This GLUT1 N-terminal domai n is insufficient for chimera tetramerization which additionally requi res GLUT1 residues 200-463. Extracellular reductants (dithiothreitol, beta-mercaptoethanol, or glutathione) reduce erythrocyte 3-O-methylglu cose uptake by up to 15-fold. This noncompetitive inhibition of sugar uptake is reversed by the cell-impermeant, oxidized glutathione. Reduc tant is without effect on sugar exit from erythrocytes. Dithiothreitol doubles the cytochalasin B binding capacity of erythrocyte-resident g lucose transporter, abolishes allosteric interactions between substrat e binding sites on adjacent subunits, and occludes tetrameric GLUT1-sp ecific GLUT1 epitopes in situ. CHO cell-resident GLUT1 structure and t ransport function are similarly affected by extracellular reductant. W e conclude that each subunit of the glucose transporter contains an ex tracellular disulfide bridge (Cys347 and Cys421) that stabilizes trans porter oligomeric structure and thereby accelerates transport function .