Biogenesis of nonspecific lipid transfer protein and sterol carrier protein x: studies using peroxisome assembly-defective pex cell mutants

Nonspecific lipid transfer protein (nsLTP; also called sterol carrier protein 2) with a molecular mass of 13 kDa is synthesized as a larger 15-kDa precursor (pre-nsLTP) with an N-terminal 20-amino acid extension presequence, as well as with the peroxisome targeting signal type 1 (PTS1), Ala-Lys-Leu, at the C terminus. The precursor pre-nsLTP is processed to mature nsLTP by proteolytic removal of the presequence, most likely after being imported into peroxisomes. Sterol carrier protein x (SCPx), a 59-kDa branched-chain fatty acid thiolase of peroxisomes, contains the entire pre-nsLTP moiety at the C-terminal part and is converted to the 46-kDa form and nsLTP after the transport to peroxisomes. We investigated which of these two potential topogenic sequences functions in biogenesis of nsLTP and SCPx. Morphological and biochemical analyses, making use of Chinese hamster ovary cell pex mutants such as the PTS1 receptor-impaired pex5 and PTS2 import-defective pex7, as well as green fluorescent protein chimeras, revealed that both pre-nsLTP and SCPx are imported into peroxisomes by the Pex5p-mediated PTS1 pathway. Nearly half of the pre-nsLTP remains in the cytosol, as assessed by subcellular fractionation of the wild-type Chinese hamster ovary cells. In an in vitro binding assay, only mature nsLTP, but not pre-nsLTP, from the cell lysates interacted with the Pex5p. It is likely, therefore, that modulation of the C-terminal PTS1 by the presequence gives rise to cytoplasmic localization of pre-nsLTP.     

Biosynthesis of prostatic acid phosphatase in a normal human cell-line

The biosynthesis of the prostatic form of human acid phosphatase was studied in normal embryonic lung cells, WI-38, by metabolic labeling with tritiated leucine and [32P]phosphate, followed by specific immunoprecipitation, gel electrophoresis, and fluorography. Of the total tartrate-inhibitable acid phosphatase activity in WI-38 cells, 30% is due to the prostatic form. The primary translation product that leads eventually to the mature prostatic enzyme is a precursor polypeptide of 112 kDa. The precursor polypeptide is processed to mature polypeptides of 59, 55, and 49 kDa via an intermediate 91-kDa precursor. WI-38 cells also secrete a 113-kDa peptide into the medium. The precursor and mature polypeptides are glycosylated and phosphorylated. Upon treatment with endo-beta-hexosaminidase H, the apparent molecular weighs of the polypeptides are reduced by approximately 4 kDa and phosphate is lost.     

The biogenesis of the cyanellae of Cyanophora paradoxa. III. In vitro synthesis of cyanellar polypeptides using separated cytoplasmic and cyanellar RNA

RNA from Cyanophora paradoxa was separated into cytoplasmic and cyanellar fractions by using a combination of subcellular fractionation and oligo-dT chromatography. In vitro translation of the separated cytoplasmic and cyanellar RNAs in a rabbit reticulocyte lysate system in the presence of [35S]methionine resulted in the incorporation of radiolabel into electrophoretically distinct sets of polypeptides. Monospecific and polyspecific antibodies that react with cyanellar polypeptides were used to probe the in vitro translation products by indirect immunoprecipitation by using Staphylococcus protein A conjugated to Sepharose beads. The results indicate that linker polypeptide L1 of the phycobilisome, the gamma subunit of coupling factor CF1, and subunit II of PS I are synthesized in the cytoplasm as precursor molecules that are 5-8 kDa larger than their mature sizes. Antibodies directed against the psbA gene product (the D1 protein) precipitated a polypeptide found in the translation products of the cyanellar RNA-directed reactions, which is about 1.5 kDa larger than the mature protein.     

Optimization of protein synthesis in isolated higher plant chloroplasts. Identification of paused translation intermediates

Protein synthesis in isolated, intact pea chloroplasts was optimized and compared to translation within chloroplasts in vivo. Many polypeptides labeled with [35S]methionine in isolated intact chloroplasts did not comigrate with polypeptides which were labeled within chloroplasts in vivo. Antibodies to the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) immunoprecipitated [35S]-labeled large subunit plus several lower-molecular-mass translation products of isolated chloroplasts. The lower-molecular-mass soluble translation products synthesized in pulse-labeled chloroplasts were converted into full-length large-subunit polypeptides during a subsequent chase period. This result suggests that many of the polypeptides observed in pulse-labeled chloroplasts are incomplete translation products which are the result of ribosome pausing at discrete points along chloroplast mRNAs. The pulse-chase technique was used to follow synthesis of the 34.5-kDa precursor of the psb A gene product and its processing to the mature 32-kDa polypeptide in isolated chloroplasts. Chloroplast translation profiles obtained using the pulse-chase assay were very similar to translation profiles obtained in vivo thus extending the utility of protein synthesis in isolated chloroplasts.     

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats.

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.     

Characterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi

The cell-free translation products of polyribosomal and post-polyribosomal mRNAs from the non-infective epimastigotes and the infective metacyclic trypomastigotes of the parasitic protozoan Trypanosoma cruzi were compared by two-dimensional polyacrylamide gel electrophoresis. The result show that although many polypeptides are conserved, quantitative and qualitative differences are observed between both differentiation stages. The results also indicate the existence of post-polyribosomal mRNAs in equilibrium with polyribosomal counterparts. The immunoprecipitation of the in vitro synthesized polypeptides with chagasic human serum and the serum raised against an 85-kDa glycoprotein (P2-WGA), potentially involved in the process of T. cruzi penetration into mammalian cells, shows that while the chagasic serum recognizes the same 72-kDa, 68-kDa and 46-kDa polypeptides in both differentiation stages, the anti-P2-WGA serum immunoprecipitates a single 48-kDa polypeptide from in vitro translation products of metacyclic trypomastigotes.     

Characterization of translation products of the polyadenylated RNA of free and membrane-bound polyribosomes of rat forebrain.

Poly(A)+ RNA (polyadenylated RNA) isolated from membrane-bound and free polyribosomes was translated in reticulocyte lysates, and the products were analysed by two-dimensional gel electrophoresis. Several translation products were specific to membrane-bound polyribosomal mRNA, including polypeptides of 47kDa, 35kDa and 21 kDa, whereas others (e.g. of 37 kDa, 17 kDa and 14 kDa) were specific to free polyribosomal mRNA. Although many products were common to both mRNA species, cross-contamination could be ruled out on the basis of the presence of these and other specific products. The common products included a 68 kDa microtubule-associated protein, tubulin, actin, the brain form of creatine kinase, neuron-specific enolase and protein 14-3-3 and calmodulin, all of which were identified on the basis of two-dimensional gel and peptide analyses. The 35 kDa protein product of membrane-specific mRNA was co-translationally processed in vitro by microsomal membranes, resulting in its cleavage to 33 kDa (and partial glycosylation). The 33 kDa processed protein (but not the 35 kDa precursor) was integrated into both dog pancreas and rat brain microsomal membranes. The occurrence of the enzymes and calmodulin as products of membrane-bound polyribosomal mRNA is discussed in the light of their presence on rat brain synaptic plasma membranes [Lim, Hall, Leung, Mahadevan & Whatley (1983) J. Neurochem. 41, 1177-1182] and their existence in a specific component of axonal flow. It is suggested that some of these translation products of the rough endoplasmic reticulum may represent proteins destined for the plasma membrane. However, the identity and location of the 35 kDa membrane-specific product (or its processed form) still remain unestablished.     

Cell-free synthesis of tumor-type poly(A) polymerase

Previous studies in this laboratory suggested that in adult liver, either the gene for the tumor-type poly(A) polymerase is poorly transcribed or the mRNA for this enzyme is largely not expressed. To test these possibilities, total RNA from rat liver and Morris hepatoma 3924A RNA were isolated by using a guanidine thiocyanate method; poly(A+) RNA and poly(A-) RNA were separated by oligo(dT)-cellulose chromatography and used for translation in a rabbit reticulocyte lysate system. After in vitro translation, the products were immunoprecipitated with either purified anti-tumor poly(A) polymerase antibodies or control immunoglobulins. When the polypeptides translated from poly(A+) or poly(A-) hepatoma RNA were precipitated with immune sera, a unique [35S]methionine-labeled 35-kilodalton (kDa) protein was observed. This band was not apparent when control serum was used for the immunoprecipitation. The radiolabeled 35-kDa polypeptide was not evident when the products were incubated with highly purified tumor nuclear poly(A) polymerase prior to immunoprecipitation. Prior incubation of the translation products with bovine serum albumin instead of poly(A) polymerase had no effect on the immunoprecipitation. This 35-kDa protein was not apparent when liver poly(A+) RNA was used to direct translation. These data demonstrate that (a) the tumor enzyme is not synthesized as a precursor, (b) tumor mRNA, but not normal liver mRNA, contains detectable sequences coding for tumor-type poly(A) polymerase, and (c) poly(A) polymerase mRNA also exists as a poly(A-) population.     

Biosynthesis and intracellular processing of carbonic anhydrase in Chlamydomonas reinhardtii

Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the CO2 concentration during photoautotrophic growth was decreased to air level. After decreasing the CO2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall-less mutant CW-15, which secretes CA into the culture medium, were pulse-labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti-CA serum. A 42-kDa polypeptide with isoelectric point (pI) of 7.1-7.3 was first synthesized. Within 5 min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)-rich RNA in a cell-free reticulocyte lysate system from a rabbit was a 38-kDa polypeptide. This was cotranslationally converted into the 42-kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42-kDa precursor had a high affinity to concanavalin A, it was assumed to have a high-mannose-type oligosaccharide. The mature enzyme had a pI of 6.1-6.2 and was composed of more than two isoforms, which had a complex-type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis.     

Primary translation products, biosynthesis, and tissue specificity of the major surfactant protein in rat

Rat lung tissue was labeled with [35S]methionine and the major surfactant-associated proteins immunoprecipitated using a specific antiserum. The protein pattern obtained was very similar to that seen in rat bronchoalveolar lavage. Rat lung mRNA was subsequently translated in an in vitro rabbit reticulocyte system, and surfactant-associated protein-related polypeptides were immunoprecipitated. A 26-kDa polypeptide was identified and characterized as follows. (a) Unlabeled surfactant proteins added to the immunoprecipitation mixture completely inhibited its immunoprecipitation. (b) Two-dimensional gel electrophoresis of the 26-kDa protein resolved it into 3 isoforms. (c) Inclusion of dog pancreatic microsomes in the translation mixture resulted in the formation of two distinct higher molecular weight groups of isoforms, suggesting that the 26-kDa protein is destined to become a glycoprotein. Immunoprecipitation of [35S]methionine-labeled rat lung tissue proteins after tunicamycin treatment resulted in 3 isoforms, identical to the ones seen in the primary translation products. In addition, expression of the surfactant proteins appears specific to the lung.     

Mosquito vitellogenin subunits originate from a common precursor

Using a cell-free translation system, we demonstrated that the two subunits of mosquito vitellogenin (VG), 200 kDa and 65 kDa, originate from a common precursor. The precursor polypeptide of 220 kDa is a translation product specific to mRNA from vitellogenic mosquitoes. In immunoprecipitation analysis, the 220-kDa polypeptide was recognized by monoclonal antibodies directed either to the large or the small VG subunit. Peptide mapping showed homology between the 220-kDa polypeptide and both subunits, thus providing further proof that the 220-kDa product of translation is the precursor for both VG subunits. In the presence of microsomal membranes, the molecular size of the VG precursor increased to 235 kDa suggesting this as a first step in co-translational modifications of VG.     

Properties, biosynthesis and processing of a sulfur-rich protein in Brazil nut (Bertholletia excelsa H.B.K.)

An abundant seed protein, which is exceptionally rich in the sulfur-containing amino acids, methionine (18%) and cysteine (8%), is synthesized in Brazil nut embryos about 9 months after flowering. This sulfur-rich protein consists of two low-molecular-mass polypeptide components, a 9-kDa polypeptide and a 3-kDa polypeptide. The two-subunit polypeptides associate through disulfide linkage(s) to form a 12-kDa protein molecule. We have demonstrated through in vitro translation studies, using RNA from 9-month-old embryos, that the sulfur-rich protein is synthesized as a larger precursor polypeptide of 18 kDa. In addition, data from in vivo labelling studies of 9-month-old Brazil nuts suggest that there are two intermediate precursors of the sulfur-rich protein, one of 15 kDa and another of 12 kDa. One of these precursors, the 12-kDa polypeptide, accumulates for a 2-month period in the developing embryos. From these data we infer that at least three stepwise cleavages are involved in the maturation of the sulfur-rich protein from its 18-kDa precursor.     

Cloning and sequencing of a cDNA encoding the major storage proteins of Theobroma cacao

The major storage proteins, polypeptides of 31 and 47 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), have been identified and partially purified by preparative gel electrophoresis. The polypeptides were both N-terminally blocked, but some N-terminal amino-acid sequence was obtained from a cyanogen bromide peptide common to both polypeptides, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy-DNA (cDNA) clone from a library made from poly(A)⁺ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 566-amino-acid polypeptide of M_r 65 612. The existence of a common precursor to the 31- and 47-kDa polypeptides of this size was confirmed by immunoprecipitation from total poly(A)⁺RNA translation products. The precursor has an N-terminal hydrophobic sequence which appears to be a typical signal sequence, with a predicted site of cleavage 20 amino acids after the start. This is followed by a very hydrophilic domain of 110 amino acids, which, by analogy with the cottonseed -globulin, is presumed to be cleaved off to leave a domain of approx. 47 kDa, very close to the observed size of the mature polypeptide. Like the hydrophilic domain of the cottonseed -globulin the cocoa hydrophilic domain is very rich in glutamine and charged residues (especially glutamate), and contains several Cys-X-X-X-Cys motifs. The cyanogen-bromide peptide common to the 47-kDa and 31-kDa polypeptides is very close to the proposed start of the mature domain, indicating that the 31-kDa polypeptide arises via further C-terminal processing. The polypeptide sequence is homologous to sequences of the vicilin class of storage proteins, previously found only in legumes and cotton. Most of these proteins have a mature polypeptide size of approx. 47 kDa, and are synthesised as precursors only slightly larger than this. Some, however, are larger polypeptides (e.g. -conglycinin from soybean is 72 kDa), usually due to an additional N-terminal domain. In cottonseed the situation appears to parallel that in cocoa in that the vicilin is synthesised as an approx. 70-kDa precursor and then processed to a 47-kDa (and in the case of cocoa also a 31-kDa) mature protein. In this context it is interesting that cotton is closer in evolutionary terms to cocoa than are the legumes, both cotton and cocoa being in the order Malvales.Abbreviations A absorbance - cDNA copy DNA - IgG immunoglobulin G - kb kilobase pairs - kDa kilodaltons - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacylamide gel electrophoresis The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies.     

Biosynthesis and processing of lysosomal acid phosphatase in cultured human cells

The biosynthesis of lysosomal acid phosphatase was studied in a normal human embryonic lung cell line, WI-38. Cells were labeled with radioactive leucine under a variety of conditions, the enzyme was immunoprecipitated using a monospecific antiserum raised against human liver lysosomal acid phosphatase, and the products were separated by electrophoresis and were visualized by fluorography. Lysosomal acid phosphatase constitutes 60% of the total tartrate-inhibitable acid phosphatase in WI-38. It is initially synthesized as a high-molecular-weight precursor polypeptide of 69 kDa. The precursor polypeptide is rapidly glycosylated and processed to a mature enzyme of 53-45 kDa via intermediates of 65 and 60 kDa in WI-38 cells. The 69-kDa precursor polypeptide is also converted to larger precursor polypeptides of 74 and 80 kDa. The multiplicity of precursor polypeptides is due at least in part to differences in the glycosylation and phosphorylation of the polypeptides. Sensitivity of phosphorylated oligosaccharide chains from precursor, mature and small polypeptides to endo-beta-hexosaminidase H-catalyzed cleavage suggests the presence of high-mannose phosphorylated oligosaccharide chains similar to those present on many other lysosomal enzymes. The effects of tunicamycin and ammonium chloride were also studied. In contrast to the effect of ammonium chloride on arylsulfatase A secretion, the lysosomal acid phosphatase in WI-38 cells was not secreted in the presence of NH4Cl. This is consistent with the existence of an alternate route for the transfer of lysosomal acid phosphatase into lysosomes. This alternate route may be the reason that I-cell fibroblasts contain a normal level of lysosomal acid phosphatase.     

Transport and metabolism of 5'-nucleotidase in a rat hepatoma cell line

The biosynthesis of the ectoenzyme 5'-nucleotidase in the rat hepatoma cell line H4S has been studied by pulse-labeling with [35S]methionine and subsequent immunoprecipitation of the cell lysate. 5'-Nucleotidase is a membrane glycoprotein with an apparent molecular mass on SDS-gels of 72 kDa. The enzyme is initially synthesized as a 68-kDa precursor which is converted to the mature 72-kDa form in 15-60 min (t1/2 = 25 min). The molecular mass of the unglycosylated enzyme is approximately 58 kDa. Culturing the cells in the presence of varying concentrations of tunicamycin, an inhibitor of N-glycosylation, revealed six species of 5'-nucleotidase after sodium dodecyl sulfate/polyacrylamide electrophoresis. This indicates the presence of five N-linked oligosaccharide chains accounting for the difference between the 58-kDa polypeptide backbone and the 68-kDa species. The 68-kDa precursor is susceptible to cleavage by endo-beta-N-acetylglycosaminidase H; the 72-kDa mature protein is converted to several bands upon this treatment. This result indicates that part of 5'-nucleotidase keeps one or two high-mannose or hybrid chains in the mature form, even after prolonged pulse-chase labeling. The newly synthesized mature enzyme reaches the cell surface after 20-30 min. The half-life of 5'-nucleotidase is about 30 h in H4S cells. No immunoprecipitable 5'-nucleosidase is released into the culture medium.     

Cholera toxin is synthesized in precursor form on free polysomes in Vibrio cholerae 569B.

Membrane-bound and free polysomes have been isolated from Vibrio cholerae 569B. Nacent polypeptide chains were completed in a cell-free translation mixture containing Escherichia coli S-300 extracts and [3H]leucine or [35S]methionine. Cholera toxin-related polypeptides synthesized in vitro were immunologically detected after treatment with either anti-subunit A or anti-subunit B serum. Immunoreactive translation products were removed from reaction mixtures with formalinized Cowan's strain of Staphylococcus aureus, electrophoresed on sodium dodecyl sulfate-polyacrylamide gels, and visualized by fluorography. Anti-subunit A serum precipitated two major polypeptide species (molecular weights 52,000 and 45,000) from translation mixtures programed with free polysomes, whereas anti-subunit B serum precipitated only the 45,000-molecular-weight polypeptide. No cholera toxin-related polypeptides were detectable in translation mixtures programed with membrane-bound polysomes. Purified subunit A and cholera toxin competed for anti-subunit A binding sites and blocked the immunoprecipitation of the 35S-labeled 52,000- and 45,000-dalton polypeptides from in vitro translation mixtures. The data presented suggest that cholera toxin is synthesized in the cytoplasm in a precursor form on free polysomes and is secreted post-translationally.     

Nonspecific lipid transfer protein (sterol carrier protein-2) defective in patients with deficient peroxisomes

The biosynthesis and intracellular localization of nonspecific lipid transfer protein (nsLTP) in control human subjects and in patients with peroxisome-deficient disorders were investigated. The molecular mass of human nsLTP was indistinguishable from that of rat nsLTP (13 kDa) by immunoblot analysis. Intracellular localization was identical with that of catalase, a marker enzyme of peroxisomal matrix, by a double immunofluorescence study. The nsLTP was deficient in liver tissues or fibroblasts from patients with peroxisome-deficient disorders such as Zellweger syndrome and neonatal adrenoleukodystrophy (ALD). Pulse-chase experiments showed that nsLTP was synthesized as a large precursor in both the control and Zellweger fibroblasts. However, the processing to the 13 kDa mature protein was disturbed and the degradation was rapid in Zellweger fibroblasts. After somatic cell fusion using Zellweger fibroblasts from different genetic groups, the processing was normalized. These results suggest that the biosynthesis and localization of human nsLTP are similar to those of rat nsLTP and that the defect of nsLTP in peroxisome-deficient disorders is a phenomenon secondary to an abnormal transport mechanism of peroxisomal proteins. The defect of nsLTP may play an important role in metabolic disturbances in bile acid synthesis and steroidogenesis in peroxisome-deficient disorders.     

Subcellular localisation and processing of non-specific lipid transfer protein are not aberrant in Rhizomelic Chondrodysplasia Punctata fibroblasts.

The import into peroxisomes and maturation of peroxisomal 3-oxoacyl-CoA thiolase are impaired in patients with the Rhizomelic form of Chondrodysplasia Punctata (RCDP). Here we show by means of immunoblotting and subcellular fractionation that non-specific lipid transfer protein (nsLTP), another peroxisomal protein synthesised as a larger precursor, is localised in peroxisomes and is present as the mature protein in RCDP fibroblasts. Thus the component of the import machinery defective in RCDP is not required for the import of nsLTP into peroxisomes.     

Biosynthesis and processing of lysosomal cathepsin L in primary cultures of rat hepatocytes

The biosynthesis and proteolytic processing of lysosomal cathepsin L was studied using in vitro translation system and in vivo pulse-chase analysis with [35S]methionine and [32P]phosphate in primary cultures of rat hepatocytes. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of a primary translation product of an immunoprecipitable 37.5-kDa cathepsin L in vitro. The 37.5-kDa form was converted to the 39-kDa form when translated in the presence of dog pancreas microsomes. During pulse-chase experiments with [35S]methionine in cultured rat hepatocytes, cathepsin L was first synthesized as a 39-kDa protein, presumably the proform, after a short time of labeling, and was subsequently processed into the mature forms of 30 and 25 kDa in the cell. On the other hand, considerable amounts of the proenzyme were found to be secreted into the culture medium without further proteolytic processing during the chase. The precursor and mature enzymes were N-glycosylated with high-mannose-type oligosaccharides, and the proenzyme molecule contained phosphorylated oligosaccharides. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, a 36-kDa unglycosylated polypeptide appeared in the cell and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results suggest that cathepsin L is initially synthesized on membrane-bound polysomes as a 37.5-kDa prepropeptide and that the cotranslational cleavage of the 1.5-kDa signal peptide and the core glycosylation convert the precursor to the 39-kDa proform, which is subsequently processed to the mature form during biosynthesis. Thus, the biosynthesis and secretion of lysosomal cathepsin L in rat hepatocytes seem to be analogous to those of the major excreted protein of transformed mouse fibroblasts [S. Gal, M. C. Willingham, and M. M. Gottesman (1985) J. Cell Biol. 100, 535-544] and the mouse cysteine proteinase of activated macrophages [D.A. Portnoy, A. H. Erickson, J. Kochan, J. V. Ravetch, and J. C. Unkeless (1986) J. Biol. Chem. 261, 14697-14703].