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-limit ed 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-me thylglucose uptake at 4 degrees C by human erythrocytes and by reseale d, hypotonically lysed erythrocytes (ghosts) is inhibited by increasin g solvent viscosity. Protein-mediated transport and transbilayer diffu sion of the slowly transported substrate 6-NBD glucosamine are unaffec ted by increasing solvent viscosity. These findings suggest that prote in-mediated 3-O-methylglucose transport is diffusion-limited in erythr ocytes. More detailed analyses of red cell 3-O-methylglucose uptake (a t 4 degrees C and at limiting extracellular sugar levels) reveal that net influx is a biexponential process characterized by rapid filling o f a small compartment (C-1 = 29 +/- 6% total cell volume; k(1) = 7.4 /- 1.7 min(-1)) and slow filling of a larger compartment (C-2 = 71 +/- 6% total cell volume; k(2) = 0.56 +/- 0.11 min(-1)). Erythrocyte D-gl ucose net uptake at 4 degrees C is also a biphasic process. Transmembr ane sugar leakage is a monoexponential process indicating that multico mponent, protein-mediated uptake does not result from sugar uptake by two cell populations of differing cellular volume. Sugar exit at limit ing 3-O-methylglucose concentrations is described by single exponentia l kinetics. This demonstrates that multicomponent sugar uptake does no t result from influx into two populations of cells with widely differe nt sugar transporter content. We conclude that biexponential sugar upt ake results from slow (relative to transport) exchange of sugars betwe en serial, intracellular sugar compartments. Biexponential sugar uptak e is observed under equilibrium exchange conditions (intracellular sug ar concentration = extracellular sugar concentration) but only at 3-O- methylglucose concentrations of less than 1 mM. Above this sugar conce ntration, exchange uptake is a monoexponential process. Because diffus ion rates are independent of diffusant concentration, this suggests th at multicomponent uptake results from high-affinity sugar binding with in the cell. The concentration of cytosolic binding sites (30 mu M, K- d(app) = 400 mu M) was estimated from the equilibrium cellular 3-O-met hylglucose space. Biexponential net 3-O-methylglucose uptake is also o bserved in human erythrocyte ghosts, in control human K562 cells, and in K562 cells induced to synthesize hemoglobin by prolonged exposure t o hemin. This demonstrates that neither membrane-bound nor free cytoso lic hemoglobin forms the sugar-binding complex. alpha-Toxin-permeabili zed cells fill rapidly (within 5 s) with 3-O-methylglucose and L-gluco se (a nontransported sugar), indicating that the glucose binding compa rtment does not extend across the entire intracellular margin of the p lasma membrane. Rather, it must be restricted to domains of locally hi gh-glucose transporter density. Immunofluorescence microscopy of eryth rocytes indicates that GLUT1 is not distributed uniformly across the c ell surface, while the anion transporter shows a uniform cell surface distribution. Red cell hexokinase land GLUT1 appear not to colocalize in hypotonically lysed erythrocytes. The kinetics of sugar uptake and exit are quantitatively mimicked by a model in which newly imported su gars enter the bulk intracellular water only following interaction wit h an intracellular, sugar-binding complex. We conclude that steady sta te sugar transport assays in human erythrocytes measure two processes: rapid sugar translocation across the bilayer and slow sugar release i nto bulk cytosol. The conclusions of previous steady state analyses wh ich assume net transport reflects only sugar translocation may require reconsideration.