Identification of the minimal lysosomal enzyme recognition domain in cathepsin D

Specific recognition of lysosomal hydrolases by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues, is governed by a common protein determinant. Previously, we generated a lysosomal enzyme recognition domain in the secretory protein glycopepsinogen by substituting in two regions (lysine 203 and amino acids 265-293 of the beta loop) from cathepsin D, a highly related lysosomal protease. Here we show that substitution of just two lysines (Lys-203 and Lys-267) stimulates mannose phosphorylation 116-fold. Substitution of additional residues in the beta loop, particularly lysines, increased phosphorylation 4-fold further, approaching the level obtained with intact cathepsin D. All the phosphorylation occurred at the carboxyl lobe glycan, indicating that additional elements are required for phosphorylation of the amino lobe glycan. These data support the proposal that as few as two lysines in the correct orientation to each other and to the glycan can serve as the minimal elements of the lysosomal enzyme recognition domain. However, our findings show that the spacing between lysines is flexible and other residues contribute to the recognition marker.     

Oligosaccharides in lysosomal enzymes. Distribution of high-mannose and complex oligosaccharides in cathepsin D and beta-hexosaminidase

The distribution of the different types of oligosaccharides in cathepsin D and in beta-hexosaminidase synthesized in cultured human fibroblasts was studied by using endo-beta-N-acetylglucosaminidase H as a probe for high-mannose oligosaccharides. The enzymes were specifically labelled in the protein or the carbohydrate moiety. In both enzymes, resistant and cleavable oligosaccharides were found. The resistant oligosaccharides prevailed in the secreted enzymes. Precursor molecules of cathepsin D contained two oligosaccharide side chains. Multiple forms of the precursor are synthesized with both, one or none of two oligosaccharides sensitive to the action of the endo-beta-N-acetylglucosaminidase H. In fibroblasts unable to phosphorylate lysosomal enzymes (mucolipidosis II) the excessively secreted lysosomal enzymes contained predominantly oligosaccharides resistant to endo-beta-N-acetylglucosaminidase H.     

Nonhuman cells correctly sort and process the human lysosomal enzyme cathepsin D

Cathepsin D, like most lysosomal enzymes, undergoes multiple proteolytic cleavages during its lifetime. Although the significance of the earliest cleavages of cathepsin D is apparent (loss of the NH2-terminal signal peptide and activation peptide), functions of the two later cleavages are not understood and do not occur in all species. To examine these later events, a cDNA coding for human cathepsin D, which is normally processed to a two-chain form, was isolated and then expressed in mammalian cells from species which do not process the enzyme to the two-chain form. Analysis of the expressed human cathepsin D demonstrated proteolytic processing identical with that seen in normal human fibroblasts. Since processing to the two-chain form of the enzyme occurs in the lysosome, these studies revealed that the human cathepsin D was correctly sorted. The data also indicated that the sorting mechanism was conserved between diverse species and that late proteolytic processing in a variety of species was not determined by the presence or absence of the processing enzymes in the cell.     

Schistosoma mansoni synthesizes novel biantennary Asn-linked oligosaccharides containing terminal beta-linked N-acetylgalactosamine.

This report describes the structure of novel complex-type Asn-linked oligosaccharides in glycoproteins synthesized by the human blood fluke, Schistosoma mansoni. Adult schistosome worm pairs (male and female) isolated from infected hamsters were metabolically radiolabelled with either [3H]glucosamine, [3H]mannose or [3H]galactose. The glycopeptides prepared by pronase digestion of the total glycoprotein fraction were isolated by affinity chromatography on columns of immobilized Concanavalin A (Con A) and Wisteria floribunda agglutinin (WFA). A subset of glycopeptides, designated IIb, that bound to both Con A and WFA was isolated. WFA has been shown to have affinity for oligosaccharides containing beta 1,4-linked N-acetylgalactosamine (GalNAc) at their non-reducing termini. Compositional analysis of IIb glycopeptides demonstrated that they contained N-acetylglucosamine (GlcNAc), GalNAc, mannose (Man) and fucose (Fuc), but no galactose (Gal) or N-acetylneuraminic acid (NeuAc). Methylation analyses and exoglycosidase digestions indicated that IIb glycopeptides were complex-type biantennary structures with branches containing the sequence GalNAc beta 1-4-[+/- Fuc alpha 1-3]GlcNAc beta 1-2Man alpha 1-R. The discovery of these unusual oligosaccharides synthesized by a human parasite, which appear to be similar to some newly discovered mammalian cell-derived oligosaccharides, may shed light on future studies related to the role oligosaccharides may play in host-parasite interactions.     

Human liver cathepsin D. Purification, crystallization and preliminary X-ray diffraction analysis of a lysosomal enzyme.

The two-chain form of active cathepsin D, a glycosylated, lysosomal aspartic proteinase, has been isolated from human liver. Isoelectric focusing revealed two major species of enzyme that differed by approximately 0.2 pI unit. Crystals suitable for X-ray diffraction analysis were prepared from acidic solutions using precipitation with ammonium sulfate. The hexagonal crystals diffracted X-rays to beyond 3.1 A resolution and belonged to space group P6(1) (or P6(5)) with cell constants a = b = 125.9 A, c = 104.1 A, gamma = 120.0 degrees. The crystals likely contain two molecules in the asymmetric unit, giving a solvent content of 56% (v/w). Biochemical analysis of crystals indicated that both isoforms were present in approximately equimolar proportions. Full structure determination of the enzyme is underway.     

Unique Asn-linked oligosaccharides of the human pathogen Entamoeba histolytica

N-Glycans of Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, are of great interest for multiple reasons. E. histolytica makes an unusual truncated N-glycan precursor (Man(5)GlcNAc(2)), has few nucleotide sugar transporters, and has a surface that is capped by the lectin concanavalin A. Here, biochemical and mass spectrometric methods were used to examine N-glycan biosynthesis and the final N-glycans of E. histolytica with the following conclusions. Unprocessed Man(5)GlcNAc(2), which is the most abundant E. histolytica N-glycan, is aggregated into caps on the surface of E. histolytica by the N-glycan-specific, anti-retroviral lectin cyanovirin-N. Glc(1)Man(5)GlcNAc(2), which is made by a UDP-Glc: glycoprotein glucosyltransferase that is part of a conserved N-glycan-dependent endoplasmic reticulum quality control system for protein folding, is also present in mature N-glycans. A swainsonine-sensitive alpha-mannosidase trims some N-glycans to biantennary Man(3)GlcNAc(2). Complex N-glycans of E. histolytica are made by the addition of alpha1,2-linked Gal to both arms of small oligomannose glycans, and Gal residues are capped by one or more Glc. In summary, E. histolytica N-glycans include unprocessed Man(5)GlcNAc(2), which is a target for cyanovirin-N, as well as unique, complex N-glycans containing Gal and Glc.     

Proteolytic processing sites producing the mature form of human cathepsin D.

1. The proteolytic processing sites of human lysosomal aspartic protease cathepsin D at which the intermediate single-chain form was converted into the mature two-chain form were determined. 2. The two chains were isolated by reversed-phase HPLC in order to investigate the cleavage sites of the enzyme. 3. Protein sequencing of the heavy chain, which was presumed to be derived from the C-terminal side in the single-chain enzyme, gave an N-terminal Leu 105. In addition, it revealed that there were also minor sequences, which commenced with Gly 106 and Gly 107. 4. A small C-terminal peptide was isolated from the light chain, which had been digested with two kinds of exogenous proteases. Sequence determination of this peptide, which was characterized as a nonapeptide by mass spectrometry, suggested that the C-terminus of the light chain was Ser 98. 5. These results indicate that a Ser 98-Ala 99 bond and an Ala 104-Leu 105 bond are cleaved to release 6 amino acid residues between the two chains.     

Biosynthesis of a lysosomal enzyme. Partial structure of two transient and functionally distinct NH2-terminal sequences in cathepsin D

Various biosynthetic forms of porcine spleen cathepsin D (Erickson, A. H. and Blobel, G. (1979) J. Biol. Chem. 254, 11771-11774), isolated by immunoprecipitation of in vivo- and in vitro-synthesized products, have been characterized by partial NH2-terminal sequence analysis. Two short lived and functionally distinct NH2-terminal sequence extensions, a "pre" sequence and a "pro" sequence, have been detected. Both sequence extensions are present in preprocathepsin D which is the primary translation product immunoprecipitated after translation of porcine spleen mRNA in a wheat germ cell-free system. Preprocathepsin D is not glycosylated and has an approximate Mr = 43,000. Its 20-residue pre sequence resembles the signal sequences of presecretory proteins in abundance of Leu residues (7 out of 20 residues). Addition of dog pancreatic microsomal vesicles to the translation system resulted in the cleavage of the pre sequence and yielded segregated and glycosylated procathepsin D (Mr = 46,000) that was indistinguishable from its in vivo-synthesized counterpart detected after pulse-labeling of cultured porcine kidney cells. Some of this in vivo-synthesized procathepsin D was secreted and persisted as such in the culture medium. The remainder was converted within a period of 15 min to 2 h to single chain cathepsin D (Mr = 44,000) by removal of a pro sequence which was estimated to be 44 residues. Its partial sequence showed considerable sequence homology to the 44-residue activation peptide of pepsinogen. It is possible, therefore, that the prosequence of procathepsin D serves as an activation peptide that keeps the enzyme inactive during intracellular transport to the lysosome. The enzymatically active single chain form of cathepsin D undergoes further cleavage into a light and a heavy chain (Mr = 15,000 and 30,000, respectively) over a period of 2-24 h after synthesis. The oligosaccharide moieties of procathepsin D and of the single chain and heavy chain forms of cathepsin D are cleaved by endoglycosidase H. Treatment of cells with tunicamycin arrests the biosynthetic pathway of cathepsin D at procathepsin D. The nonglycosylated procathepsin D is not proteolytically processed and its secretion is greatly inhibited.     

Granulocytic differentiation of HL-60 cells is associated with increase of poly-N-acetyllactosamine in Asn-linked oligosaccharides attached to human lysosomal membrane glycoproteins

HL-60 cells were induced to differentiate into granulocytic cells by dimethyl sulfoxide, and structures of Asn-linked oligosaccharides attached to lysosomal membrane glycoproteins (lamp-1 and lamp-2) were elucidated before and after differentiation. Lamp-1 and lamp-2 were immunoprecipitated from the cells after labeling with radioactive sugars, and glycopeptides were prepared. The structures of glycopeptides obtained after serial lectin-affinity chromatography were elucidated by endo-beta-galactoside and methylation analysis. Glycopeptides bound to tomato lectin-Sepharose were found to be tetraantennary oligosaccharides that contain two or three poly-N-acetyllactosaminyl chains, of which one side chain contains three or more N-acetyllactosaminyl repeats, whereas those bound to Datura stramonium agglutinin-Sepharose were found to be tetraantennary oligosaccharides containing one or two short poly-N-acetyllactosaminyl side chains. Glycopeptides that were not bound to concanavalin A, tomato lectin, or D. stramonium agglutinin were found to be triantennary oligosaccharides with a negligible amount of poly-N-acetyllactosaminyl side chains. Comparison of Asn-linked oligosaccharides from undifferentiated and differentiated HL-60 cells reveals the following features. First, the number of Asn-linked oligosaccharides containing poly-N-acetyllactosaminyl side chains increases dramatically with a concomitant decrease in less complex Asn-linked oligosaccharides after differentiation. Second, the number of poly-N-acetyllactosaminyl side chains per Asn-linked oligosaccharides increases significantly. These increases in poly-N-acetyllactosamine were associated with increased activity of UDP-GlcNAc:beta-D-Gal-beta 1----3-N-acetylglucosaminyltransferase "extension enzyme," a key enzyme in the formation of poly-N-acetyllactosamines. Furthermore, the increased amount of poly-N-acetyllactosamine in lamp-1 and lamp-2 resulted in longer half-lives of lamp-1 and lamp-2 in differentiated HL-60 cells. These results suggest strongly that the differentiation of HL-60 cells into more phagocytic cells is associated with an increase in the complexity of Asn-linked oligosaccharides attached to lysosomal membrane glycoproteins, which in turn may play a role in stabilizing lysosomes.     

Several cooperating binding sites mediate the interaction of a lysosomal enzyme with phosphotransferase.

Lysosomal targeting of soluble lysosomal hydrolases is mediated by mannose 6-phosphate receptors, which recognize and bind mannose 6-phosphate residues in the oligosaccharide chains of proteins destined for delivery to lysosomes. This recognition marker is generated by the sequential action of two enzymes, the first of which, UDP-N-acetylglucosamine phosphotransferase, recognizes lysosomal enzymes on the basis of a structural determinant in their polypeptide chains. This recognition event is a key step in lysosomal targeting of soluble proteins, but the exact nature of the recognition determinant is not well understood. In this study we have characterized the phosphotransferase recognition signals of human lysosomal aspartylglucosaminidase (AGA) using transient expression of polypeptides carrying targeted amino acid substitutions. We found that three lysine residues and a tyrosine residing in three spatially distinct regions of the AGA polypeptide are necessary for phosphorylation of the oligosaccharides. Two of the lysines are especially important for the lysosomal targeting efficiency of AGA, which seems to be mostly dictated by the degree of phosphorylation of the alpha subunit oligosaccharide. On the basis of the results of this and previous studies we suggest a general model for recognition of lysosomal enzymes by the phosphotransferase.     

Two crystal structures for cathepsin D: the lysosomal targeting signal and active site.

Two crystal structures are described for the lysosomal aspartic protease cathepsin D (EC 3.4.23.5). The molecular replacement method was used with X-ray diffraction data to 3 A resolution to produce structures for human spleen cathepsin D and for bovine liver cathepsin D complexed with the 6-peptide inhibitor pepstatin A. The lysosomal targeting region of cathepsin D defined by previous expression studies [Barnaski et al. (1990) Cell, 63, 281-219] is located in well defined electron density on the surface of the molecules. This region includes the putative binding site of the cis-Golgi phosphotransferase which is responsible for the initial sorting step for soluble proteins destined for lysosomes by phosphorylating the carbohydrates on these molecules. Carbohydrate density is visible at both expected positions on the cathepsin D molecules and, at the best defined position, four sugar residues extend towards the lysosomal targeting region. The active site of the protease and the active site cleft substrate binding subsites are described using the pepstatin inhibited structure. The model geometry for human cathepsin D has rms deviations from ideal of bonds and angles of 0.013 A and 3.2 degrees respectively. For bovine cathepsin D the corresponding figures are 0.014 A and 3.3 degrees. The crystallographic residuals (R factors) are 16.1% and 15.8% for the human and inhibited bovine cathepsin D models respectively. The free R factors, calculated with 10% of the data reserved for testing the models and not used for refinement, are 25.1% and 24.1% respectively.     

Isolation and characterization of a stable activation intermediate of the lysosomal aspartyl protease cathepsin D.

Procathepsin D is the intracellular aspartyl protease precursor of cathepsin D, a major lysosomal enzyme. Procathepsin D is rapidly processed inside the cell, and, thus, examination of its proteolyic activation and structure has been difficult. To study this proenzyme, a nonglycosylated form of the human fibroblast procathepsin D was expressed in Escherichia coli, refold in vitro, and purified by affinity chromatography on pepstatinyl agarose. Sequence analysis of the refolded, autoactivated enzyme allowed determination of the autoproteolytic cleavage site. The sequence surrounding this cleavage site between residues LeuP26 and IleP27 (in the "pro" region) resembled the first cleavage site found during activation of other aspartyl proteases. Thus, the autoactivated procathepsin D is analogous to the pepsin activation intermediate, which has been termed pseudopepsin. The enzymatic activity, thermal and pH stability, and fluorescence spectra of pseudocathepsin D were compared to mature, predominantly two-chain, cathepsin D isolated from human placenta. The results indicated that pseudocathepsin D and mature enzyme have a similar Km toward a peptide substrate and cleave a protein substrate at identical sites. Temperature stability of the recombinant enzyme was similar to that of the tissue-derived enzyme. However, the recombinant enzyme had increased stability at low pH when compared to the glycosylated tissue-derived two-chain cathepsin D. Fluorescence spectra of the recombinant and tissue-derived enzymes were identical. Thus, the absence of asparagine-linked oligosaccharides and the presence of the remaining segment of propeptide did not significantly alter the structural and enzymatic properties of the enzyme.     

Role of thiols, pH and cathepsin D in the lysosomal catabolism of serum albumin.

Attempts were made to assess the role of thiols and to determine the cathepsins involved in the degradation of serum albumin in mouse liver and kidney lysosomes. Unlike cysteine or beta-mercaptoethanol, reduced glutathione (GSH) did not stimulate the degradation of formaldehyde-treated albumin in liver lysosomes, suggesting that the tripeptide did not penetrate the membrane. However, GSH was a much more effective stimulant of proteolysis in kidney lysosomes than was cysteine at low concentrations, and the effect was saturable at 1-2 mM concentrations. Thiols did not stimulate proteolysis in lysosomes when the disulphide bonds of albumin were reduced and alkylated, suggesting that the stimulatory effects were solely due to disulphide-bond reduction in protein substrates. Results obtained with thiols and iodoacetamide suggested that albumins denatured by disulphide-bond reduction and alkylation, disulphide-bond reduction without alkylation, or by treatment with 8 M-urea, were all degraded primarily by cathepsin D in lysosomes, but formaldehyde-denatured albumin was attacked by thiol proteinases. These findings correlated well with studies on the degradation of these proteins by rat liver lysosome (tritosome) extracts. Studies with the proteinase inhibitors leupeptin and pepstatin and the stimulatory effects of thiols in these extracts suggested that formaldehyde-denatured albumin was degraded primarily by the thiol proteinases, but that native albumin or albumins denatured by disulphide-bond reduction or by treatment with 8 M-urea were attacked by cathepsin D. Denaturation of serum albumin by any of the methods used caused a shift in the pH optimum of albumin catabolism by tritosome extracts or by purified cathepsin D from approx. 3-4 to 5-6. These results were discussed in terms of a possible mechanism for the catabolic aspect of serum albumin turnover.     

Role of N-linked oligosaccharide flexibility in mannose phosphorylation of lysosomal enzyme cathepsin L

Mannose phosphorylation of N-linked oligosaccharides by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is a key step in the targeting of lysosomal enzymes in mammalian cells and tissues. The selectivity of this process is determined by lysine-based phosphorylation signals shared by lysosomal enzymes of diverse structure and function. By introducing new glycosylation sites at several locations on the surface of mouse procathepsin L and modeling oligosaccharide conformations for sites that are phosphorylated, it was shown that the inherent flexibility of N-linked oligosaccharides can account for the specificity of the transferase for oligosaccharides at different locations on the protein. By using this approach, the physical relationship between the lysine-based signal and the site of phosphorylation of mannose residues was determined. The analysis also revealed the existence of additional independent lysine-based phosphorylation signals on procathepsin L, which account for the low level of phosphorylation observed when the primary Lys-54/Lys-99 signal is ablated. Mutagenesis of residues that surround Lys-54 and Lys-99 and demonstration of mannose phosphorylation of a glycosylated derivative of green fluorescent protein provide strong evidence that the cathepsin L phosphorylation signal is a simple structure composed of as few as two well placed lysine residues.     

Lysosomal enzyme phosphorylation. Recognition of a protein-dependent determinant allows specific phosphorylation of oligosaccharides present on lysosomal enzymes

We have investigated the basis for the specific recognition of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. This enzyme is responsible for the selective phosphorylation of mannose residues on lysosomal enzymes. Two mammalian lysosomal enzymes, cathepsin D and uteroferrin, and two nonlysosomal glycoproteins were treated with endo-beta-N-acetylglucosaminidase H to remove those high mannose oligosaccharide units which are accessible on the native protein. These proteins were then tested as inhibitors of three different glycosyltransferases. The endo H-treated lysosomal enzymes were shown to be specific inhibitors of the phosphorylation of intact lysosomal enzymes. Proteolytic fragments of cathepsin D, including the entire light chain and heavy chain, did not retain the ability to be recognized by the N-acetylglucosaminylphosphotransferase. These findings indicate that the intact protein portion of lysosomal enzymes contains a specific recognition determinant which leads to high-affinity binding to the N-acetylglucosaminylphosphotransferase. The expression of this determinant appears to be dependent on the conformation of the protein.     

Transformation by oncogenic ras-p21 alters the processing and subcellular localization of the lysosomal protease cathepsin D.

The expression, processing, and intracellular localization of cathepsin D (CD), an endosomal-lysosomal protease involved in malignancy, were studied in rat embryo fibroblasts transformed with an active mutant of c-Ha-ras oncogene. The pattern of the processed molecular forms of CD, comprising two single-chain mature forms of 45 and 43 kDa and two double-chain mature forms of 34 + 9 kDa and 30 + 14 kDa, expressed by the parental cell line was similar to that found in normal rat liver cells. By contrast, in the ras-transfected counterpart this pattern was profoundly altered in that the 45 kDa species was much less represented and the 30 + 14 kDa species virtually absent. In both untransformed and ras-transformed cells the conversion of proCD into mature forms was not inhibited by ammonium chloride, which is known to increase the intravacuolar pH of post-Golgi compartments. Yet, this drug induced the accumulation of the 43 and 45 kDa molecular forms of mature CD in ras-transformed cells and of the 34 kDa molecule in untransformed cells. As compared to controls, in ras-transformed fibroblasts vacuolar compartments containing CD were reduced in number and mostly located toward the periphery of the cell. This contrasted with the perinuclear distribution of CD-positive granules in untransformed cells. Serum deprivation did not affect the growth, nor the intra- and extracellular accumulation of CD activity in ras-transformed cultures, while it blocked the growth and strongly stimulated the accumulation of CD in the medium in cultures of control fibroblasts. Altogether these data are indicative for a crucial role of ras GTPase in the regulation of the transport between post-Golgi organelles.     

The asparagine-linked oligosaccharides at individual glycosylation sites in human thyrotrophin.

The asparagine-linked carbohydrate structures at each of the three glycosylation sites of human thyrotrophin were investigated by 400 MHz 1H-NMR spectroscopy. Highly purified, biologically active human thyrotrophin (hTSH) was dissociated into its subunits hTSH alpha (glycosylated at Asn 52 and Asn 78) and hTSH beta (glycosylated at Asn 23). The alpha-subunit was further treated with trypsin which gave two glycopeptides that were subsequently separated by reverse-phase HPLC and identified by amino acid sequence analysis. The oligosaccharides were liberated from hTSH alpha glycopeptides and from intact hTSH beta by hydrazinolysis, and were fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC preparatory to structural analysis. The N-glycans present on hTSH were mainly diantennary complex-type structures with a common Man alpha 1-3 branch that terminated with 4-O-sulphated GalNAc. The Man alpha 1-6 branch displayed structural heterogeneity in the terminal sequence, with chiefly alpha 2-3-sialylated Gal and/or 4-O-sulphated GalNAc. The relative amounts of the two major complete diantennary oligosaccharides and their core fucosylation differed according to glycosylation site; the sulphated/sialylated diantennary oligosaccharide was most abundant at the two sites on the alpha-subunit, whereas the disulphated, core-fucosylated oligosaccharide was more plentiful on the beta-subunit. Some interesting structural features, not previously reported for the N-glycans of hTSH, included 3-O-sulphated galactose (SO4-3Gal) and peripheral fucose (Fuc alpha 1-3GlcNAc) in the Man alpha 1-6 branch of some diantennary structures; the former suggests the presence of a hitherto uncharacterized galactose-3-O-sulphotransferase in thyrotroph cells of the human anterior pituitary gland.     

Endolysosomal transport of newly-synthesized cathepsin D in a sucrose model of lysosomal storage

Newly-synthesized soluble lysosomal enzymes are transported from the trans-Golgi network to lysosomes by a mannose 6-phosphate receptor-mediated pathway. Lysosomal storage of indigestible material has been reported to perturb the biosynthesis and the fate of lysosomal hydrolases. In this study, we have focused our attention on the last steps in the transport of newly-synthesized cathepsin D to lysosomes in sucrose-treated WI-38 fibroblasts. Pulse-chase experiments indicate that, in sucrose-treated cells, cathepsin D maturation is delayed by 2 to 4 h. By subcellular fractionation, we show that newly-synthesized cathepsin D precursors transit through organelles endowed with a high sedimentation coefficient. These organelles are recovered in the dense region of a self-forming Percoll density gradient while the bulk of hydrolytic activities is redistributed to the low density region. Only later, are the precursors delivered to organelles containing the bulk of active hydrolases. There, procathepsin D is proteolytically processed into its 31 kDa-mature form. Our results suggest that when sucrose is present, the delayed maturation of procathepsin D is related to the delivery of the polypeptides into an organelle behaving in centrifugation like lysosomes but which is poorly efficient in proteolytic processing of procathepsin D. This low proteolytic activity of this organelle could be due to its poor ability to interact with hydrolase-containing structures.