ATP-dependent substrate occlusion by the human erythrocyte sugar transporter.

Human erythrocyte sugar transport presents a functional complexity that is not explained by existing models for carrier-mediated transport. It has been suggested that net sugar uptake is the sum of three serial processes: sugar translocation, sugar interaction with an intracellular binding complex, and the release from this complex into bulk cytosol. The present study was carried out to identify the erythrocyte sugar binding complex, to determine whether sugar binding occurs inside or outside the cell, and to determine whether this binding complex is affected by cytosolic ATP or transporter quaternary structure. Sugar binding assays using cells and membrane protein fractions indicate that sugar binding to erythrocytes is quantitatively accounted for by sugar binding to the hexose transport protein, GluT1. Kinetic analysis of net sugar fluxes indicates that GluT1 sugar binding sites are cytoplasmic. Intracellular ATP increases GluT1 sugar binding capacity from 1 to 2 mol of 3-O-methylglucose/mol GluT1 and inhibits the release of bound sugar into cytosol. Reductant-mediated, tetrameric GluT1 dissociation into dimeric GluT1 is associated with the loss of ATP and 3-O-methylglucose binding. We propose that sugar uptake involves GluT1-mediated, extracellular sugar translocation into an ATP-dependent cage formed by GluT1 cytoplasmic domains. Caged or occluded sugar has three possible fates: (1) transport out of the cell (substrate cycling); (2) interaction with sugar binding sites within the cage, or (3) release into bulk cytosol. We show how this hypothesis can account for the complexity of erythrocyte sugar transport and its regulation by cytoplasmic ATP.

Membrane-bound glyceraldehyde-3-phosphate dehydrogenase and multiphasic erythrocyte sugar transport.

Net sugar import by human erythrocytes consists of ATP-modulated rapid and slow phases while sugar export consists of a single slow phase. We have proposed that this behaviour results from obligate substrate tunnelling from transporter to bulk cytosol through a complex containing high-affinity, low-capacity sugar binding sites (Cloherty, Sultzman, Zottola & Carruthers, 1995). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to compartmentalize ATP delivery to erythrocyte membrane ATPases and interact directly with the erythrocyte glucose transporter in vitro. The present study examines the possibility that GAPDH is an obligate component of the hypothesized sugar-binding complex. GAPDH remains associated with the erythrocyte membrane following cell lysis and remains associated with the cytoskeleton under conditions where more than 99% of the membrane glucose transport protein (GLUT1) is released by detergent (Triton X-100). GAPDH is released from erythrocyte membranes upon exposure to Mg.ATP or to NADH. ATP displacement of membrane-bound GAPDH is half-maximal at 200 microM ATP and appears to involve ATP interaction with multiple, co-operative sites. GAPDH interaction with purified tetrameric GLUT1 is saturable, co-operative and also inhibited by ATP. ATP inhibition of GAPDH binding to purified tetrameric GLUT1 is less effective than ATP inhibition of GAPDH binding to intact erythrocyte membranes. Removal of cellular GAPDH by exposing erythrocyte membranes to NADH prior to membrane resealing neither affects ATP modulation of sugar transport nor reduces biphasic net sugar uptake to a single phase. We conclude that ATP-sensitive GAPDH interaction with the cytoplasmic surface of erythrocyte membranes and GLUT1 is responsible neither for ATP modulation of sugar transport nor for multiphasic net sugar import by human red cells.

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.

The 2.0 A crystal structure of Scapharca tetrameric hemoglobin: cooperative dimers within an allosteric tetramer.

The crystal structure of the allosteric tetrameric hemoglobin from Scapharca inaequivalvis (HbII) has been determined in the carbonmonoxy liganded state using a combination of anomalous scattering and molecular replacement. The molecular model has been refined at 2.0 A resolution to a conventional R-factor of 0.173 and a free R-factor of 0.244. The tetramer is formed from two identical heterodimers. Each heterodimer is assembled with intersubunit contacts involving the E and F helices and heme groups in a manner that is very similar to that of the cooperative Scapharca homodimeric hemoglobin. In addition, the ordered water structure observed in these dimeric interfaces is quite similar. These structural similarities strongly suggest that the dimers within the Scapharca tetramer are cooperative. Subunits assemble into a tetramer in a distinctly non-tetrahedral arrangement, with the pseudo 2-fold axes of the heterodimer oriented at an angle of 74.5 degrees relative to the molecular 2-fold. This arrangement requires that two subunit types have distinct locations and contacts, despite the very similar tertiary structures. HbII polymerizes to higher-order assemblages in a ligand, proton and anion dependent fashion. The lattice contacts in the HbII-CO crystal suggest possible modes for this association.