Intracellular trafficking of lysosomal membrane proteins

Lysosomes are the site of degradation of obsolete intracellular material during autophagy and of extracellular macromolecules following endocytosis and phagocytosis. The membrane of lysosomes and late endosomes is enriched in highly glycosylated transmembrane proteins of largely unknown function. Significant progress has been made in recent years towards elucidating the pathways by which these lysosomal membrane proteins are delivered to late endosomes and lysosomes. While some lysosomal membrane proteins follow the constitutive secretory pathway and reach lysosomes indirectly via the cell surface and endocytosis, others exit the trans-Golgi network in clathrin-coated vesicles for direct delivery to endosomes and lysosomes. Sorting from the Golgi or the plasma membrane into the endosomal system is mediated by signals encoded by the short cytosolic domain of these proteins. This review will discuss the role of lysosomal membrane proteins in the biogenesis of the late endosomal and lysosomal membranes, with particular emphasis on the structural features and molecular mechanisms underlying the intracellular trafficking of these proteins.     

Intracellular activity and secretion of various lysosomal glycosidases in cultured human skin fibroblasts

The intracellular activities of four lysosomal glycosidases (alpha-L-fucosidase, beta-D-hexosaminidase, beta-D-galactosidase and beta-D-glucuronidase) in human skin fibroblasts cultured in a medium with 0.1% serum increased in a greater degree than that in a medium with 10% serum. Only two glycosidases (alpha-L-fucosidase and beta-D-hexosaminidase) were secreted by fibroblasts in the culture medium. The extracellular activity of alpha-L-fucosidase and beta-D-hexosaminidase was equivalent to 80 and 25% of their intracellular activity in serum-sufficient fibroblasts and 40 and 15%--in serum-restricted fibroblasts. These results suggest that the observer phenomena are controlled by the levels of autophagy, endocytosis and membrane recycling.     

Intracellular transport and processing of lysosomal cathepsin B

Intracellular transport and processing of lysosomal cathepsin B was investigated in the subcellular fractions of rat liver by pulse-labeling experiments with [35S]methionine in vivo. A newly synthesized procathepsin B with a molecular weight of 39 kDa firstly appeared in the rough microsomal fraction at 10 min postinjection of label. This procathepsin B moved from the microsomal fractions to the Golgi subfractions at 30 min postinjection, and then a processed mature enzyme appeared in the lysosomal fraction at 60 min. These results suggest that the propeptide-processing of procathepsin B takes place in lysosomes in the course of intracellular transport from endoplasmic reticulum through Golgi complex to lysosomes.     

Intracellular pH of stimulated thymocytes measured with a new fluorescent indicator

A new fluorescent intracellular pH indicator is described ("quene 1") which is related to the tetracarboxylate Ca2+ indicator based on the quinoline fluorophor ("quin 2"). Quene 1 has excitation and emission maxima at 390 and 530 nm, respectively, and shows a 30-fold increase in fluorescence between pH 5 and 9 with a pK alpha of 7.3. The fluorescence is insensitive to Ca2+ and Mg2+ at free concentrations up to 10(-4) M and to the proportions of Na+ and K+ at total concentrations of Na+ and K+ from 100 to 200 mM. The indicator is loaded into thymocytes using the tetraacetoxymethyl ester derivative which is hydrolyzed in the cells to give the tetracarboxylate anion. Intracellular pH can be measured at intracellular quene 1 concentrations of approximately 0.1 mM and quene 1 does not perturb glycolysis or the ATP level in resting cells at concentrations up to 0.8 mM. The intracellular pH of mouse thymocytes indicated by quene 1 is 7.15 +/- 0.04 and it is insensitive to the concentration of Ca2+ or Mg2+ in the extracellular medium. The intracellular pH decreased when the pH of the medium was lowered by addition of HCl, but was insensitive to NaOH at extracellular pH values up to 8.0. Rapid transient changes in intracellular pH are induced by NH4Cl, NaCO2CH3, or HCO3-/CO2. The thymocytes showed no early changes (within 30 min) in intracellular pH in response to mitogenic concentrations of lectins or 4 beta-phorbol-12-myristate-13-acetate.     

Cathepsin M: a lysosomal proteinase with aldolase-inactivating activity

A proteinase, designated cathepsin M, that catalyzes the limited modification and inactivation of fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) and fructose 1,6-bisphosphatase (EC 3.1.3.11) has been partially purified from rabbit liver. On the basis of its molecular size (M_r = 30,000), activation by sulfhydryl compounds and inhibition by leupeptin it has been characterized as a B-type cathepsin, but other properties distinguish it from cathepsins B, L, and H. Approximately 50% of the total cathepsin M activity is associated with membranes prepared from disrupted lysosomes; this fraction of the activity is also expressed by intact lysosomes. In the membrane-bound form the enzyme is active at neutral pH, but the soluble enzyme and the activity eluted from the membranes are maximally active at pH 5.0. Fasting increases the activity of cathepsin M; the increase is almost entirely in the membrane-bound fraction.     

Identification of the non-lysosomal glucosylceramidase as beta-glucosidase 2

The primary catabolic pathway for glucosylceramide is catalyzed by the lysosomal enzyme glucocerebrosidase that is defective in Gaucher disease patients. A distinct non-lysosomal glucosylceramidase has been described but its identity remained enigmatic for years. We here report that the non-lysosomal glucosylceramidase is identical to the earlier described bile acid beta-glucosidase, being beta-glucosidase 2 (GBA2). Expressed GBA2 is identical to the native non-lysosomal glucosylceramidase in various enzymatic features such as substrate specificity and inhibitor sensitivity. Expression of GBA2 coincides with increased non-lysosomal glucosylceramidase activity, and GBA2-targeted RNA interference reduces endogenous non-lysosomal glucosylceramidase activity in cells. GBA2 is found to be located at or close to the cell surface, and its activity is linked to sphingomyelin generation. Hydrophobic deoxynojirimycins are extremely potent inhibitors for GBA2. In mice pharmacological inhibition of GBA2 activity is associated with impaired spermatogenesis, a phenomenon also very recently reported for GBA2 knock-out mice (Yildiz, Y., Matern, H., Thompson, B., Allegood, J. C., Warren, R. L., Ramirez, D. M., Hammer, R. E., Hamra, F. K., Matern, S., and Russell, D. W. (2006) J. Clin. Invest. 116, 2985-2994). In conclusion, GBA2 plays a role in cellular glucosylceramide metabolism.     

Deficient lysosomal carboxypeptidase activity in galactosialidosis

In the lysosome, the glycosidases neuraminidase (EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23) are associated to a 52 kDa "protective protein" to form a large multi-enzymatic complex. Deficient synthesis or inactivation of this protective protein causes galactosialidosis, a lysosomal storage disorder in man in which both neuraminidase and beta-galactosidase activities are deficient. Since the protective protein possesses extensive sequence homology with carboxypeptidase Y (carb Y) and the KEX 1 gene product from yeast, we have used the artificial substrate N-CBZ-Phe-Leu to detect and characterize the peptidase activity of the lysosomal carboxypeptidase (carb L). Using both a purified preparation of the lysosomal multi-enzymatic complex and cultured skin fibroblasts of patients affected with galactosialidosis, we demonstrate that the 52 kDa protective protein is responsible for carb L activity. The fibroblasts of three patients affected with late infantile and juvenile galactosialidosis were found to be deficient in carb L activity (1.4% of normal mean value).     

Molecular basis of reduced glucosylceramidase activity in the most common Gaucher disease mutant, N370S

Gaucher disease is caused by the defective activity of the lysosomal hydrolase, glucosylceramidase. Although the x-ray structure of wild type glucosylceramidase has been resolved, little is known about the structural features of any of the >200 mutations. Various treatments for Gaucher disease are available, including enzyme replacement and chaperone therapies. The latter involves binding of competitive inhibitors at the active site to enable correct folding and transport of the mutant enzyme to the lysosome. We now use molecular dynamics, a set of structural analysis tools, and several statistical methods to determine the flexible behavior of the N370S Gaucher mutant at various pH values, with and without binding the chaperone, N-butyl-deoxynojirimycin. We focus on the effect of the chaperone on the whole protein, on the active site, and on three important structural loops, and we demonstrate how the chaperone modifies the behavior of N370S in such a way that it becomes more active at lysosomal pH. Our results suggest a mechanism whereby the binding of N-butyl-deoxynojirimycin helps target correctly folded glucosylceramidase to the lysosome, contributes to binding with saposin C, and explains the initiation of the substrate-enzyme complex. Such analysis provides a new framework for determination of the structure of other Gaucher disease mutants and suggests new approaches for rational drug design.     

Intracellular metabolism of 4-hydroxynonenal

4-hydroxynonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert a multitude of biological, cytotoxic, and signal effects. Mammalian cells possess highly active pathways of HNE metabolism. The metabolic fate of HNE was investigated in various mammalian cells and organs such as hepatocytes, intestinal enterocytes, renal tubular cells, aortic and brain endothelial cells, synovial fibroblasts, neutrophils, thymocytes, heart, and tumor cells. The experiments were carried out at 37 degrees C at initial HNE concentrations between 1 microM--that means in the range of physiological and pathophysiologically relevant HNE levels--to 100 microM. In all cell types which were investigated, 90-95% of 100 microM HNE were degraded within 3 min of incubation. At 1 microM HNE the physiological blood serum level of about 0.1-0.2 microM was restored already after 10-30 s. As primary products of HNE in hepatocytes and other cell types the glutathione-HNE-1:1-conjugate, the hydroxynonenoic acid and the corresponding alcohol of HNE, the 1,4-dihydroxynonene, were identified. Furthermore, the beta-oxidation of hydroxynonenoic acid including the formation of water was demonstrated. The quantitative share of HNE binding to proteins was low with about 2-8% of total HNE consumption. The glycine-cysteine-HNE, cysteine-HNE adducts and the mercapturic acid from glutathione-HNE adduct were not formed in the most cell types, but in kidney cells and neutrophils. The rapid metabolism underlines the role of HNE degrading pathways in mammalian cells as important part of the secondary antioxidative defense mechanisms in order to protect proteins from modification by aldehydic lipid peroxidation products.     

Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes

Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22 degrees C reduces the dextran diffusion rates by approximately 30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature.     

Intracellular distribution of lysosomal enzymes in the mouse pigment mutants pale ear and pallid

Summary The size distribution of lysosomes was determined in kidney proximal tubule cells of two mouse pigment mutants, pale ear and pallid, which have an increase in kidney lysosomal enzyme content caused by a decreased rate of secretion of lysosomal enzymes into urine. Both mutations have larger lysosomes when compared with normal mice. However, neither mutant contains the giant lysosomes (up to 11 micron diameter) common to the well-characterized beige mutant, which has a kidney secretory defect similar to the pale ear and pallid mutants. Subcellular distribution studies, performed by the osmotic shock technique, likewise suggested differences among the pigment mutants. A very high content of soluble enzyme, indicative of lysosomal fragility during homogenization, was found in extracts from the beige mutation. By comparison, the percent of soluble enzyme became progressively lower in extracts of the pallid and pale ear mutants and was lowest in extracts from normal mice. All 3 pigment mutants had normal concentrations of osmotically resistant membrane-bound lysosomal enzymes. This indicates that the excess, non-secreted, lysosomal enzyme in all three pigment mutants likely is present in classical lysosomal organelles rather than in other non-lysosomal subcellular membrane fractions. The results also illustrate that mammalian mutants which exhibit decreased lysosomal secretory rates can have strikingly different effects on morphology of lysosomes.Supported in Part by National Science Foundation Grant PCM 77-24804. E. K. N. was supported in part by United States Public Health Service Grant GM 007093-03.