Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor

Many peroxisomal proteins are imported into peroxisomes via recognition of the peroxisomal targeting signal (PTS1) present at the C-termini by the PTS1 receptor (Pex5p). Catalase, a peroxisomal protein, has PTS1-like motifs around or at the C-terminus. However, it remains unclear whether catalase is imported into peroxisome via the PTS1 system. In this work, we analyzed the PTS of pumpkin catalase (Cat1). A full or truncated pumpkin Cat1 cDNA fused at the 3' end of the green fluorescent protein (GFP) coding sequence was introduced and stably expressed in tobacco BY-2 (Nicotiana tabacum cv. Bright Yellow 2) cells or Arabidopsis thaliana by Agrobacterium-mediated transformation. The cellular localization of GFP was analyzed by fluorescence microscopy. The results showed that the C-terminal 10-amino acid region containing an SKL motif-like tripeptide (SHL) was not required for the import into peroxisomes. Surprisingly, the C-terminal 3-amino acid region was required for the import when the fusion proteins were transiently expressed by using particle gun bombardment, suggesting that the transient expression system is inadequate to analyze the targeting signal. We proposed that the C-terminal amino acid region from 13 to 11 (QKL), which corresponds with the PTS1 consensus sequence, may function as an internal PTS1. Analysis of the binding of Cat1 to PTS1 receptor (Pex5p) by the yeast two-hybrid system revealed that Cat1 can bind with the PTS1 receptor (Pex5p), indicating that Cat1 is imported into peroxisomes by the PTS1 system.     

Obstruction of polyubiquitination affects PTS1 peroxisomal matrix protein import

Pex4p is an ubiquitin-conjugating enzyme that functions at a late stage of peroxisomal matrix protein import. Here we show that in the methylotrophic yeast Hansenula polymorpha production of a mutant form of ubiquitin (Ub(K48R)) has a dramatic effect on PTS1 matrix protein import. This effect was not observed in cells lacking Pex4p, in which the peroxisome biogenesis defect was largely suppressed. These findings provide the first indication that the function of Pex4p in matrix protein import involves polyubiquitination. We also demonstrate that the production of Ub(K48R) in H. polymorpha results in enhanced Pex5p degradation. A similar observation was made in cells in which the PEX4 gene was deleted. We demonstrate that in both strains Pex5p degradation was due to ubiquitination and subsequent degradation by the proteasome. This process appeared to be dependent on a conserved lysine residue in the N-terminus of Pex5p (Lys21) and was prevented in a Pex5p(K21R) mutant. We speculate that the degradation of Pex5p by the proteasome is important to remove receptor molecules that are stuck at a late stage of the Pex5p-mediated protein import pathway.     

Novel peroxisomal protease Tysnd1 processes PTS1- and PTS2-containing enzymes involved in beta-oxidation of fatty acids

Peroxisomes play an important role in beta-oxidation of fatty acids. All peroxisomal matrix proteins are synthesized in the cytosol and post-translationally sorted to the organelle. Two distinct peroxisomal signal targeting sequences (PTSs), the C-terminal PTS1 and the N-terminal PTS2, have been defined. Import of precursor PTS2 proteins into the peroxisomes is accompanied by a proteolytic removal of the N-terminal targeting sequence. Although the PTS1 signal is preserved upon translocation, many PTS1 proteins undergo a highly selective and limited cleavage. Here, we demonstrate that Tysnd1, a previously uncharacterized protein, is responsible both for the removal of the leader peptide from PTS2 proteins and for the specific processing of PTS1 proteins. All of the identified Tysnd1 substrates catalyze peroxisomal beta-oxidation. Tysnd1 itself undergoes processing through the removal of the presumably inhibitory N-terminal fragment. Tysnd1 expression is induced by the proliferator-activated receptor alpha agonist bezafibrate, along with the increase in its substrates. A model is proposed where the Tysnd1-mediated processing of the peroxisomal enzymes promotes their assembly into a supramolecular complex to enhance the rate of beta-oxidation.     

pex5 Mutants that differentially disrupt PTS1 and PTS2 peroxisomal matrix protein import in Arabidopsis

PEX5 and PEX7 are receptors required for the import of peroxisome-bound proteins containing one of two peroxisomal targeting signals (PTS1 or PTS2). To better understand the role of PEX5 in plant peroxisomal import, we characterized the Arabidopsis (Arabidopsis thaliana) pex5-10 mutant, which has a T-DNA insertion in exon 5 of the PEX5 gene. Sequencing results revealed that exon 5, along with the T-DNA, is removed in this mutant, resulting in a truncated pex5 protein. The pex5-10 mutant has germination defects and is completely dependent on exogenous Suc for early seedling establishment, based on poor utilization of seed-storage fatty acids. This mutant also has delayed development and reduced fertility, although adult pex5-10 plants appear normal. Peroxisomal metabolism of indole-3-butyric acid, propionate, and isobutyrate also is disrupted. The pex5-10 mutant has reduced import of both PTS1 and PTS2 proteins, and enzymatic processes that occur in peroxisomes are disrupted. To specifically study the import and importance of PTS1 proteins, we made a truncated PEX5 construct lacking the PTS1-binding region (PEX5(454)). Transformation of this construct into pex5-10 resulted in the rescue of PTS2 import, thereby creating a line with PTS1-specific import defects. The pex5-10 (PEX5(454)) plants still had developmental defects, although restoring PTS2 import resulted in a less severe mutant phenotype. Comparison of pex5-10 and pex5-10 (PEX5(454)) phenotypes can separate the import mechanisms for enzymes acting in different peroxisomal processes, including indole-3-butyric acid/2,4-dichlorophenoxybutyric acid oxidation, isobutyrate and propionate metabolism, and photorespiration.     

PTS1-independent sorting of peroxisomal matrix proteins by Pex5p

Most peroxisomal matrix proteins contain a peroxisomal targeting signal 1 (PTS1) for sorting to the correct organelle. This signal is located at the extreme C-terminus and generally consists of only three amino acids. The PTS1 is recognized by the receptor protein Pex5p. Several examples have been reported of peroxisomal matrix proteins that are sorted to peroxisomes via Pex5p, but lack a typical PTS1 tripeptide. In this contribution we present an overview of these so-called non-PTS1 proteins and discuss the current knowledge of the molecular mechanisms involved in their sorting.     

The deubiquitination of the PTS1-import receptor Pex5p is required for peroxisomal matrix protein import

Peroxisomal biogenesis depends on the correct import of matrix proteins into the lumen of the organelle. Most peroxisomal matrix proteins harbor the peroxisomal targeting-type 1 (PTS1), which is recognized by the soluble PTS1-receptor Pex5p in the cytosol. Pex5p ferries the PTS1-proteins to the peroxisomal membrane and releases them into the lumen. Finally, the PTS1-receptor is monoubiquitinated on the conserved cysteine 6 in Saccharomyces cerevisiae. The monoubiquitinated Pex5p is recognized by the peroxisomal export machinery and is retrotranslocated into the cytosol for further rounds of protein import. However, the functional relevance of deubiquitination has not yet been addressed.In this study, we have analyzed a Pex5p-truncation lacking Cys6 [(Δ6)Pex5p], a construct with a ubiquitin-moiety genetically fused to the truncation [Ub-(Δ6)Pex5p], as well as a construct with a reduced susceptibility to deubiquitination [Ub(G75/76A)-(Δ6)Pex5p]. While the (Δ6)Pex5p-truncation is not functional, the Ub-(Δ6)Pex5p chimeric protein can facilitate matrix protein import. In contrast, the Ub(G75/76A)-(Δ6)Pex5p chimera exhibits a complete PTS1-import defect. The data show for the first time that not only ubiquitination but also deubiquitination rates are tightly regulated and that efficient deubiquitination of Pex5p is essential for peroxisomal biogenesis.     

Identification of Pex13p a peroxisomal membrane receptor for the PTS1 recognition factor

We have identified an S. cerevisiae integral peroxisomal membrane protein of M of 42,705 (Pex13p) that is a component of the peroxisomal protein import apparatus. Pex13p's most striking feature is an src homology 3 (SH3) domain that interacts directly with yeast Pex5p (former Pas10p), the recognition factor for the COOH-terminal tripeptide signal sequence (PTS1), but not with Pex7p (former Pas7p), the recognition factor for the NH2-terminal nonapeptide signal (PTS2) of peroxisomal matrix proteins. Hence, Pex13p serves as peroxisomal membrane receptor for at least one of the two peroxisomal signal recognition factors. Cells deficient in Pex13p are unable to import peroxisomal matrix proteins containing PTS1 and, surprisingly, also those containing PTS2. Pex13p deficient cells retain membranes containing the peroxisomal membrane protein Pex11p (former Pmp27p), consistent with the existence of independent pathways for the integration of peroxisomal membrane proteins and for the translocation of peroxisomal matrix proteins.     

Identification of the peroxisomal targeting signal for cottonseed catalase

Catalase is a ubiquitous peroxisomal matrix enzyme, yet the molecular targeting signal(s) for sorting it in plant cells has not been defined. The most common peroxisome targeting signal (PTS) is a C-terminal tripeptide composed of a conserved SKL motif (type 1 PTS). The PTS for cottonseed catalase (Ccat) was elucidated in this study from immunofluorescence microscopic analyses of tobacco BY-2 suspension cells serving as an in vivo import system. To distinguish biolistically introduced Ccat from endogenous tobacco catalase, Ccat was hemagglutinin (HA) epitope-tagged at its N-terminus. Bombardment with HA-Ccat resulted in the import of Ccat into glyoxysomes, the specialized type of peroxisome in BY-2 cells. The C-terminal tripeptide of Ccat, PSI, is necessary for import. Evidence for this were mislocalizations to the cytosol of PSI-truncated Ccat and AGV-substituted (for PSI) Ccat. PSI-COOH, however, was not sufficient to re-route chloramphenicol acetyltransferase (CAT) from the cytosol to glyoxysomes, whereas the Ccat tetrapeptide RPSI-COOH was sufficient. Surprisingly, substitution of K (common at the fourth position in other plant catalases) for the R (CAT-KPSI) decreased import efficiency. However, substitution of K did not affect import, when additional upstream residues in Ccat were included (e.g. CAT-NVKPSI). Other evidence for the importance of upstream residues comprised abolishment of Ccat import due to substitutions with non-conserved residues (e.g. -AGVNVRPSI for -SRLNVRPSI). These data indicate that Ccat is sorted to plant peroxisomes by a degenerate type 1 PTS (PSI-COOH) whose residues are functionally dependent on a strict context of adjacent C-terminal amino acid residues.     

Functional similarity between the peroxisomal PTS2 receptor binding protein Pex18p and the N-terminal half of the PTS1 receptor Pex5p

Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor Pex5p comprises cargo recognition and transport. While the C-terminal half (Pex5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of Pex5p (Pex5p-N) to the receptor cycle has been less clear. Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae Pex5p-N alone facilitates the import of the major matrix protein Fox1p. This finding suggests that Pex5p-N is sufficient for receptor docking and cargo transport into peroxisomes. Moreover, we found that Pex5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway. A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to Pex5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain. On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of Pex5p in the PTS1 import pathway.     

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation

Catalase activity was inhibited by aminotriazole administration to rats in order to evaluate the influence of catalase on the peroxisomal fatty acyl-CoA beta-oxidation system. 2 h after the administration of aminotriazole, peroxisomes were prepared from rat liver, and the activities of catalase, the beta-oxidation system and individual enzymes of beta-oxidation (fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase) were determined. Catalase activity was decreased to about 2% of the control. Among the individual enzymes of the beta-oxidation system, thiolase activity was decreased to 67%, but the activities of fatty acyl-CoA oxidase, crotonase and beta-hydroxybutyryl-CoA dehydrogenase were almost unchanged. The activity of the peroxisomal beta-oxidation system was assayed by measuring palmitoyl-CoA-dependent NADH formation, and the activity of the purified peroxisome preparation was found to be almost unaffected by the administration of aminotriazole. The activity of the system in the aminotriazole-treated preparation was, however, significantly decreased to 55% by addition of 0.1 mM H2O2 to the incubation mixture. Hydrogen peroxide (0.1 mM) reduced the thiolase activity of the aminotriazole-treated peroxisomes to approx. 40%, but did not affect the other activities of the system. Thiolase activity of the control preparation was decreased to 70% by addition of hydrogen peroxide (0.1 mM). The half-life of 0.1 mM H2O2 added to the thiolase assay mixture was 2.8 min in the case of aminotriazole-treated peroxisomes, and 4 s in control peroxisomes. The ultraviolet spectrum of acetoacetyl-CoA (substrate of thiolase) was clearly changed by addition of 0.1 mM H2O2 to the thiolase assay mixture without the enzyme preparation; the absorption bands at around 233 nm (possibly due to the thioester bond of acetoacetyl-CoA) and at around 303 nm (due to formation of the enolate ion) were both significantly decreased. These results suggest that H2O2 accumulated in peroxisomes after aminotriazole treatment may modify both thiolase and its substrate, and consequently suppress the fatty acyl-CoA beta-oxidation. Therefore, catalase may protect thiolase and its substrate, 3-ketoacyl-CoA, by removing H2O2, which is abundantly produced during peroxisomal enzyme reactions.     

Immunoelectron microscopy of peroxisomes employing the antibody for the SKL sequence PTS1 C-terminus common to peroxisomal enzymes.

Immunohistochemistry employing a new hapten antibody that detects the SKL sequence and its variants of the PTS1 C-terminus of peroxisomal enzymes was attempted to visualize peroxisomes across species. Rabbits were immunized with the SKL sequence coupled with KLH, between which an arm molecule was interposed. IgG fractions of antisera were affinity-purified against the hapten and employed for immunochemical analyses and immunoelectron microscopy. The specificity of the antibody was examined by immunoblot analyses for various purified enzymes of rat liver peroxisomes and by dot-blot analyses inhibited by SKL peptide and its variants. Various animal and plant tissues were subjected to immunoelectron microscopy with the protein A-gold technique. The antibody reacted with various enzymes in the peroxisome with the SKL motif. The affinity of the antibody for tripeptides, which varied depending on their structures, was higher for SKL than for its variants. Hepatic and renal peroxisomes of vertebrates, peroxisomes in the fat body of an insect, and the cotyledon of a plant were visualized by immunoelectron microscopy. Immunohistochemistry employing this SKL antibody may provide specific staining that can detect peroxisomes across different species.     

Degradation of peroxisomal catalase and urate oxidase of rat liver

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5–9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation.After [³H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37°C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process.From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.     

Interaction with deoxycholate of rat liver peroxisomal and cytosolic catalase

Rat liver catalase was found to interact with deoxycholate (DOC). When purified, the peroxisomal catalase was precipitated at pH 6 in the presence of DOC, whereas in the peroxisomal extract (with DOC) it was unsedimentable at pH 6. The membrane fraction in the extract interacted with the catalase instead of DOC, and prevented the precipitation of catalase with DOC at pH 6. The peroxisomal catalase seemed to be easily modified by lysosomal protease during manipulation, and this proteolytic cleavage rendered the molecule able to interact with the membrane. On the other hand, the cytosolic catalase, both in the cytosol fraction and in the purified preparation, sedimented at pH 6 in the presence of DOC. The cytosolic catalase was far more resistant to proteolytic modification than the peroxisomal catalase. The molecule of peroxisomal catalase is assumed to have a site for recognizing the membrane, whereas such a structure may be absent in the cytosolic catalase or may not be easily exposed by proteolytic cleavage.     

Targeting signal of the peroxisomal catalase in the methylotrophic yeast Hansenula polymorpha.

The methylotrophic yeast, Hansenula polymorpha, harbours a unique catalase (EC 1.11.1.6), which is essential for growth on methanol as a carbon source and is located in peroxisomes. Its corresponding gene has been cloned and the nucleotide sequence determined. The deduced amino acid sequence displayed the tripeptide serine-lysine-isoleucine at the extreme C-terminus, which is similar to sequences of other peroxisomal targeting signals. Exchange of the ultimate amino acid, isoleucine, of catalase for serine revealed a cytosolic enzyme activity and a concomitant loss of peroxisome function. We concluded that the tripeptide is essential for targeting of catalase in H. polymorpha.     

Degradation of peroxisomal catalase and urate oxidase of rat liver.

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5--9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation. After [3H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37 degrees C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process. From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.     

Lack of peroxisomal catalase causes a progeric phenotype in Caenorhabditis elegans

Studies using the nematode Caenorhabditis elegans as a model system to investigate the aging process have implicated the insulin/insulin-like growth factor-I signaling pathway in the regulation of organismal longevity through its action on a subset of target genes. These targets can be classified into genes that shorten or extend life-span upon their induction. Genes that shorten life-span include a variety of stress response genes, among them genes encoding catalases; however, no evidence directly implicates catalases in the aging process of nematodes or other organisms. Using genetic mutants, we show that lack of peroxisomal catalase CTL-2 causes a progeric phenotype in C. elegans. Lack of peroxisomal catalase also affects the developmental program of C. elegans, since Deltactl-2 mutants exhibit decreased egg laying capacity. In contrast, lack of cytosolic catalase CTL-1 has no effect on either nematode aging or egg laying capacity. The Deltactl-2 mutation also shortens the maximum life-span of the long lived Deltaclk-1 mutant and accelerates the onset of its egg laying period. The more rapid aging of Deltactl-2 worms is apparently not due to increased carbonylation of the major C. elegans proteins, although altered peroxisome morphology in the Deltactl-2 mutant suggests that changes in peroxisomal function, including increased production of reactive oxygen species, underlie the progeric phenotype of the Deltactl-2 mutant. Our findings support an important role for peroxisomal catalase in both the development and aging of C. elegans and suggest the utility of the Deltactl-2 mutant as a convenient model for the study of aging and the human diseases acatalasemia and hypocatalasemia.     

Two independent peroxisomal targeting signals in catalase A of Saccharomyces cerevisiae

In contrast to many other peroxisomal proteins catalase A contains at least two peroxisomal targeting signals each sufficient to direct reporter proteins to peroxisomes. One of them resides at the extreme carboxy terminus constituting a new variant of this signal, -SSNSKF, not active in monkey kidney cells (Gould, S. J., G. A. Keller, N. Hosken, J. Wilkinson, and S. Subramani 1989. J. Cell Biol. 108:1657- 1664). However, this signal is completely dispensable for import of catalase A itself. In its amino-terminal third this protein contains another peroxisomal targeting signal sufficient to direct reporter proteins into microbodies. This internal signal depends on the context. The nature of this targeting signal might be a short defined sequence or a structural feature recognized by import factors. In addition, we have demonstrated that the carboxy-terminal seven amino acids of citrate synthase of Saccharomyces cerevisiae encoded by CIT2 and containing the canonical -SKL represents a targeting signal sufficient to direct reporter proteins to peroxisomes.     

Purification of glyoxysomal catalase and immunochemical comparison of glyoxysomal and leaf peroxisomal catalase in germinating pumpkin cotyledons

As a step to study the mechanism of the microbody transition (glyoxysomes to leaf peroxisomes) in pumpkin (Cucurbita sp. Amakuri Nankin) cotyledons, catalase was purified from glyoxysomes. The molecular weight of the purified catalase was determined to be 230,000 to 250,000 daltons. The enzyme was judged to consist of four identical pieces of the monomeric subunit with molecular weight of 55,000 daltons. Absorption spectrum of the catalase molecule gave two major peaks at 280 and 405 nanometers, showing that the pumpkin enzyme contains heme. The ratio of absorption at 405 and 280 nanometers was 1.0, the value being lower than that obtained for catalase from other plant sources. These results indicate that the pumpkin glyoxysomal catalase contains the higher content of heme in comparison with other plant catalase.

The immunochemical resemblance between glyoxysomal and leaf peroxisomal catalase was examined by using the antiserum specific against the purified enzyme preparation from pumpkin glyoxysomes. Ouchterlony double diffusion and immunoelectrophoretic analysis demonstrated that catalase from both types of microbodies cross-reacted completely whereas the immunotitration analysis showed that the specific activity of the glyoxysomal catalase was 2.5-fold higher than that of leaf peroxisomal catalase. Single radial immunodiffusion analysis showed that the specific activity of catalase decreased during the greening of pumpkin cotyledons.

     

Restoration of peroxisomal catalase import in a model of human cellular aging

Peroxisomes play an important role in human cellular metabolism by housing enzymes involved in a number of essential biochemical pathways. Many of these enzymes are oxidases that transfer hydrogen atoms to molecular oxygen forming hydrogen peroxide. The organelle also contains catalase, which readily decomposes the hydrogen peroxide, a potentially damaging oxidant. Previous work has demonstrated that aging compromises peroxisomal protein import with catalase being particularly affected. The resultant imbalance in the relative ratio of oxidases to catalase was seen as a potential contributor to cellular oxidative stress and aging. Here we report that altering the peroxisomal targeting signal of catalase to the more effective serine-lysine-leucine (SKL) sequence results in a catalase molecule that more strongly interacts with its receptor and is more efficiently imported in both in vitro and in vivo assays. Furthermore, catalase-SKL monomers expressed in cells interact with endogenous catalase subunits resulting in altered trafficking of the latter molecules. A dramatic reduction in cellular hydrogen peroxide levels accompanies this increased peroxisomal import of catalase. Finally, we show that catalase-SKL stably expressed in cells by retroviral-mediated transduction repolarizes mitochondria and reduces the number of senescent cells in a population. These results demonstrate the utility of a catalase-SKL therapy for the restoration of a normal oxidative state in aging cells.