Cathepsin D precursors in clathrin-coated organelles from human fibroblasts

Coated vesicles were isolated from metabolically labeled human fibroblasts with the aid of affinity-purified antibodies against human brain clathrin and Staphylococcus aureus cells. The material adsorbed to the S. aureus cells was enriched in clathrin. When the S. aureus cells bearing the immunoadsorbed material were treated with 0.5% saponin, extracts containing the precursor form of cathepsin D were obtained. The extraction of the precursor was promoted in the presence of mannose 6-phosphate. Material adsorbed to S. aureus cells coated with control immunoglobulins was nearly free of clathrin and contained a small amount of the cathepsin D precursor (less than 20% of that adsorbed with anti-clathrin antibodies). The extraction of this cathepsin D precursor was independent of mannose 6-phosphate and was complete after a brief exposure to saponin. The amount of cathepsin D precursor in coated membranes varied between 0.4 and 2.5% of total precursor. Analysis of pulse chase-labeled fibroblasts revealed that cathepsin D was only transiently associated with coated membranes. The mean residence time of cathepsin D precursor in coated membranes was estimated to be 2 min. These observations support the view that coated membranes participate in the transfer of precursor forms of endogenous lysosomal enzymes to lysosomes.     

Molecular forms of cathepsin D in coated vesicle preparations

We have studied the polypeptide pattern of cathepsin D associated with coated vesicle fractions prepared from human placenta. In these fractions cathepsin D was about 35-fold enriched in the precursor polypeptides as compared to the unfractionated tissue extract. The enrichment was more prominent if the vesicles were fractionated in the presence of Triton X-100. Adsorption of exogenously added metabolically labelled cathepsin D precursor to the fractionated material was negligible. It is likely that the precursor and may be also the mature cathepsin D polypeptides are present in the matrix of the coated vesicles. This finding substantiates the idea that coated vesicles participate in the transport of newly synthesized cathepsin D into the lysosomes.     

Endocytosis and the recycling of plasma membrane

For study of the time order of glycosylation, formation of complex oligosaccharides and proteolytic maturation as well as the site of proteolytic maturation of cathepsin D, fibroblasts were subjected to pulse-chase labeling, and cathepsin D was isolated from either total cell extracts or subcellular fractions by immune precipitation and analyzed for its molecular forms and sensitivity to endo-beta-N- acetylglucosaminidase H. After a 10-min pulse, cathepsin D was detected in its glycosylated precursor form, indicating an early, probably a cotranslational, N-glycosylation of cathepsin D. Conversion of the high- mannose oligosaccharide side chains into forms resistant to endo-beta-N- acetylglucosaminidase H started after approximately 40 min, indicating that transport of cathepsin D from the endoplasmic reticulum to the trans-Golgi apparatus requires approximately 40 min. Processing of the 53-kdalton precursor polypeptide of cathepsin D to a 47-kdalton intermediate followed about 20 min after the formation of complex oligosaccharides, and, another 30 min later, 31-kdalton mature forms of cathepsin D were detected. Processing of cathepsin D was first observed in light membranes as a partial conversion of the 53-kdalton precursor into the 47-kdalton intermediate. Both the precursor and the intermediate are transferred into the high density-class lysosomes. After 8 h, the processing to the mature 31-kdalton form of cathepsin D is mostly completed.     

Cathepsin D and beta-hexosaminidase synthesized in the presence of 1-deoxynojirimycin accumulate in the endoplasmic reticulum

Biosynthesis, transport, and maturation of cathepsin D and beta-hexosaminidase was examined in fibroblasts exposed to 1-deoxynojirimycin, a glucose analogue known to inhibit trimming glucosidases (Saunier, B., Kilker, R. D., Jr., Tkacz, J. S., Quaroni, A., and Herscovics, A. (1982) J. Biol. Chem. 257, 14155-14161; Hettkamp, H., Bause, E., and Legler, G. (1982) Biosci. Rep. 2, 899-906). Cells treated with 1-deoxynojirimycin contained precursors of cathepsin D and beta-hexosaminidase larger by about 1-2 kDa than control cells. The shift in molecular size was probably due to glucose residues that were rapidly removed from the precursors in the absence but not in the presence of 1-deoxynojirimycin. In addition, 1-deoxynojirimycin inhibited the glycosylation of the beta-chain precursor of beta-hexosaminidase and the synthesis of glycoproteins, including that of cathepsin D. The proteolytic processing of the larger precursors was retarded by several hours. The delay in proteolytic maturation was secondary to the accumulation of the larger precursors in organelles, which fractionated with membranes of the endoplasmic reticulum and Golgi complex. The accumulated cathepsin D precursor contained neither mannose 6-phosphate residues nor complex type oligosaccharides, which are formed in the cis and trans aspects of the Golgi complex. Cathepsin D precursors eventually released from the site of accumulation were apparently deglucosylated, acquired mannose 6-phosphate residues and complex type oligosaccharides, and were transferred into lysosomes as efficiently as in control cells. Our results suggest that transport of cathepsin D from the endoplasmic reticulum to the Golgi complex depends on removal of glucose residues from its carbohydrate.     

Biosynthesis of the multiple forms of rabbit cardiac cathepsin D

Rabbit cardiac cathepsin D exists as multiple isomeric forms of Mr = 48,000 within cardiac tissue. Their mechanism of formation and their functional role in cardiac protein degradation are unknown. We have previously demonstrated that cathepsin D is initially synthesized as an Mr = 53,000 precursor that is processed by limited proteolysis within cardiac lysosomes to the Mr = 48,000 active forms of the enzyme. To determine if the multiple forms of active cathepsin D originate from a common precursor, isolated perfused Langendorff rabbit hearts were labeled in pulse (15 or 30 min) and pulse-chase (30 or 150 min) experiments with [35S]methionine. Newly synthesized cathepsin D was isolated by butanol/Triton X-100 extraction and immunoadsorption with anti-cathepsin D IgG-Sepharose, and the isomeric forms were separated by two-dimensional electrophoresis and fluorography. After 15- and 30-min pulse perfusions, 35S-labeled cathepsin D appeared as a single precursor form (Mr = 53,000, pI = 6.6). After 30-min pulse and 30-min chase, the precursor was modified to yield multiple precursor forms, all with molecular weight 53,000, but with differing pI values (6.6-6.0). After 30-min pulse and 150-min chase perfusion, multiple forms of both precursor and proteolytically processed active cathepsin D (Mr = 48,000, pI = 6.2-5.6) were detected. The 35S-labeled, proteolytically processed forms of active cathepsin D co-migrated with the major cathepsin D forms present in cardiac tissue. Subcellular fractionation and perfusions in the presence of chloroquine demonstrated that the multiple precursor forms of cathepsin D originated in a nonlysosomal intracellular compartment. Thus, the multiple forms of active cathepsin D originate from a common high molecular weight precursor, and their synthesis occurs prior to the limited proteolysis of the precursor in cardiac lysosomes.     

Lysosomal enzyme precursors in human fibroblasts. Activation of cathepsin D precursor in vitro and activity of beta-hexosaminidase A precursor towards ganglioside GM2

Precursors of cathepsin D and beta-hexosaminidase were isolated from secretions of human fibroblasts and their activity was studied with natural substrates. The immunoprecipitated precursor of cathepsin D, Mr 53000, was inactive with radioactive hemoglobin as substrate. At pH 3.8-4.2 an activation of the precursor took place, which was correlated by a reduction in size to Mr 51500. The observed cleavage of cathepsin D precursor in vitro resembles the autocatalytic activation of pepsinogen. The precursor of beta-hexosaminidase A is able to cleave the natural substrate GM2 ganglioside. This reaction, like that of the mature enzyme, depends on the presence of a protein activator, which interacts with the substrate and the enzyme.     

Progestin and estrogen control of cathepsin D expression and processing in rat uterine luminal epithelium and stroma-myometrium.

Progestins increase the activity and rate of synthesis of cathepsin D, a lysosomal aspartyl protease, in the uterine luminal epithelium in ovariectomized rats. Western blot analysis of luminal epithelial proteins determined that the progestin, medroxyprogesterone acetate (MPA) increased the 43-kDa form of cathepsin D by 7-fold in 24 hr, whereas estradiol increased the amount of the same form by only 2-fold. To examine the precursor-product relationship between cathepsin D proteins in the luminal epithelium and stroma-myometrium after progestin or estradiol treatment, uterine proteins were prelabeled by incubation with [35S]methionine in vitro, cathepsin D was isolated by immunoprecipitation, and equal amounts of labeled cathepsin D were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After each hormonal treatment in each uterine tissue, a 48-kDa precursor was processed into a 44-kDa cathepsin D product. Endoglycosidase H digestion of [35S]methionine-labeled cathepsin D from the luminal epithelium and stroma-myometrium of medroxyprogesterone-treated rats shifted the molecular masses of the cathepsin D proteins by approximately 5.7 kDa. To examine the contribution of increased mRNA to increased rates of cathepsin D synthesis, we measured levels of cathepsin D mRNA in uterine tissues after progestin and estrogen treatment. Total RNA was isolated from the uterine luminal epithelium and from the stroma-myometrium. Northern blot analysis identified a single 2.2-kb RNA band corresponding to the size expected for cathepsin D mRNA. Medroxyprogesterone increased levels of cathepsin D mRNA in the luminal epithelium (greater than 17-fold) and in the stroma myometrium (3-fold), with maximum increases at 9 hr after treatment. Estradiol also increased cathepsin D mRNA levels in both uterine tissues, but by only 2-fold. No hormonal effects on liver cathepsin D mRNA were observed. Increases in cathepsin D synthesis and activity in uterine tissues in response to progestin and estrogen appear to depend in part upon increased levels of mRNA.     

Molecular forms of beta-hexosaminidase and cathepsin D in serum and urine of healthy subjects and patients with elevated activity of lysosomal enzymes.

A procedure is described that allows the characterization of the molecular forms of beta-hexosaminidase and cathepsin D in controls and pathological specimens of human serum and human urine. The following observations were made. (1) In human serum, beta-hexosaminidase (alpha- and beta-chain) and cathepsin D are present predominantly in their high-molecular-weight precursor forms. In human urine, these enzymes exist as both precursor and mature forms. (2) Cathepsin D precursor from serum and urine differs in the number of oligosaccharides that are sensitive to endo-beta-N-acetylglucosaminidase H. Therefore the urine enzyme is not likely to originate from the serum. (3) The presence exclusively of precursors of beta-hexosaminidase and of cathepsin D in the sera of patients with hepatitis suggests that in hepatitis secretion of lysosomal enzymes is elevated, rather than the enzymes leaking from damaged cells. (4) In the urine of patients with nephrotic syndrome, beta-hexosaminidase and cathepsin D are present in grossly elevated amounts, but do not differ in the polypeptide patterns from controls. (5) In urine from a patient with mucolipidosis II, the elevated activity of beta-hexosaminidase is accounted for mainly by the precursor forms. Mature beta-chain of beta-hexosaminidase is lacking, and incompletely processed beta-hexosaminidase polypeptides are present. Both the precursor and the mature forms of cathepsin D are increased. They contain only complex oligosaccharides.     

Xenopsin-related peptide(s) are formed from xenopsin precursor by leukocyte protease(s) and cathepsin D

Lysates of isolated rat polymorphonuclear leukocytes and macrophages were found to generate xenopsin-related peptides when incubated with a liver extract used as a source of precursor. The lysosomal enzyme, cathepsin D, was also shown to display this property and to share with the lysate a similar pH dependence (optimum, approximately pH 3.5) and sensitivity to the acid protease inhibitor, pepstatin A (ID50: lysate, 10 nM; cathepsin D, 30 nM). When subjected to HPLC on mu-Bondapak C-18, the xenopsin-related peptides generated by the lysate eluted near to those formed by cathepsin D and when tested in a radioreceptor assay for neurotensin, they displayed similar cross-reactivities (peak 2, approximately 50%; peak 1, approximately 100%). These results indicate that cathepsin D from lysed granulocytes can process precursor protein(s) to form radioreceptor-active xenopsin-related peptides.     

Acid hydrolases in early and late endosome fractions from rat liver.

We have examined the distribution of the cation-independent mannose 6-phosphate receptor and five acid hydrolases in early and late endosomes and a receptor-recycling fraction isolated from livers of estradiol-treated rats. Enrichment of mannose 6-phosphate receptor mass relative to that of crude liver membranes was comparable in membranes of early and late endosomes but was even greater in membranes of the receptor-recycling fraction. Enrichment of acid hydrolase activities (aryl sulfatase, N-acetyl-beta-glucosaminidase, tartrate-sensitive acid phosphatase, and cholesteryl ester acid hydrolase) and cathepsin D mass was also comparable in early and late endosomes but was considerably lower in the receptor-recycling fraction. The enrichment of two acid hydrolases, acid phosphatase and cholesteryl ester acid hydrolase, in endosomes was severalfold greater than that of the other three examined, about 40% of that found in lysosomes. Acid phosphatase and cholesteryl ester acid hydrolase were partially associated with endosome membranes, whereas cathepsin D was found entirely in the endosome contents. These findings raise the possibility that lysosomal enzymes traverse early endosomes during transport to lysosomes in rat hepatocytes and suggest that the greater enrichment of some acid hydrolases in endosomes is related to their association with endosome membranes. Despite the substantial enrichment of lysosomal enzymes in hepatocytic endosomes, we found that two, cholesteryl ester acid hydrolase and cathepsin D, did not degrade cholesteryl esters and apolipoprotein B-100 of endocytosed low density lipoproteins in vivo, presumably because they are inactive at the pH within endosomes.     

Cathepsin D is membrane-associated in macrophage endosomes

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.     

Mitogenic effects of certain cathepsins and calciferin on the intact liver in vivo

Calciferin, a new parathyroid hormone stimulating the release of cathepsins D and L (but not B) from isolated lysosomes, or the release of cathepsin D from erythrocytes or ghosts in vitro, elevated free cathepsin D in the blood, and at the same time stimulated DNA synthesis in the intact liver when it was injected into mice. Both calciferin and free cathepsin D in the blood (rats) were elevated concomitantly soon after 70% hepatectomy, reaching a peak around 5 hr. The cathepsin D-elevation was almost proportional to fractional hepatectomies. Cathepsin L (but not B), when injected intraperitoneally into mice, stimulated DNA synthesis and mitosis in the intact liver much like cathepsin D, the effect of which was reported earlier. In contrast to the mitogenic effects of calciferin or cathepsins (D and L) in vivo, only cathepsin L (but not cathepsin D or calciferin) in low concentrations appeared to stimulate DNA synthesis in the cultured liver cells, and also stimulated adenylate cyclase of isolated liver plasma membranes in vitro. Dibutyryl-cyclic AMP in concentrations lower than 10(-5) M also stimulated DNA synthesis in cultured liver cells.     

The catalytically inactive precursor of cathepsin D induces apoptosis in human fibroblasts and HeLa cells

In several reports cathepsin D has been implicated in apoptosis. In some systems the effects of agents considered to be mediated by cathepsin D were inhibited in the presence of pepstatin A, an inhibitor of the enzyme. In other studies the effect of a mutant cathepsin D deprived of activity was indistinguishable from that of the normal enzyme. Here we show that in human fibroblasts and in HeLa cells apoptosis can be induced by microinjecting into cytosol either mature cathepsin D or its inactive precursor procathepsin D. The microinjected precursor remains in the uncleaved form. These results confirm that the proapoptotic effect of cathepsin D in the cytosol is independent of its catalytic activity and suggest that the interaction of cathepsin D with the downstream effector does not involve the active site of the enzyme, since in the proenzyme the active site is masked by the prosequence.     

Synthesis, transport and processing of cathepsin C in Morris hepatoma 7777 cells and rat hepatocytes

The synthesis, transport and processing of cathepsin C was studied in Morris hepatoma 7777 cells by metabolic labelling, immunoprecipitation and characterization of labelled polypeptides by gel electrophoresis and fluorography. The largest detectable precursor of cathepsin C was a polypeptide of Mr = 92 500. Even 3 min after synthesis this precursor was accompanied by four polypeptides with Mr values ranging from 63 000 to 54 000, indicating cleavage of the precursors within the endoplasmic reticulum. The early forms of cathepsin C were associated with low-buoyant-density organelles containing the markers of endoplasmic reticulum and Golgi complex. About 30% of these early forms were secreted within 3 h after synthesis. The remaining 70% were transferred into dense lysosomes and processed between 2 and 3 h after synthesis to a mixture of the least five major and nine minor polypeptides with Mr values ranging from 73 000 to 12 000. These forms remained stable for at least 3 days. In freshly isolated hepatocytes cathepsin C was processed to forms closely related to those found in the hepatoma cells. Cathepsin C was synthesized in Morris hepatoma 7777 cells as a glycoprotein with mannose-6-phosphate residues that mediated mannose-6-phosphate-specific receptor-dependent uptake in human skin fibroblasts. In contrast to hepatocytes, synthesis of mannose-6-phosphate receptors in Morris hepatoma 7777 cells was below the limit of detection. The hepatoma cells did not express at the cell surface these or other receptors mediating endocytosis of lysosomal enzymes. Further, processing and transport of newly synthesized cathepsin C was largely resistant to NH4Cl. Apparently, cathepsin C is transferred in Morris hepatoma 7777 cells by a mechanism independent of mannose-6-phosphate-specific receptors.     

Cell type dependent inhibition of transport of cathepsin D in HepG2 cells and fibroblasts exposed to deoxy-manno-nojirimycin and deoxynojirimycin

The synthesis, transport and processing of lysosomal enzymes was examined in human hepatoma HepG2 cells and in human fibroblasts exposed to the Golgi alpha-mannosidase I inhibitor 1-deoxy-manno-nojirimycin. In HepG2 cells cathepsin D, beta-hexosaminidase and arylsulfatase B synthesized in the presence of 5 mM 1-deoxy-manno-nojirimycin contained exclusively endo-beta-N-acetylglucosaminidase H-cleavable oligosaccharides, indicating that alpha-mannosidase I had been inhibited efficiently. The proteolytic processing of intracellularly retained cathepsin D was retarded and the fraction of secreted cathepsin D was increased two-fold. In fibroblasts neither segregation nor maturation of cathepsin D were affected by 1-deoxy-manno-nojirimycin in spite of the inhibition of oligosaccharide processing. In the presence of the glucosidase I inhibitor 1-deoxynojirimycin, the precursor of cathepsin D (larger by about 1 kDa than the secreted form) accumulated transiently in light membranes in HepG2 cells. Release from the site of accumulation was accompanied by a decrease in size by about 1 kDa. This change was attributed to the removal of glucose residues. In fibroblasts the transient accumulation of larger precursors in the presence of 1-deoxynojirimycin was more pronounced than in HepG2 cells. The differential effects of alpha-mannosidase I and glucosidase I inhibitors on the transport of cathepsin D in HepG2 cells and fibroblasts may indicate that different intermediates in the biosynthetic pathway of asparagine-linked oligosaccharides participate in the transport of lysosomal enzymes in the two cell types.     

Ultrastructural localization of the mannose 6-phosphate receptor in rat liver

An affinity-purified rabbit antibody against rat liver mannose 6- phosphate receptor (MP-R) was prepared. The antibody was directed against a 215 kd-polypeptide and it recognized both ligand-occupied and free receptor. Anti-MP-R was used for immunofluorescence and immunoelectron microscopy of cryosections from rat liver. MP-R was demonstrated in all parenchymal liver cells, but not in endothelial lining cells. MP-R labeling was found at the entire plasma membrane, in coated pits and coated vesicles, in the compartment of uncoupling receptor and ligand, and in the Golgi complex. Lysosomes showed only scarce MP-R label. In double-labeling immunoelectron microscopy, MP-R co-localized with albumin in the Golgi cisternae and in secretory vesicles with lipoprotein particles. Cathepsin D was associated with MP- R in the Golgi cisternae. This finding indicates that MP-R/cathepsin D complexes traverse the Golgi complex on their way to the lysosomes. The possible involvement of CURL in lysosomal enzyme targeting is discussed.     

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking.     

Putative convertase involved in the proteolytic conversion of rat proalbumin to serum albumin

We have found a proteolytic activity in Golgi membranes which efficiently converts [35S]methionine-labeled proalbumin, isolated from pulse-labeled rat hepatocytes in culture, to serum albumin in an in vitro assay system. The proalbumin-converting activity was dependent on Ca2+ and the maximum activity was observed at pH 5.5-6.0. Since the enzyme activity was found to be resistant not only to both leupeptin and E-64 but also to thiol-blocking reagents, it is unlikely that cathepsin B is involved in the proteolytic conversion of proalbumin occurring in the Golgi complex.     

Mammalian GGAs act together to sort mannose 6-phosphate receptors

The GGAs (Golgi-localized, gamma ear-containing, ADP ribosylation factor-binding proteins) are multidomain proteins implicated in protein trafficking between the Golgi and endosomes. We examined whether the three mammalian GGAs act independently or together to mediate their functions. Using cryo-immunogold electron microscopy, the three GGAs were shown to colocalize within coated buds and vesicles at the trans-Golgi network (TGN) of HeLa cells. In vitro binding experiments revealed multidomain interactions between the GGAs, and chemical cross-linking experiments demonstrated that GGAs 1 and 2 form a complex on Golgi membranes. RNA interference of each GGA resulted in decreased levels of the other GGAs and their redistribution from the TGN to cytosol. This was associated with impaired incorporation of the cation-independent mannose 6-phosphate receptor into clathrin-coated vesicles at the TGN, partial redistribution of the receptor to endosomes, and missorting of cathepsin D. The morphology of the TGN was also altered. These findings indicate that the three mammalian GGAs cooperate to sort cargo and are required for maintenance of TGN structure.     

Enhanced degradation of cathepsin D synthesized in the presence of the threonine analog beta-hydroxynorvaline

The threonine analog beta-hydroxynorvaline is an inhibitor of asparagine-linked glycosylation. In the presence of the analog human fibroblasts synthesized cathepsin D molecules containing two, one, or no oligosaccharides. The nonglycosylated cathepsin D precursor was but a minor species and was degraded within 45 min of its synthesis, presumably in the lumen of the endoplasmic reticulum. The polypeptides with one or two oligosaccharides were normally segregated into lysosomes and their proteolytic maturation was not affected. The stability of mature glycosylated and nonglycosylated cathepsin D polypeptides within the lysosomes, however, was markedly decreased. The recovery of cathepsin D polypeptides was increased in the presence of inhibitors of cysteine and aspartyl-proteinases. These data suggest that the absence of carbohydrate side chains in cathepsin D results in an enhancement of the degradation rate of the precursor in the endoplasmic reticulum, and the replacement of threonine by beta-hydroxynorvaline in an enhanced degradation of the mature cathepsin D in lysosomes.