Translocation of sugars across the lysosome membrane

Abstract

Lysosomes are intracellular sites of macromolecule degradation confined by a membrane that localizes hydrolytic enzymes in close proximity to their macromolecular substrates. The monomeric products of digestion then cross the lysosome membrane and enter the cytosol to participate in further cellular metabolism. The mode of metabolite translocation was formerly considered to be diffusion, but latterly evidence has indicated the presence of carriers in the lysosome membrane (for reviews, see Reijngoud & Tager, 1977; Forster & Lloyd, 1988).In comparison with the transport of amino acids, for which kinetic studies have been performed on seven carriers to date, little is known about the mechanisms of sugar translocation across the lysosome membrane. Carriers for only two sugars, glucose and sialic acid, have so far been suggested. Evidence for the sialic acid porter comes mainly from the existence of Salla diseasc and sialic acid storage disease, in which dysfunction of the putative sialic acid carrier occurs (for example, see Renlund et ul., 1986). Studies on glucose (and ribose) transport across the lysosome membrane have been performed on modified lysosomes (Tritosomes), where glucose translocation was attributed to carrier-mediated transport ( K , 50 mM) and possibly free diffusion (Maguire et al., 1983). Sugars are known to cross other membranes by more than one mechanism: for example, erythritol enters erythrocytes by an approximately equal combination of transport and diffusion (W. D. Stein, personal communication).Our aim is to establish the diffusional component of glucose and ribose translocation across the lysosome membrane. The experimental approach chosen was to compare the uptake of these sugars with the uptake of a range of substances that, like the sugars, contain only carbon, hydrogen and oxygen. Most of the molecules studied are non-physiological and therefore unlikely to be substrates for transport systems; they were chosen to encompass a range of physicochemical parameters, particularly molecular size and hydrogen-bonding capacity.Relative net uptake rates were measured using the osmotic-protection method (Bird et al., 1 987), in which lyso-44 1 somes are suspended in 0.25 M solutions of the solute under investigation. Non-latent lysosomal hexosaminidase activity, assayed with a non-permeant substrate, indicates the proportion of broken lysosomes in the resuspension. A nonpermeant solute maintains lysosomal enzyme latency. Influx of the solute leads to osmotic imbalance and subsequent lysosome breakage. The osmotic-protection method does not require highly purified lysosome preparations and allows the study of solutes that are not available in radiolabelled form. However, the information gained on uptake rates is indirect and cannot fully establish the mechanism of uptake.The net uptake rates of the non-physiological compounds correlated well with their physico-chemical features. The data showed that an increase in hydrogen-bonding capacity is more important than an increase in molecular mass in ...

Lysosomal Sequestration of Amine-Containing Drugs: Analysis and Therapeutic Implications

“…Alternatively, if they are suspended in a solution containing a molecule that is not permeable across the membrane at 250 mM concentration, the organelles will remain stable. Lloyd utilized this behavior to evaluate the permeability of lysosomes to various compounds of interest 25,26. In order to evaluate the rupturing of the organelle, the activity of luminal lysosomal enzymes was assessed in the media containing the organelle after addition of a variety of analytes.…”

Neutral-sugar transport by rat liver lysosomes

“…It has been suggested that simple diffusion may play a role in the lysosomal transport of neutral sugars [12,14]. This may be the case for L-fucose transport, accounting for the extremely large Km.…”

“…These studies have been limited to examination of uptake of sugars, and little information is available about efflux of sugars from lysosomes. Argument persists over the existence of this transport system, and it has been suggested that neutral sugars merely diffuse across the lysosomal membrane [12][13][14]. In addition, transport of L-fucose, a glycoprotein constituent, has not been well studied.…”