Characterization of the Late Endosomal ESCRT Machinery in Trypanosoma brucei

The multivesicular body (MVB) is a specialized Rab7+ late endosome (LE) containing multiple intralumenal vesicles that function in targeting ubiquitinylated cell surface proteins to the lysosome for degradation. African trypanosomes lack a morphologically well‐defined MVB, but contain orthologs of the ESCRT (Endosomal Sorting Complex Required for Transport) machinery that mediates MVB formation. We investigate the role of TbVps23, an early ESCRT component, and TbVps4, the terminal ESCRT ATPase, in lysosomal trafficking in bloodstream form trypanosomes. Both localize to the TbRab7+ LE and RNAi silencing of each rapidly blocks growth. TbVps4 silencing results in approximately threefold accumulation of TbVps23 at the LE, consistent with blocking terminal ESCRT disassembly. Trafficking of endocytic and biosynthetic cargo, but not default lysosomal reporters, is also negatively affected. Others reported that TbVps23 mediates ubiquitin‐dependent lysosomal degradation of invariant surface glycoproteins (ISG65) (Leung et al., Traffic 2008;9:1698–1716). In contrast, we find that TbVps23 ablation does not affect ISG65 turnover, while TbVps4 silencing markedly enhances lysosomal degradation. We propose several models to accommodate these results, including that the ESCRT machinery actually retrieves ISG65 from the LE to earlier endocytic compartments, and in its absence ISG65 traffics more efficiently to the lysosome. Overall, these results confirm that the ESCRT machinery is essential in Trypanosoma brucei and plays important and novel role(s) in LE function in trypanosomes .     

Multiple motifs regulate trafficking of the LAMP-like protein p67 in the ancient eukaryote Trypanosoma brucei

p67 is a lysosome-associated membrane protein-like lysosomal type I transmembrane glycoprotein in African trypanosomes. The p67 cytoplasmic domain (CD) is both necessary and sufficient for lysosomal targeting in procyclic insect-stage parasites. The p67CD contains two [DE]XXXL[LI]-type dileucine motifs, which function as lysosomal targeting signals in mammalian cells. Using a green fluorescent protein fusion to the p67 transmembrane and cytoplasmic domains as a reporter system, we investigated the role of these motifs in lysosomal targeting in procyclic trypanosomes. Pulse-chase turnover studies, steady-state immunolocalization and quantitative flow cytometry all gave consistent results. Mutagenesis of the membrane-distal dileucine motif impairs lysosomal trafficking leading to partial appearance of the reporter on the cell surface. Mutagenesis of the membrane-proximal motif has little effect on proper targeting. Simultaneous mutagenesis of both motifs results in quantitative delivery to the cell surface. Thus, the distal motif plays a dominant role, but both dileucine motifs are necessary for maximal lysosomal targeting. Additional studies suggest that the upstream acidic residues in each motif influence lysosomal targeting and may also affect forward trafficking in the early secretory pathway. These results strongly suggest an evolutionary conservation in lysosomal trafficking mechanisms in the ancient eukaryote Trypanosoma brucei.     

Human serum lyses Trypanosoma brucei by triggering uncontrolled swelling of the parasite lysosome

Trypanosoma brucei brucei infects a wide range of mammals, but is unable to infect humans because this subspecies is lysed by normal human serum (NHS). The phenotype of cellular lysis is debated. For some authors the lysosome undergoes osmotic swelling due to massive influx of chloride ions from the cytoplasmic compartment, but others describe multiple small cytoplasmic vacuoles and general swelling of the cellular body. Using population-level imaging of live immobilized trypanosomes throughout the lysis process, we report that specific swelling of the lysosome is a genuine and major characteristic of NHS-mediated lysis and that this phenotype is independent of the strain of trypanosomes and of NHS aging or damaging. Thus, irrespective of experimental conditions NHS reproducibly induced the swelling of the parasite lysosome.     

Trypanosome Lytic Factor-1 Initiates Oxidation-stimulated Osmotic Lysis of Trypanosoma brucei brucei

Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. brucei gambiense and T. brucei rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate-specific apolipoproteins: the ion channel-forming toxin ApoL1 (apolipoprotein L1) and the hemoglobin (Hb) scavenger Hpr (haptoglobin-related protein). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. brucei brucei. The lipophilic antioxidant N,N′-diphenyl-p-phenylenediamine protected TLF-1-treated T. brucei brucei from lysis. Conversely, lysis of TLF-1-treated T. brucei brucei was increased by the addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the iron-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knock-out of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1-treated T. brucei brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1-treated T. brucei brucei became rapidly susceptible to hypotonic lysis. T. brucei brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane, which sensitizes T. brucei brucei to oxidation-stimulated osmotic lysis.     

Changes in oxidative metabolism and ultrastructure accompanying differentiation of the mitochondrion in Trypanosoma brucei

A method is described for obtaining optimal growth and morphological transformation of Trypanosoma brucei 792G in a monophasic blood lysate medium starting from populations of bloodstream trypanosomes containing over 90 per cent intermediate and stumpy forms. Transformation was accompanied by: (1) an increase in length of the trypanosomes from 13·8 (± 3·0) μm to 23·0 (± 2·5) μm; (2) an increase in the kinetoplastic index from 1·0 to greater than 2·0; (3) development of the mitochondrion from a single abflagellar canal to a network of subpellicular canals, with outgrowth of the post-kineto-plastic region of the mitochondrion; (4) replacement of some of the tubular mitochondrial cristae by plate-like cristae; (5) the acquisition of succinoxidase, succinate-cytochrome c reductase, and glycerophosphate-cytochrome c reductase activities; (6) a marked increase in proline oxidase and NADH-cytochrome c reductase activities; (7) loss of the surface coat of the flagellate and concomitant reduction in the smooth-membrane systems lying between the nucleus and flagellar pocket; (8) reduction in infectivity of the trypanosomes to the mammalian host. Although growth in primary culture continued up to 100 h, transformation was completed in 48 h. All the respiratory enzyme activities tested were insensitive to cyanide throughout transformation. Division of trypanosomes appeared to be taking place throughout the transformation process. Cyanide sensitivity developed only after subculture of the trypanosomes into a biphasic medium.     

Cloning and characterization of a basic cysteine-like protease (cathepsin L1) expressed in the gut of larval Diaprepes abbreviatus L. (Coleoptera: Curculionidae)

Diaprepes abbreviatus is an important pest that causes extensive damage to citrus in the USA. Analysis of an expressed sequence tag (EST) library from the digestive tract of larvae and adult D. abbreviatus identified cathepsins as major putative digestive enzymes. One class, sharing amino acid sequence identity with cathepsin L’s, was the most abundant in the EST dataset representing 14.4% and 3.6% of the total sequences in feeding larvae and adults, respectively. The predominant cathepsin (Da-CTSL1) among this class was further studied. Three dimensional modeling of the protein sequence showed that the mature Da-CTSL1 protein folds into an expected cathepsin L structure producing a substrate binding pocket with appropriate positioning of conserved amino acid residues. A full-length cDNA was obtained and the proCTSL1 encoding sequence was expressed in Rosetta™ Escherichia coli cells engineered to express tRNAs specific for eukaryotic codon usage. The Da-CTSL1 was expressed as a fusion protein with GST and His₆ tags and purified in the presence of 1% Triton X-100 by Ni-NTA affinity and size exclusion chromatography. Recombinant mature Da-CTSL1 (23 KDa) exhibits optimal activity at pH 8, rather than at acidic pH that was shown of all previously characterized cathepsins L. Substrate specificity supports the hypothesis that Da-CTSL1 is a unique basic cathepsin L and protease inhibitor studies also suggest unique activity, unlike other characterized acidic cathepsin Ls. This paper describes for the first time a prokaryotic expression system for the production of a functional eukaryotic cathepsin L1 from larval gut of D. abbreviatus.     

Stabilization of immobilized cathepsin L in non-aqueous medium

A lysosomal cysteine protease cathepsin L (3.4.22.15) purified from goat brain has been immobilized in calcium alginate beads in the presence of BSA through entrapment. Most favorable conditions for the entrapment were standardized as 3.0%(w/v) alginate and 1.5%(w/v) calcium chloride. Comparing the properties of free and immobilized enzyme using Z-Phe-Arg-4mβNA as chromogenic substrate, it was found that the immobilized enzyme could retain～70% of the original activity after five successive batch reactions. Vis-à-vis the free enzyme, immobilization conferred high stability to the enzyme both in the acidic and alkaline range, the enzyme lost no activity up to 60°C (Temperature stability for free enzyme is only up to 50°C). The pH optima for the enzyme shifted from 6.2 to 6.6 on entrapment. The increase in activity and stability of the enzyme in immobilized form even in the presence of high concentration of DMSO and ethanol is surprising and may make it useful for catalyzing organic reactions like trans-esterification and trans-amidation.     

Substrate inhibition and allosteric regulation by heparan sulfate of Trypanosoma brucei cathepsin L

The cysteine protease brucipain is an important drug target in the protozoan Trypanosoma brucei, the causative agent of both Human African trypanosomiasis and Animal African trypanosomiasis. Brucipain is closely related to mammalian cathepsin L and currently used as a framework for the development of inhibitors that display anti-parasitic activity. We show that recombinant brucipain lacking the C-terminal extension undergoes inhibition by the substrate benzyloxycarbonyl-FR-7-amino-4-methylcoumarin at concentrations above the K(m), but not by benzyloxycarbonyl-VLR-7-amino-4-methylcoumarin. The allosteric modulation exerted by the substrate is controlled by temperature, being apparent at 25°C but concealed at 37°C. The behavior of the enzyme in vitro can be explained by discrete conformational changes caused by the shifts in temperature that render it less susceptible to substrate inhibition. Enzyme inhibition by the di-peptydyl substrate impaired the degradation of human fibrinogen at 25°C, but not at 37°C. We also found that heparan sulfate acts as a natural allosteric modulator of the enzyme through interactions that prevent substrate inhibition. We propose that brucipain shifts between an active and an inactive form as a result of temperature-dependent allosteric regulation.     

Disruption of the Man-6-P targeting pathway in mice impairs osteoclast secretory lysosome biogenesis

Osteoclasts are specialized cells that secrete lysosomal acid hydrolases at the site of bone resorption, a process critical for skeletal formation and remodeling. However, the cellular mechanism underlying this secretion and the organization of the endo-lysosomal system of osteoclasts have remained unclear. We report that osteoclasts differentiated in vitro from murine bone marrow macrophages contain two types of lysosomes. The major species is a secretory lysosome containing cathepsin K and tartrate-resistant acid phosphatase (TRAP), two hydrolases critical for bone resorption. These secretory lysosomes are shown to fuse with the plasma membrane, allowing the regulated release of acid hydrolases at the site of bone resorption. The other type of lysosome contains cathepsin D, but little cathepsin K or TRAP. Osteoclasts from Gnptab(-/-) (gene encoding GlcNAc-1-phosphotransferase α, β-subunits) mice, which lack a functional mannose 6-phosphate (Man-6-P) targeting pathway, show increased secretion of cathepsin K and TRAP and impaired secretory lysosome formation. However, cathepsin D targeting was intact, showing that osteoclasts have a Man-6-P-independent pathway for selected acid hydrolases.     

The Effects of the Methylating Agent 1,2-Bis(methylsulfonyl)-1-methylhydrazine on Morphology, DNA Content and Mitochondrial Function of Trypanosoma brucei Subspecies

Repeated exposure of trypanosomes in vitro or in vivo to low concentrations of the methylating agent 1,2-bis(methylsulfonyl)-1-methylhydrazine induces a series of moderately synchronous morphological and biochemical changes. Cell division halts and the long-slender bloodstream forms transform to short-stumpy forms via larger intermediate-stage cells which contain approximately double the normal G₂ content of DNA. In common with naturally occurring short-stumpy trypanosomes, drug-induced short-stumpy forms do not infect rodents and when transferred to Cunningham's medium, transform to and replicate as procylics. Furthermore, these short-stumpy forms exhibit α-ketoglutarate supported motility and oxygen consumption, acquire the ability to reduce nitroblue tetrazolium (NADH diaphorase positivity) and appear to be in the G₁ or G₀ stage of the cell cycle based upon DNA content.     

Cell penetrable,clickable and tagless activity-based probe of human cathepsin L

Human cathepsin L is a ubiquitously expressed endopeptidase and is known to play critical roles in a wide variety of cellular signaling events. Its overexpression has been implicated in numerous human diseases, including highly invasive forms of cancer. Inhibition of cathepsin L is therefore considered a viable therapeutic strategy. Unfortunately, several redundant and even opposing roles of cathepsin L have recently emerged. Selective cathepsin L probes are therefore needed to dissect its function in context-specific manner before significant resources are directed into drug discovery efforts. Herein, the development of a clickable and tagless activity-based probe of cathepsin L is reported. The probe is highly efficient, active-site directed and activity-dependent, selective, cell penetrable, and non-toxic to human cells. Using zebrafish model, we demonstrate that the probe can inhibit cathepsin L function in vivo during the hatching process. It is anticipated that the probe will be a highly effective tool in dissecting cathepsin L biology at the proteome levels in both normal physiology and human diseases, thereby facilitating drug-discovery efforts targeting cathepsin L.     

Structure-based design of specific cathepsin inhibitors and their application to protection of bone metastases of cancer cells.

We report the antihypercalcemic and antimetastatic effects of CLIK-148 in vivo, which is a specific inhibitor of cathepsin L. The decalcification during bone absorption is followed by the degradation of type-1 collagen by osteoclastic cathepsins. Tumor-bearing osteoclasts or TNF-alpha-activated osteoclasts secrete large amounts of cysteine proteases, especially procathepsin L, which powerfully degrade type-1 collagen leading to tumor-associated bone absorption and release of bone calcium. The bone pit formations in vitro, which are caused by osteoclasts derived from human bone marrow cells activated by RANKL and M-CSF and also by mice osteoclasts activated by TNF-alpha, are significantly prevented by CLIK-148 treatment. We evaluated the in vivo inhibitory effect of malignant hypercalcemia induced by LJC-1 human mandibular cancer inoculation by CLIK-148 treatment, and the CLIK-148 treatment significantly protected against the tumor-induced hypercalcemia. On the protection of bone metastasis of colon 26 PMF-15 implanted to mouse calvaria, CLIK-148 treatment significantly inhibited calvaria bone absorption (direct metastasis). The CLIK-148 treatment also reduced distant bone metastasis to the femur and tibia of melanoma A375 tumors implanted into the left ventricle of the heart.     

RNA interference of Trypanosoma brucei cathepsin B and L affects disease progression in a mouse model

We investigated the roles played by the cysteine proteases cathepsin B and cathepsin L (brucipain) in the pathogenesis of Trypansoma brucei brucei in both an in vivo mouse model and an in vitro model of the blood-brain barrier. Doxycycline induction of RNAi targeting cathepsin B led to parasite clearance from the bloodstream and prevent a lethal infection in the mice. In contrast, all mice infected with T. brucei containing the uninduced Trypanosoma brucei cathepsin B (TbCatB) RNA construct died by day 13. Induction of RNAi against brucipain did not cure mice from infection; however, 50% of these mice survived 60 days longer than uninduced controls. The ability of T. b. brucei to cross an in vitro model of the human blood-brain barrier was also reduced by brucipain RNAi induction. Taken together, the data suggest that while TbCatB is the more likely target for the development of new chemotherapy, a possible role for brucipain is in facilitating parasite entry into the brain.     

Trypanosoma brucei RNA editing protein TbMP42 (band VI) is crucial for the endonucleolytic cleavages but not the subsequent steps of U-deletion and U-insertion

Trypanosome mitochondrial mRNAs achieve their coding sequences through RNA editing. This process, catalyzed by approximately 20S protein complexes, involves large numbers of uridylate (U) insertions and deletions within mRNA precursors. Here we analyze the role of the essential TbMP42 protein (band VI/KREPA2) by individually examining each step of the U-deletional and U-insertional editing cycles, using reactions in the approximately linear range. We examined control extracts and RNA interference (RNAi) extracts prepared soon after TbMP42 was depleted (when primary effects should be most evident) and three days later (when precedent shows secondary effects can become prominent). This analysis shows TbMP42 is critical for cleavage of editing substrates by both the U-deletional and U-insertional endonucleases. However, on simple substrates that assess cleavage independent of editing features, TbMP42 is similarly required only for the U-deletional endonuclease, indicating TbMP42 affects the two editing endonucleases differently. Supplementing RNAi extract with recombinant TbMP42 partly restores these cleavage activities. Notably, we find that all the other editing steps (the 3'-U-exonuclease [3'-U-exo] and ligation steps of U-deletion and the terminal-U-transferase [TUTase] and ligation steps of U-insertion) remain at control levels upon RNAi induction, and hence are not dependent on TbMP42. This contrasts with an earlier report that TbMP42 is a 3'-U-exo that may act in U-deletion and additionally is critical for the TUTase and/or ligation steps of U-insertion, observations our data suggest reflect indirect effects of TbMP42 depletion. Thus, trypanosomes require TbMP42 for both endonucleolytic cleavage steps of RNA editing, but not for any of the subsequent steps of the editing cycles.     

Rab11 mediates selective recycling and endocytic trafficking in Trypanosoma brucei

Trypanosoma brucei possesses a streamlined secretory system that guarantees efficient delivery to the cell surface of the critical glycosyl‐phosphatidylinositol (GPI)‐anchored virulence factors, variant surface glycoprotein (VSG) and transferrin receptor (TfR). Both are thought to be constitutively endocytosed and returned to the flagellar pocket via TbRab11+ recycling endosomes. We use conditional knockdown with established reporters to investigate the role of TbRab11 in specific endomembrane trafficking pathways in bloodstream trypanosomes. TbRab11 is essential. Ablation has a modest negative effect on general endocytosis, but does not affect turnover, steady state levels or surface localization of TfR. Nor are biosynthetic delivery to the cell surface and recycling of VSG affected. TbRab11 depletion also causes increased shedding of VSG into the media by formation of nanotubes and extracellular vesicles. In contrast to GPI‐anchored cargo, TbRab11 depletion reduces recycling of the transmembrane invariant surface protein, ISG65, leading to increased lysosomal turnover. Thus, TbRab11 plays a critical role in recycling of transmembrane, but not GPI‐anchored surface proteins. We proposed a two‐step model for VSG turnover involving release of VSG‐containing vesicles followed by GPI hydrolysis. Collectively, our results indicate a critical role of TbRab11 in the homeostatic maintenance of the secretory/endocytic system of bloodstream T. brucei.      

A new nuclear protease with cathepsin L properties is present in HeLa and Caco‐2 cells

Recently many authors have reported that cathepsin L can be found in the nucleus of mammalian cells with important functions in cell‐cycle progression. In previous research, we have demonstrated that a cysteine protease (SpH‐protease) participates in male chromatin remodeling and in cell‐cycle progression in sea urchins embryos. The gene that encodes this protease was cloned. It presents a high identity sequence with cathepsin L family. The active form associated to chromatin has a molecular weight of 60 kDa, which is higher than the active form of cathepsin L described until now, which range between 25 and 35 kDa. Another difference is that the zymogen present in sea urchin has a molecular weight of 75 and 90 kDa whereas for human procathepsin L has a molecular weight of 38–42 kDa. Based on these results and using a polyclonal antibody available in our laboratory that recognizes the active form of the 60 kDa nuclear cysteine protease of sea urchin, ortholog to human cathepsin L, we investigated the presence of this enzyme in HeLa and Caco‐2 cells. We have identified a new nuclear protease, type cathepsin L, with a molecular size of 60 kDa, whose cathepsin activity increases after a partial purification by FPLC and degrade in vitro histone H1. This protease associates to the mitotic spindle during mitosis, remains in the nuclei in binuclear cells and also translocates to the cytoplasm in non‐proliferative cells. J. Cell. Biochem. 111: 1099–1106, 2010. © 2010 Wiley‐Liss, Inc.     

Clathrin-dependent targeting of receptors to the flagellar pocket of procyclic-form Trypanosoma brucei

In trypanosomatids, endocytosis and exocytosis occur exclusively at the flagellar pocket, which represents about 0.43% of the pellicle membrane and is a deep invagination of the plasma membrane where the flagellum extends from the cell. Receptor molecules are selectively retained at the flagellar pocket. We studied the function of clathrin heavy chain (TbCLH) in the trafficking of the flagellar pocket receptors in Trypanosoma brucei by using the double-stranded RNA interference approach. It appears that TbCLH is essential for the survival of both the procyclic form and the bloodstream form of T. brucei, even though structures resembling large coated endocytic vesicles are absent in procyclic-form trypanosomes. Down-regulation of TbCLH by RNA interference (RNAi) for 24 h rapidly and drastically reduced the uptake of macromolecules via receptor-mediated endocytosis in procyclic-form trypanosomes. This result suggested the importance of TbCLH in receptor-mediated endocytosis of the procyclic-form trypanosome, in which the formation of large coated endocytic vesicles may not be required. Surprisingly, induction of TbCLH RNAi in the procyclic T. brucei for a period of 48 h prohibited the export of the flagellar pocket-associated transmembrane receptor CRAM from the endoplasmic reticulum to the flagellar pocket, while trafficking of the glycosylphosphatidylinositol-anchored procyclin coat was not significantly affected. After 72 h of induction of TbCLH RNAi, procyclics exhibited morphological changes to an apolar round shape without a distinct structure of the flagellar pocket and flagellum. Although trypanosomes, like other eukaryotes, use similar organelles and machinery for protein sorting and transport, our studies reveal a novel role for clathrin in the secretory pathway of trypanosomes. We speculate that the clathrin-dependent trafficking of proteins to the flagellar pocket may be essential for the biogenesis and maintenance of the flagellar pocket in trypanosomes.     

Over‐expression of an inactive mutant cathepsin D increases endogenous alpha‐synuclein and cathepsin B activity in SH‐SY5Y cells

Parkinson's disease is a neurodegenerative movement disorder. The histopathology of Parkinson's disease comprises proteinaceous inclusions known as Lewy bodies, which contains aggregated α‐synuclein. Cathepsin D (CD) is a lysosomal protease previously demonstrated to cleave α‐synuclein and decrease its toxicity in both cell lines and mouse brains in vivo. Here, we show that pharmacological inhibition of CD, or introduction of catalytically inactive mutant CD, resulted in decreased CD activity and increased cathepsin B activity, suggesting a possible compensatory response to inhibition of CD activity. However, this increased cathepsin B activity was not sufficient to maintain α‐synuclein degradation, as evidenced by the accumulation of endogenous α‐synuclein. Interestingly, the levels of LC3, LAMP1, and LAMP2, proteins involved in autophagy‐lysosomal activities, as well as total lysosomal mass as assessed by LysoTracker flow cytometry, were unchanged. Neither autophagic flux nor proteasomal activities differs between cells over‐expressing wild‐type versus mutant CD. These observations point to a critical regulatory role for that endogenous CD activity in dopaminergic cells in α‐synuclein homeostasis which cannot be compensated for by increased Cathepsin B. These data support the potential need to enhance CD function in order to attenuate α‐synuclein accumulation as a therapeutic strategy against development of synucleinopathy.

     

An alternate targeting pathway for procathepsin L in mouse fibroblasts

In transformed mouse fibroblasts, a significant proportion of the lysosomal cysteine protease cathepsin L remains in cells as an inactive precursor which associates with membranes by a mannose phosphate-independent interaction. When microsomes prepared from these cells were resolved on sucrose gradients, this procathepsin L was localized in dense vesicles distinct from those enriched for growth hormone, which is secreted constitutively when expressed in fibroblasts. Ultrastructural studies using antibodies directed against the propeptide to avoid detection of the mature enzyme in lysosomes revealed that the proenzyme was concentrated in dense cores within small vesicles and multivesicular endosomes which labeled with antibodies specific for CD63. Consistent with the resemblance of these cores to those of regulated secretory granules, secretion of procathepsin L from fibroblasts was modestly stimulated by phorbol, 12-myristate, 13-acetate. When protein synthesis was blocked with cycloheximide and lysosomal proteolysis inhibited with leupeptin, procathepsin L was found to gradually convert to the active single-chain protease. The data suggest that when synthesis levels are high, a portion of the procathepsin L is packaged in dense cores within multivesicular endosomes localized near the plasma membrane. Gradual activation of this proenzyme achieves targeting of the proenzyme to lysosomes by a mannose phosphate receptor-independent pathway.