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PubMed1992

Expression of mouse cathepsin L cDNA in Saccharomyces cerevisiae: evidence that cathepsin L is sorted for targeting to yeast vacuole.

To investigate the intracellular transport mechanism of lysosomal cathepsin L in yeast cells, we attempted to produce mouse cathepsin L in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin L revealed that the yeast cells carrying the cathepsin L coding sequence produced 39- and 30-kDa products, which correspond to the rat procathepsin L and the single-chain form of mature cathepsin L, respectively. The precursor polypeptide showed sensitivity toward endoglycosidase H treatment. Cell fractionation experiments demonstrated that the processed form of 30-kDa cathepsin L was found to be colocalized to the yeast vacuole with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. In the prepared vacuolar fraction, a considerable amount of cathepsin L was revealed to be cofractionated with the vacuolar membranes. Furthermore, the phase separation experiments with Triton X-114 provide the first evidence showing that the mature form of cathepsin L polypeptide is strongly associated with the vacuolar membranes. Therefore, the present results suggest that the mouse cathepsin L precursor polypeptide is initially synthesized as the proenzyme in the yeast cells and then correctly delivered to the vacuole. During the intracellular sorting pathway, the procathepsin L would undergo the post-translational proteolytic processing step to generate the mature enzyme. Based on these lines of evidence, we propose that cathepsin L is recognized by mechanisms similar to those for the intracellular sorting and processing of vacuolar proteins in the yeast cells.

Animals↗

PubMed2002

Vacuolar type H+ pumping pyrophosphatases of parasitic protozoa.

Trans-membrane proton pumping is responsible for a myriad of physiological processes including the generation of proton motive force that drives bioenergetics. Among the various proton pumping enzymes, vacuolar pyrophosphatases (V-PPases) form a distinct class of proton pumps, which are characterised by their ability to translocate protons across a membrane by using the potential energy released by hydrolysis of the phosphoanhydride bond of inorganic pyrophosphate. Until recently, V-PPases were known to be the purview of only plant vacuoles and plasma membranes of phototrophic bacteria. Recent discoveries of V-PPases in kinetoplastid and apicomplexan parasites, however, have expanded our view of the evolutionary reach of these enzymes. The lack of V-PPases in the vertebrate hosts of these parasites makes them potentially excellent targets for developing broad-spectrum antiparasitic agents. This review surveys the current understanding of V-PPases in parasitic protozoa with an emphasis on malaria parasites. Topological predictions suggest remarkable similarity of the parasite enzymes to their plant homologues with 15-16 membrane spanning domains and conserved sequences shown to constitute critical catalytic residues. Remarkably, malaria parasites have been shown to possess two V-PPase genes, one is an apparent orthologue of the canonical plant enzyme, whereas the other is a more distantly related paralogue with homology to a recently identified new class of K+-insensitive plant V-PPases. V-PPases appear to localise both to the plasma membrane and cytoplasmic organelles believed to be acidocalcisomes or polyphosphate bodies. Gene transfer experiments suggest that one of the malarial V-PPases is predominantly localised to the surface of intraerythrocytic parasites. We suggest a model in which V-PPase localised to the malaria parasite plasma membrane may serve as an electrogenic pump utilising pyrophosphate as an energy source, thus sparing the more precious ATP. Searching for V-PPase inhibitors could prove fruitful as a novel means of antiparasitic chemotherapy.

Amino Acid Sequence↗

PubMed1999

Post-translational maturation of natural and drug-induced missorted phytohemagglutinin.

The bean lectin phytohemagglutinin (PHA) was expressed in transgenic suspension-cultured BY-2 tobacco cells simultaneously with another recombinant vacuolar protein, the sweet potato sporamin. In contrast to previous observations in different transgenic plant systems when expressed in BY-2 tobacco cells, phytohemagglutinin is mostly but not exclusively targeted to the vacuole. Indeed, a small amount of recombinant phytohemagglutinin is secreted into the culture medium of tobacco cells. Furthermore part of this extracellular phytohemagglutinin has no lectin activity and presents an abnormal glycosylation consistent with higher accessibility of glycans N-linked to these extracellular phytohemagglutinin forms. Phytohemagglutinin secretion occurs regardless of recombinant protein expression level. Consequently, missorting in this case is due to an abnormal phytohemagglutinin conformation or oligomerization rather than to receptor saturation. The treatment of BY-2 cells with drugs, such as monensin and wortmannin, increases even more the transport of phytohemagglutinin to the cell surface through a general inhibition of the sorting mechanisms of vacuolar proteins. The sensitivity to wortmannin is similar for the sorting of phytohemagglutinin and endogenous tobacco chitinase and beta-1,3-glucanase, suggesting that phytohemagglutinin and COOH-terminal propeptide mediated vacuolar sorting share similar mechanisms. A characterization of glycans N-linked to extracellular phytohemagglutinin secreted by monensin- or wortmannin-treated transgenic tobacco cells illustrates that in contrast with monensin, wortmannin completely inhibits the sorting of vacuolar proteins without having any effect on the efficiency of Golgi processing enzymes.

Journal Article↗

PubMed2004

A new kind of prion: a modified protein necessary for its own modification.

We recently described an infectious protein (prion) unrelated to amyloid formation, that is an enzyme whose precursor can only be activated by the active form of the enzyme. All previously described infectious proteins are self-propagating amyloid forms of chromosomally encoded proteins. The infectious enzyme, vacuolar protease B (PrB), can activate its own precursor in an indefinitely self-propagating process. Transfer from cell to cell of cytoplasm containing active protease B transmits this non-chromosomal gene. The importance of this system is that many protein-modifying enzymes may act on themselves, and if conditions are right, may become prions as well.

Amyloid↗

PubMed1994

Ultrastructural analysis of the autophagic process in yeast: detection of autophagosomes and their characterization.

Under nutrient-deficient conditions, the yeast S. cerevisiae sequesters its own cytoplasmic components into vacuoles in the form of "autophagic bodies" (Takeshige, K., M. Baba, S. Tsuboi, T. Noda, and Y. Ohsumi. 1992. J. Cell Biol. 119:301-311). Immunoelectron microscopy showed that two cytosolic marker enzymes, alcohol dehydrogenase and phosphoglycerate kinase, are present in the autophagic bodies at the same densities as in the cytosol, but are not present in vacuolar sap, suggesting that cytosolic enzymes are also taken up into the autophagic bodies. To understand this process, we performed morphological analyses by transmission and immunological electron microscopies using a freeze-substitution fixation method. Spherical structures completely enclosed in a double membrane were found near the vacuoles of protease-deficient mutant cells when the cells were shifted to nutrient-starvation media. Their size, membrane thickness, and contents of double membrane-structures corresponded well with those of autophagic bodies. Sometimes these double membrane structures were found to be in contact with the vacuolar membrane. Furthermore their outer membrane was occasionally seen to be continuous with the vacuolar membrane. Histochemical staining of carbohydrate strongly suggested that the structures with double membranes fused with the vacuoles. These results indicated that these structures are precursors of autophagic bodies, "autophagosomes" in yeast. All the data obtained suggested that the autophagic process in yeast is essentially similar to that of the lysosomal system in mammalian cells.

Alcohol Dehydrogenase↗

PubMed2004

Structure and function of plant aspartic proteinases.

Aspartic proteinases of the A1 family are widely distributed among plant species and have been purified from a variety of tissues. They are most active at acidic pH, are specifically inhibited by pepstatin A and contain two aspartic residues indispensible for catalytic activity. The three-dimensional structure of two plant aspartic proteinases has been determined, sharing significant structural similarity with other known structures of mammalian aspartic proteinases. With a few exceptions, the majority of plant aspartic proteinases identified so far are synthesized with a prepro-domain and subsequently converted to mature two-chain enzymes. A characteristic feature of the majority of plant aspartic proteinase precursors is the presence of an extra protein domain of about 100 amino acids known as the plant-specific insert, which is highly similar both in sequence and structure to saposin-like proteins. This insert is usually removed during processing and is absent from the mature form of the enzyme. Its functions are still unclear but a role in the vacuolar targeting of the precursors has been proposed. The biological role of plant aspartic proteinases is also not completely established. Nevertheless, their involvement in protein processing or degradation under different conditions and in different stages of plant development suggests some functional specialization. Based on the recent findings on the diversity of A1 family members in Arabidopsis thaliana, new questions concerning novel structure-function relationships among plant aspartic proteinases are now starting to be addressed.

Amino Acid Sequence↗

PubMed1993

Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast.

The fate of a mutant form of each of the two yeast vacuolar enzymes proteinase yscA (PrA) and carboxypeptidase yscY (CPY) has been investigated. Both mutant proteins are rapidly degraded after entering the secretory pathway. Mutant PrA is deleted in 37 amino acids spanning the processing site region of the PrA pro-peptide. The mutant enzyme shows no activity towards maturation of itself or other vacuolar hydrolases, a function of wild-type PrA. Mutant CPY carries an Arg instead of a Gly residue in a highly conserved region, two positions distant from the active-site Ser. In contrast to wild-type CPY, the mutant form was quickly degraded by trypsin in vitro, indicating an altered structure. Using antisera specific for alpha-1-->6 and alpha-1-->3 outer-chain mannose linkages, no Golgi-specific carbohydrate modification could be detected on either mutant protein. Subcellular fractionation studies located both mutant enzymes in the endoplasmic reticulum. Degradation kinetics of both proteins show the same characteristics, indicating similar degradation pathways. The degradation process was shown to be independent of a functional sec18 gene product and takes place before Golgi-specific carbohydrate modifications occur. The proteasome, the major proteolytic activity of the cytoplasm, is not involved in this degradation event. All degradation characteristics of the two mutant proteins are consistent with a degradation process within the endoplasmic reticulum ('ER degradation').

Adenosine Triphosphatases↗

PubMed1993

An aspartic proteinase present in seeds cleaves Arabidopsis 2 S albumin precursors in vitro.

The Arabidopsis thaliana 2 S albumins are examples of vacuolar proteins which undergo intensive posttranslational processing. An in vitro processing assay to screen for processing enzymes present in seeds was developed using an in vitro synthesized 2 S albumin precursor as the substrate. A protease was characterized which cleaved the substrate into two fragments with molecular weights (as determined from their migration distance on SDS-polyacrylamide gel) corresponding to those of the small and large subunits of Arabidopsis 2 S albumin. The pH optimum of this protease activity, its inhibition by pepstatin A, and partial sequence data led to the conclusion that the protease under study was an aspartic proteinase. Synthetic peptides representing two 2 S albumin propeptides allowed the determination of the in vitro cleavage sites, and suggested that the protease activity is capable, in vitro, of cleaving the amino-terminal propeptide as well as the internal propeptide linking the two subunits. Alterations of the amino acids in and around the cleavage sites, made to study the specificity of the protease activity, suggest that both structural and sequence determinants are important in cleavage site recognition.

Albumins↗

PubMed1994

Prohormone processing in permeabilized cells: endoproteolytic cleavage of prosomatostatin in the trans-Golgi network.

Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence has implicated either the trans-Golgi network (TGN) or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin, proSRIF, (Stoller TJ, Shields D (1988) J Cell Biol 107, 2087-2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident. Following the 20 degrees C block, the cells were permeabilized and proSRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by pre-incubating the permeabilized cells with low concentrations of N-ethylmaleimide or bafilomycin, both inhibitors of vacuolar-type ATP-dependent proton pumps. These data suggest that ATP is required for generation of an acidic pH in the lumen of the TGN which is necessary for the activity of prohormone processing enzymes. By exploiting a permeabilized cell system, we have demonstrated that proSRIF cleavage is initiated in the TGN, in a reaction which is facilitated by a Golgi-associated vacuolar type ATPase.

Adenosine Triphosphate↗

PubMed1996

ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway.

Secretion of proteins is initiated by their uptake into the endoplasmic reticulum (ER), which possesses a proteolytic system able to degrade misfolded and nonassembled proteins. The ER degradation system was studied with yeast mutants defective in the breakdown of a mutated soluble vacuolar protein, carboxypeptidase yscY (CPY*). The ubiquitin-conjugating enzyme Ubc7p participated in the degradation process, which was mediated by the cytosolic 26S proteasome. It is likely that CPY* entered the ER, was glycosylated, and was then transported back out of the ER lumen to the cytoplasmic side of the organelle, where it was conjugated with ubiquitin and degraded.

Biological Transport↗

PubMed1986

Overproduction-induced mislocalization of a yeast vacuolar protein allows isolation of its structural gene.

Using an immunological screening procedure that allows the detection of yeast cells aberrantly secreting vacuolar proteins, we have isolated a cloned DNA fragment containing the structural gene for the vacuolar enzyme proteinase A (PrA; EC 3.4.23.6). A large portion of PrA is misdirected to the cell surface in cells harboring the PrA structural gene on a multicopy plasmid. This mislocalized PrA traverses the late stages of the secretory pathway and differs slightly in apparent molecular weight from the vacuolar form. A deletion in the genomic copy of the PrA structural gene eliminates immunoreactive PrA as well as the enzymatic activities of at least three other vacuolar hydrolases. In the case of the vacuolar enzyme carboxypeptidase Y (EC 3.4.16.1), the lack of activity is due to the absence of proteolytic activation of the zymogen. Thus, PrA may be required for in vivo processing of a number of yeast vacuolar hydrolases.

Aspartic Acid Endopeptidases↗

PubMed1996

Pathogenesis-related functions of plant beta-1,3-glucanases investigated by antisense transformation--a review.

Plant beta-1,3-glucanases (beta Glu) have been implicated in several physiological and developmental processes, e.g., cell division, microsporogenesis, pollen germination, fertilization and seed germination. These enzymes, particularly the antifungal class-I vacuolar isoforms, are also believed to be part of the defences of plants against fungal infection. The function of beta Glu in tobacco and Nicotiana sylvestris has been investigated by antisense transformation. Transformation with GLA, the gene encoding the A isoform of tobacco class-I beta Glu, in reverse orientation regulated by the strong cauliflower mosaic virus 35S RNA promoter effectively and specifically blocked the induction of class-I beta Glu. This induction was in response to ethylene treatment and following infection with the pathogenic fungus, Cercospora nicotianae, tobacco mosaic virus (TMV) and tobacco necrosis virus (TNV). Nevertheless, the plants compensated for this deficiency by producing a functionally equivalent (i.e., "ersatz') enzyme or enzymes. The fact that compensation occurred specifically in response to infection suggests that beta Glu activity has an important role in pathogenesis. Antisense transformation substantially reduced lesion size and number in virus-infected local-lesion hosts. These results suggest novel antisense-based strategies for protecting plants against virus infection. They also raise the intriguing possibility that viruses use a defence mechanism of the host, production of antifungal beta Glu, to promote their own replication and spread.

DNA, Antisense↗

PubMed2005

Assembly of the yeast vacuolar H+-ATPase and ATP hydrolysis occurs in the absence of subunit c''.

The V-ATPases are ubiquitous enzymes of eukaryotes. They are involved in many cellular processes via their ability to pump protons across biological membranes. They are two domain enzymes comprising an ATP hydrolysing sector and a proton translocating sector. Both sectors are functionally coupled. The proton tanslocating sector, V0, is comprised of five polypeptides in an as yet undetermined stoichiometry. In V0 three homologous proteins, subunit c, c' and c'' have previously been reported to be essential for assembly of the enzyme. However, we report that subunit c'' is not essential for assembly but is for functional coupling of the enzyme.

Adenosine Triphosphate↗

PubMed1995

Molecular probing of the secretory pathway in peptide hormone-producing cells.

The biosynthetic machinery in the melanotrope cells of the Xenopus intermediate pituitary is primarily dedicated to the generation of proopiomelanocortin (POMC)-derived, melanophore-stimulating peptides. Transfer of the animal to a black background stimulates the production of these peptides and causes a dramatic increase in POMC mRNA levels. To identify genes involved in the biosynthesis and regulated release of peptide hormones, we differentially screened an intermediate pituitary cDNA library of toads adapted to a black background with cDNA probes derived from intermediate pituitary mRNA of black- and white-adapted animals. Here we report the identification of twelve distinct genes whose expression levels in the melanotropes are regulated in coordination with that of POMC. Four of these genes are novel while the others code for translocon-associated proteins, a lumenal cysteine protease of the endoplasmic reticulum, prohormone-processing enzymes, members of the granin family and a transmembrane protein presumably involved in the assembly and/or specific functioning of vacuolar H(+)-ATPase from secretory granules. Our results indicate that a wide variety of both soluble and membrane-associated components of the secretory pathway is recruited in physiologically activated, peptide hormone-producing cells.

Amino Acid Sequence↗

PubMed1995

Purification, cDNA cloning and characterization of proteinase B, an asparagine-specific endopeptidase from germinating vetch (Vicia sativa L.) seeds.

Proteinase B, an asparagine-specific endopeptidase, has been purified from germinating vetch (Vicia sativa L.) seeds. The final preparation consists of two enzymically active proteins with molecular masses of approximately 39 kDa and 37 kDa. Synthetic substrates were used to confirm cleavage specificity of the proteinase B preparation. As expected, the enzyme cleaves the substrates at the C-terminal side of Asn residues. The octapeptide ETRNGVEE was digested most efficiently. When Gly was replaced by Ile or Glu, cleavage took place with lower efficiency. Polyclonal antibodies displayed both proteins in cotyledon extracts of germinated vetch seeds. In addition, a strong cross-reacting protein band was found in cotyledon extracts of developing seeds, indicating the presence of a very similar enzyme during seed development. cDNA clones encoding proteinase B precursor have been obtained on the basis of the N-terminal amino acid sequence DDDFEGTRWAILLAGS, by means of the polymerase chain reaction. The cDNA clones contain an open reading frame of 1479 bp encoding a polypeptide of 493 amino acids. The precursor displayed 59% sequence identity to the cDNA-derived amino acid sequence of a vacuolar Asn-specific enzyme from the developing castor beam endosperm which is thought to catalyze the post-translational processing of pro-proteins into the mature forms. Proteinase B is synthesized de novo during seed germination. The results of Southern-blot analyses suggested that there are at least two genes for proteinase B.

Amino Acid Sequence↗

PubMed1993

Posttranslational processing of a new class of hydroxyproline-containing proteins. Prolyl hydroxylation and C-terminal cleavage of tobacco (Nicotiana tabacum) vacuolar chitinase.

The fungicidal class I chitinases (EC 3.2.1.14) are believed to be important in defending plants against microbial pathogens. The vacuolar isoforms of tobacco (Nicotiana tabacum), chitinases A and B, are the first examples of a new type of hydroxyproline-containing protein with intracellular location, enzymic activity, and a small number of hydroxyprolyl residues restricted to a single, short peptide sequence. We have investigated the posttranslational processing and intracellular transport of transgene-encoded chitinase A in callus cultures of Nicotiana tabacum L. cv Havana 425 and leaves of Nicotiana sylvestris Spegazzini and Comes. Pulse-chase experiments and cell fractionation show that chitinase A is processed in two distinct steps. In the first step, the nascent protein undergoes an increase in apparent M(r) of approximately 1500 detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with the inhibitor of prolyl hydroxylation, alpha,alpha'-dipyridyl, and pulse-chase labeling of cells expressing recombinant forms of chitinase A indicate that the anomalous increase in M(r) is due to hydroxylation of prolyl residues. This step occurs in the endomembrane system before sorting for secretion and vacuolar transport and does not appear to be required for correct targeting of chitinase A to the vacuole. The second step is a proteolytic cleavage. Sequencing of tryptic peptides of the mature proteins indicates that during processing essentially all molecules of chitinase A and B lose a C-terminal heptapeptide, which has been shown to be a vacuolar targeting signal. This appears to occur primarily in the endomembrane system late in intracellular transport. A model for the posttranslational modification of chitinase A is proposed.

Amino Acid Sequence↗

PubMed1990

Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells.

We have shown that highly purified vacuoles of suspension-cultured tomato (Lycopersicon esculentum) cells contain RNA-oligonucleotides, using two different approaches to label and detect RNA: (a) in vivo labeling of cellular RNA with [5-(3)H]uridine, followed by preparation of vacuoles from protoplasts and by quantification of radioactively labeled material; and (b) in vitro labeling and analysis on sequencing gels of nucleic acids prepared from tomato vacuoles and their identification as RNA. The intravacuolar location of the RNA found in vacuolar preparations was concluded from analyzing for RNA intact organelles after repeated flotation steps as well as ribonuclease A treatment. About 3% of the RNA in protoplasts was localized within vacuoles, exceeding by severalfold the contribution made by contamination with unlysed protoplasts and subcellular organelles. Investigation of the size distribution of vacuolar RNA revealed an oligonucleotide pattern strikingly different from that which would arise from contaminating protoplasts; vacuolar RNA fragments are considerably shorter than 80 nucleotides. Characterization of these oligoribonucleotides (3'-phosphorylated termini; relatively rich in pyrimidines) as possible products of tomato vacuolar ribonuclease I action, and, in addition, enzymatic hydrolysis of vacuolar RNA by inherent enzyme activities in lysed vacuole preparations support the hypothesis that plant vacuoles are involved in cellular nucleolytic processes.

Journal Article↗

PubMed2004

Increased expression and translocation of lysosomal cathepsins contribute to macrophage apoptosis in atherogenesis.

It has been recently reported that atherosclerotic lesions in both humans and mice express several lysosomal proteases, including cathepsins B, D, L, and S, which may affect plaque development and stability. The mechanisms responsible for the extralysosomal expression of lysosomal cathepsins and the related atherogenic implications remain unknown. We find that the lesion-dependent expression of cathepsins B and L is mainly in macrophage-infiltrated areas of human carotid atheroma. These enzymes appear in the cytoplasm and nuclei of apoptotic macrophages (normally confined to the lysosomal compartment) and in the extracellular areas. After exposure to oxidized low-density lipoprotein (oxLDL) or 7beta-hydroxycholesterol (7beta-OH), macrophages initially transform into foam cells and then undergo apoptotic cell death. The oxidized lipids induce lysosomal destabilization, with leakage to the cytosol of lysosomal enzymes (cathepsins B, D, and L), as detected by cytochemistry and immunocytochemistry. A remarkable increase in cathepsin D mRNA levels was observed after 7beta-OH exposure. Like macrophages within atheroma, intralysosomal cathepsins B and L are translocated to the cytoplasm and nuclei of 7beta-OH-exposed cells. Our results suggest that endocytosed oxLDL and oxysterols not only destabilize the acidic vacuolar compartment but also cause the upregulation and translocation of lysosomal cathepsins, which may act as cleaving enzymes during the apoptotic process. The increased macrophage apoptosis and nuclear and matrix degradation by lysosomal enzymes in atheroma may play important roles in plaque development and rupture.

Apoptosis↗