The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites.

At any instant, the human erythrocyte sugar transporter presents at least one sugar export site but multiple sugar import sites. The present study asks whether the transporter also presents more than one sugar exit site. We approached this question by analysis of binding of [3H]cytochalasin B (an export conformer ligand) to the human erythrocyte sugar transporter and by analysis of cytochalasin B modulation of human red blood cell sugar uptake. Phloretin-inhibitable cytochalasin B binding to human red blood cells, to human red blood cell integral membrane proteins, and to purified human red blood cell glucose transport protein (GluT1) displays positive cooperativity at very low cytochalasin B levels. Cooperativity between sites and K(d(app)) for cytochalasin B binding are reduced in the presence of intracellular ATP. Red cell sugar uptake at subsaturating sugar levels is inhibited by high concentrations of cytochalasin B but is stimulated by lower (<20 nM) concentrations. Increasing concentrations of the e1 ligand forskolin also first stimulate then inhibit sugar uptake. Cytochalasin D (a cytochalasin B analogue that does not interact with GluT1) is without effect on sugar transport over the same concentration range. Cytochalasin B and ATP binding are synergistic. ATP (but not AMP) enhances [3H]cytochalasin B photoincorporation into GluT1 while cytochalasin B (but not cytochalasin D) enhances [gamma-32P]azidoATP photoincorporation into GluT1. We propose that the red blood cell glucose transporter is a cooperative tetramer of GluT1 proteins in which each protein presents a translocation pathway that alternates between uptake (e2) and export (e1) states but where, at any instant, two subunits must present uptake (e2) and two subunits must present exit (e1) states.

The human erythrocyte sugar transporter presents two sugar import sites.

The human erythrocyte sugar transporter presents sugar import (e2) and sugar export (e1) sites simultaneously. This study asks whether the sugar transporter exposes only one or multiple import sites. We approached this question by analysis of cytochalasin B binding to the human erythrocyte sugar export site in the presence of sugars that bind to the sugar import site. Extracellular maltose does not enter human erythrocytes. High concentrations of maltose (1-100 mM) inhibit cytochalasin B binding to human red cells. Low concentrations (25-500 microM) increase the level of erythrocyte cytochalasin B binding. Maltose modulation of cytochalasin B binding is mediated by altered affinity of sugar export sites for cytochalasin B. Similar results are obtained with other cell-impermeant inhibitors of sugar uptake. Extracellular D-glucose (a transported sugar) stimulates cytochalasin B binding at low D-glucose concentrations (10-250 microM), but this effect is lost at higher concentrations. Intracellular D-glucose inhibits cytochalasin B binding. Low concentrations of extracellular maltose and other nontransported inhibitors stimulate 3-O-methylglucose uptake in erythrocytes. Higher sugar concentrations (1-100 mM) inhibit transport. These data support the hypothesis that the erythrocyte sugar transporter presents two sugar import sites and at least one sugar export site. This conclusion is consistent with the proposed oligomeric structure of the sugar transporter, a complex of four GluT1 proteins in which each subunit presents a translocation pathway.

Structural and physiologic determinants of human erythrocyte sugar transport regulation by adenosine triphosphate.

Human erythrocyte sugar transport is mediated by the integral membrane protein GLUT1 and is regulated by cytosolic ATP [Carruthers, A., and Helgerson, A. L. (1989) Biochemistry 28, 8337-8346]. This study asks the following questions. (1) Where is the GLUT1 ATP binding site? (2) Is ATP-GLUT1 interaction sufficient for sugar transport regulation? (3) Is ATP modulation of transport subject to metabolic control? GLUT1 residues 301-364 were identified as one element of the GLUT1 ATP binding domain by peptide mapping and N-terminal sequence analysis of proteolytic fragments of azidoATP-photolabeled GLUT1. Nucleotide binding and sugar transport experiments undertaken with dimeric and tetrameric forms of GLUT1 indicate that only tetrameric GLUT1 binds and is subject to modulation by ATP. Reconstitution experiments indicate that nucleotide and tetrameric GLUT1 are sufficient for ATP modulation of sugar transport. Feedback control of GLUT1 regulation by ATP was investigated by measuring sugar uptake into erythrocyte ghosts containing or lacking ATP and glycolytic intermediates. Only AMP and ADP modulate ATP regulation of transport. Reduced cytosolic pH inhibits ATP modulation of GLUT1-mediated 3OMG uptake and increases Kd(app) for ATP interaction with GLUT1. We conclude that tetrameric but not dimeric GLUT1 is subject to direct regulation by cytosolic ATP and that this regulation is antagonized by intracellular AMP and acidification.

Regulation of GLUT1-mediated sugar transport by an antiport/uniport switch mechanism.

Avian erythrocyte sugar transport is stimulated during anoxia and during exposure to inhibitors of oxidative phosphorylation. This stimulation results from catalytic desuppression of the cell surface glucose transporter GLUT1 [Diamond, D., & Carruthers, A. (1993) J. Biol. Chem. 268, 6437-6444]. The present study was undertaken to investigate the mechanisms of GLUT1 suppression/desuppression. Sugar uniport (sugar uptake or exit in the absence of sugar at the opposite side of the membrane) is absent in normoxic avian erythrocytes, but sugar antiport (sugar uptake coupled to sugar exit) is present. Exposure to cyanide and/or to FCCP (mitochondrial inhibitors) stimulates erythrocyte sugar uniport but not sugar antiport. K(m)(app) for 3-O-methylglucose uniport and antiport are unaffected by metabolic poisoning. Ki(app) for inhibitions of 3-O-methylglucose uniport by cytochalasin B and forskolin (sugar export site ligands) are unaffected by progressive stimulation of sugar uniport. Cyanide and FCCP stimulation of 3-O-methylglucose uniport are associated with increased AMP-activated protein kinase activity. Purified human GLUT1 is not phosphorylated by exposure to cytosol extracted from poisoned avian erythrocytes. FCCP does not stimulate GLUT1-mediated 3-O-methylglucose uptake in K562 cells but does increase K562 AMP-activated protein kinase activity. FCCP stimulation of 3-O-methylglucose uniport in resealed erythrocyte ghosts requires cytosolic ATP and/or glutathione. The nonmetabolizable ATP analog AMP-PNP cannot be substituted for ATP in this action. These results are contrasted with allosteric regulation of human erythrocyte sugar transport and suggest that avian erythrocyte sugar transport suppression results from inhibition of carrier uniport function. Uniport suppression is not mediated by interaction with cytosolic molecular species that bind to the sugar export site. The antiport to uniport switch mechanism requires ATP hydrolysis, is associated with elevated AMP-activated kinase function, and, if triggered by this kinase, is mediated by factors absent in K562 cells and downstream from the kinase.

Human erythrocyte sugar transport is incompatible with available carrier models.

GLUT1-mediated, passive D-glucose transport in human erythrocytes is asymmetric, Vmax and K(m)(app) for D-glucose uptake at 4 degrees C are 10-fold lower than Vmax and K(m)(app) for D-glucose export. Transport asymmetry is not observed for GLUT1-mediated 3-O-methylglucose transport in rat, rabbit, and avian erythrocytes and rat adipocytes where Vmax for sugar uptake and exit are identical. This suggests that transport asymmetry is either an intrinsic catalytic property of human GLUT1 or that factors present in human erythrocytes affect GLUT1-mediated sugar transport. In the present study we assess human erythrocyte sugar transport asymmetry by direct measurement of sugar transport rates and by analysis of the effects of intra- and extracellular sugars on cytochalasin B binding to the sugar export site. We also perform internal consistency tests to determine whether the measured, steady-state 3-O-methylglucose transport properties of human erythrocytes agree with those expected of two hypothetical models for protein-mediated sugar transport. The simple-carrier hypothesis describes a transporter that alternately exposes sugar import and sugar export pathways. The fixed-site carrier hypothesis describes a sugar transporter that simultaneously exposes sugar import and sugar export pathways. Steady-state 3-O-methylglucose transport in human erythrocytes at 4 degrees C is asymmetric. Vmax and K(m)(app) for sugar uptake are 10-fold lower than Vmax and K(m)(app) for sugar export. Phloretin-inhibitable cytochalasin B binding to intact red cells is unaffected by extracellular D-glucose but is competitively inhibited by intracellular D-glucose. This inhibition is reduced by 13% +/- 4% when saturating extracellular D-glucose levels are also present. Assuming transport is mediated by a simple-carrier and that cytochalasin B and intracellular D-glucose binding sites are mutually exclusive, the cytochalasin B binding data are explained only if transport is almost symmetric (Vmax exit = 1.4 Vmax entry). The cytochalasin B binding data are consistent with both symmetric and asymmetric fixed-site carriers. Analysis of 3-O-methylglucose, 2-deoxy-D-glucose, and D-glucose uptake in the presence of intracellular 3-O-methylglucose, demonstrates significant divergence in experimental and theoretical transport behaviors. We conclude either that human erythrocyte sugar transport is mediated by a carrier mechanism that is fundamentally different from those considered previously or that human erythrocyte-specific factors prevent accurate determination of GLUT1-mediated sugar translocation across the cell membrane. We suggest that GLUT1-mediated sugar transport in all cells is an intrinsically symmetric process but that intracellular sugar complexation in human red cells prevents accurate determination of transport rates.

Net sugar transport is a multistep process. Evidence for cytosolic sugar binding sites in erythrocytes.

Human erythrocyte net sugar transport is hypothesized to be rate-limited by reduced cytosolic diffusion of sugars and/or by reversible sugar association with intracellular macromolecules [Naftalin, R.J., Smith, P.M., & Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235-249]. The present study examines these hypotheses. Protein-mediated 3-O-methylglucose uptake at 4 degrees C by human erythrocytes and by resealed, hypotonically lysed erythrocytes (ghosts) is inhibited by increasing solvent viscosity. Protein-mediated transport and transbilayer diffusion of the slowly transported substrate 6-NBD glucosamine are unaffected by increasing solvent viscosity. These findings suggest that protein-mediated 3-O-methylglucose transport is diffusion-limited in erythrocytes. More detailed analyses of red cell 3-O-methylglucose uptake (at 4 degrees C and at limiting extracellular sugar levels) reveal that net influx is a biexponential process characterized by rapid filling of a small compartment (C1 = 29 +/- 6% total cell volume; k1 = 7.4 +/- 1.7 min-1) and slow filling of a larger compartment (C2 = 71 +/- 6% total cell volume k2 = 0.56 +/- 0.11 min-1). Erythrocyte D-glucose net uptake at 4 degrees C is also a biphasic process. Transmembrane sugar leakage is a monoexponential process indicating that multicomponent, protein-mediated uptake does not result from sugar uptake by two cell populations of differing cellular volume. Sugar exit at limiting 3-O-methylglucose concentrations is described by single exponential kinetics. This demonstrates that multicomponent sugar uptake does not result from influx into two populations of cells with widely different sugar transporter content. We conclude that biexponential sugar uptake results from slow (relative to transport) exchange of sugars between serial, intracellular sugar compartments. Biexponential sugar uptake is observed under equilibrium exchange conditions (intracellular sugar concentration = extracellular sugar concentration) but only at 3-O-methylglucose concentrations of less than 1 mM. Above this sugar concentration, exchange uptake is a monoexponential process. Because diffusion rates are independent of diffusant concentration, this suggests that multicomponent uptake results from high-affinity sugar binding within the cell. The concentration of cytosolic binding sites (30 microM, Kd(app) = 400 microM) was estimated from the equilibrium cellular 3-O-methylglucose space. Biexponential net 3-O-methylglucose uptake is also observed in human erythrocyte ghosts, in control human K562 cells, and in K562 cells induced to synthesize hemoglobin by prolonged exposure to hemin. This demonstrates that neither membrane-bound nor free cytosolic hemoglobin forms the sugar-binding complex.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Rapid substrate translocation by the multisubunit, erythroid glucose transporter requires subunit associations but not cooperative ligand binding.

The human erythroid glucose transporter is a GLUT1 homotetramer whose structure and function are stabilized by noncovalent, cooperative subunit interactions. The present study demonstrates that exofacial tryptic digestion of GLUT1 abolishes cooperative interactions between substrate binding sites on adjacent subunits under circumstances where subunit associations and high catalytic turnover are maintained. Extracellular trypsin produces rapid, quantitative cleavage of the human red cell-resident sugar transport protein, GLUT1. One major carboxyl-terminal peptide of M(r)(app) 25,000 is detected by immunoblot analysis. Endofacial tryptic digestion of GLUT1 results in the complete loss of GLUT1 carboxyl-terminal structure. GLUT1-mediated erythrocyte sugar uptake, transport inhibition by cytochalasin B, and GLUT1 oligomeric structure are unaffected by exofacial GLUT1 proteolysis. In contrast, the cytochalasin B binding capacity of GLUT1 and the Kd(app) for cytochalasin B binding to the transporter are doubled following exofacial tryptic digestion of GLUT1. Photoaffinity labeling experiments show that increased cytochalasin B binding results from increased ligand binding to the 25 kDa carboxyl-terminal GLUT1 peptide. Proteolysis abolishes allosteric interactions between sugar import (maltose binding) and sugar export (cytochalasin B binding) sites that normally exist on adjacent subunits within the transporter complex, but interact with negative cooperativity. Following exofacial proteolysis, these sites become mutually exclusive. Dithiothreitol disrupts GLUT1 quaternary structure, inhibits 3-O-methylglucose transport, and abolishes cooperative interactions between sugar import and export sites in control cells. Studies with reconstituted purified GLUT1 confirm that the action of trypsin on cytochalasin B binding is direct, show that proteolysis increases the apparent affinity of the sugar efflux site for transported sugars, and suggest that the membrane bilayer stabilizes GLUT1 noncovalent structure and catalytic function following GLUT1 proteolysis. Collectively, these findings demonstrate that GLUT1 does not require an intact polypeptide backbone for catalytic function. They show that the multisite sugar transporter mechanism is converted to a simple ping-pong carrier mechanism following exofacial GLUT1 proteolysis. They reveal that subunit cooperativity can be lost under circumstances where cohesive structural interactions between transporter subunits are maintained. They also refute the hypothesis [Hebert, D. N., & Carruthers, A. (1992) J. Biol. Chem. 267, 23829-23838] that rapid substrate translocation by the multisubunit erythroid glucose transporter requires cooperative interactions between subunit ligand binding sites.