cDNA-derived amino acid sequence of rat mitochondrial 3-oxoacyl-CoA thiolase with no transient presequence: structural relationship with peroxisomal isozyme.

The sorting of homologous proteins between two separate intracellular organelles is a major unsolved problem. 3-Oxoacyl-CoA thiolase is localized in mitochondria and peroxisomes, and provides a good system for the study on the problem. Unlike most mitochondrial matrix proteins, mitochondrial 3-oxoacyl-CoA thiolase in rats is synthesized with no transient presequence and possess information for mitochondrial targeting and import in the mature protein. Two overlapping cDNA clones contained an open reading frame encoding a polypeptide of 397 amino acid residues (predicted Mr = 41,868), a 5' untranslated sequence of 164 bp, a 3' untranslated sequence of 264 bp and a poly(A) tract. The amino acid sequence of the mitochondrial thiolase is 37% identical with that of the mature portion of rat peroxisomal 3-oxoacyl-CoA thiolase precursor. These results suggest that the two thiolases have a common origin and obtained information for targeting to respective organelles during evolution. Two portions in the mitochondrial thiolase that may serve as a mitochondrial targeting signal are presented.     

Signal peptide for peroxisomal targeting: replacement of an essential histidine residue by certain amino acids converts the amino-terminal presequence of peroxisomal 3-ketoacyl-CoA thiolase to a mitochondrial signal peptide.

The role of the histidine residue at position -17 of the amino-terminal signal peptide of rat peroxisomal 3-ketoacyl-CoA thiolase was studied in vivo, employing site-directed mutagenesis. Among the nine amino acids tested, only glutamine could partially substitute for the histidine. Mutants carrying basic amino acids, arginine and lysine, and hydrophobic residues, leucine and valine, in place of histidine were all translocated to mitochondria, but not to peroxisomes. These results indicate that the signal peptide of the thiolase is recognized by a mechanism totally different from that for the SKL motif, a known peroxisomal targeting signal. Relationship of the thiolase signal peptide to those of mitochondrial proteins is discussed.     

Transport of peroxisomal proteins synthesized as large precursors in plants

Plant peroxisomes contain at least four proteins, namely, citrate synthase, malate dehydrogenase, long-chain acyl-CoA oxidase, and 3-ketoacyl-CoA thiolase, which are synthesized as large precursors with an N-terminal cleavable presequence. Each presequence has a conserved domain (R[I/L./Q]-X₅-HL) that is homologous to peroxisomal targeting signal 2 from mammals and yeasts. In addition, a cysteine residue is found at the C-terminal ends of the presequences, whose function has not yet been described. The authors analyzed the function of the presequences and the conserved amino acids using transgenic Arabidopsis plants, which accumulate β-glucuronidase carrying the presequence of the peroxisomal proteins from plants. Immunological and immunocytochemical studies on the transgenic plants showed that a conserved sequence in the extrapeptides is essential for targeting to peroxisomes, and a cysteine residue at the cleavage site is involved in the processing of the presequence. These results suggest that the presequences of the peroxisomal proteins function as targeting signals, and are necessary for the recognition of the processing.     

Molecular cloning and sequence analysis of the cDNA for rat mitochondrial enoyl-CoA hydratase. Structural and evolutionary relationships linked to the bifunctional enzyme of the peroxisomal beta-oxidation system

To elucidate structural relationships between the mitochondrial and peroxisomal isozymes of beta-oxidation systems, cDNA of the mitochondrial enoyl-CoA hydratase was cloned and sequenced. The 1454-bp cDNA sequence contained a 870 bp of open reading frame, encoding a polypeptide of 290 amino acid residues. When compared with the amino-terminal sequence of the mature enzyme, the predicted sequence contained a 29-residue presequence at the amino terminus. This presequence had characteristics typical of a mitochondrial signal peptide. The primary structure of this enzyme showed significant similarity with the amino-terminal portion of sequence of the peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. The carboxy-terminal part of the latter enzyme has sequence similarity with mitochondrial 3-hydroxyacyl-CoA dehydrogenase [Ishii, N., Hijikata, M., Osumi, T. & Hashimoto, T. (1987) J. Biol. Chem. 262, 8144-8150]. These findings suggest that the peroxisomal bifunctional enzyme has the hydratase and dehydrogenase functions on the amino- and carboxy-terminal sides, respectively. The mitochondrial beta-oxidation enzymes and the peroxisomal bifunctional enzyme may have common evolutionary origins.     

Up-regulation of the peroxisomal beta-oxidation system occurs in butyrate-grown Candida tropicalis following disruption of the gene encoding peroxisomal 3-ketoacyl-CoA thiolase

In the yeast Candida tropicalis, two thiolase isozymes, peroxisomal acetoacetyl-CoA thiolase and peroxisomal 3-ketoacyl-CoA thiolase, participate in the peroxisomal fatty acid beta-oxidation system. Their individual contributions have been demonstrated in cells grown on butyrate, with C. tropicalis able to grow in the absence of either one. In the present study, a lack of peroxisomal 3-ketoacyl-CoA thiolase protein resulted in increased expression (up-regulation) of acetoacetyl-CoA thiolase and other peroxisomal proteins, whereas a lack of peroxisomal acetoacetyl-CoA thiolase produced no corresponding effect. Overexpression of the acetoacetyl-CoA thiolase gene did not suppress the up-regulation or the growth retardation on butyrate in cells without peroxisomal 3-ketoacyl-CoA thiolase, even though large amounts of the overexpressed acetoacetyl-CoA thiolase were detected in most of the peroxisomes of butyrate-grown cells. These results provide important evidence of the greater contribution of 3-ketoacyl-CoA thiolase to the peroxisomal beta-oxidation system than acetoacetyl-CoA thiolase in C. tropicalis and a novel insight into the regulation of the peroxisomal beta-oxidation system.     

A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase.

Several peroxisomal proteins do not contain the previously identified tripeptide peroxisomal targeting signal (PTS) at their carboxy-termini. One such protein is the peroxisomal 3-ketoacyl CoA thiolase, of which two types exist in rat [Hijikata et al. (1990) J. Biol. Chem., 265, 4600-4606]. Both rat peroxisomal thiolases are synthesized as larger precursors with an amino-terminal prepiece of either 36 (type A) or 26 (type B) amino acids, that is cleaved upon translocation of the enzyme into the peroxisome. The prepieces are necessary for import of the thiolases into peroxisomes because expression of an altered cDNA encoding only the mature thiolase, which lacks any prepiece, results in synthesis of a cytosolic enzyme. When appended to an otherwise cytosolic passenger protein, the bacterial chloramphenicol acetyltransferase (CAT), the prepieces direct the fusion proteins into peroxisomes, demonstrating that they encode sufficient information to act as peroxisomal targeting signals. Deletion analysis of the thiolase B prepiece shows that the first 11 amino acids are sufficient for peroxisomal targeting. We conclude that we have identified a novel PTS that functions at amino-terminal or internal locations and is distinct from the C-terminal PTS. These results imply the existence of two different routes for targeting proteins into the peroxisomal matrix.     

cDNA cloning and expression of a gene for 3-ketoacyl-CoA thiolase in pumpkin cotyledons

A cDNA clone for 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was isolated from a gt11 cDNA library constructed from the poly(A)⁺ RNA of etiolated pumpkin cotyledons. The cDNA insert contained 1682 nucleotides and encoded 461 amino acid residues. A study of the expression in vitro of the cDNA and analysis of the amino-terminal sequence of the protein indicated that pumpkin thiolase is synthesized as a precursor which has a cleavable amino-terminal presequence of 33 amino acids. The amino-terminal presequence was highly homologous to typical amino-terminal signals that target proteins to microbodies. Immunoblot analysis showed that the amount of thiolase increased markedly during germination but decreased dramatically during the light-inducible transition of microbodies from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA increased temporarily during the early stage of germination. In senescing cotyledons, the levels of the thiolase mRNA and protein increased again with the reverse transition of microbodies from leaf peroxisomes to glyoxysomes, but the pattern of accumulation of the protein was slightly different from that of malate synthase. These results indicate that expression of the thiolase is regulated in a similar manner to that of other glyoxysomal enzymes, such as malate synthase and citrate synthase, during seed germination and post-germination growth. By contrast, during senescence, expression of the thiolase is regulated in a different manner from that of other glyoxysomal enzymes.     

Cloning and sequence determination of cDNA encoding a second rat liver peroxisomal 3-ketoacyl-CoA thiolase

3-Ketoacyl-coenzyme A thiolase (thiolase) catalyzes the final step of the fatty acid beta-oxidation pathway in peroxisomes. Thiolase is unique among rat liver peroxisomal enzymes in that it is synthesized as a precursor possessing a 26-amino acid (aa) N-terminal extension which is cleaved to generate the mature enzyme. To facilitate further examination of the synthesis, intracellular transport and processing of this enzyme, cDNA clones were selected from a lambda gt11 rat liver library using antiserum raised against peroxisomal thiolase. Upon sequencing several cDNA clones, it was revealed that there are at least two distinct thiolase enzymes localized to rat liver peroxisomes, one identical to the previously published rat liver peroxisomal thiolase (thiolase 1) [Hijikata et al., J. Biol. Chem. 262 (1987) 8151-8158] and a novel thiolase (thiolase 2). The THL2 cDNA possesses a single open reading frame of 1302 nucleotides (nt) encoding a protein of 434 aa (Mr 44790). The coding region of THL2 cDNA exhibits 94.6% nt sequence identity with THL1 and 95.4% identity at the level of aa sequence. Northern-blot analysis indicates that the mRNA encoding thiolase 2 is approx. 1.7 kb in size. The mRNA encoding thiolase 2 is induced approx. twofold upon treatment of rats with the peroxisome-proliferating drug, clofibrate. In contrast, the thiolase 1 mRNA is induced more than tenfold under similar conditions.     

Thiolase mRNA translated in vitro yields a peptide with a putative N-terminal presequence

Thiolase is part of the fatty acid oxidation machinery which in plants is located within glyoxysomes or peroxisomes. In cucumber cotyledons, proteolytic modification of thiolase takes place during the transfer of the cytosolic precursor into glyoxysomes prior to the intraorganellar assembly of the mature enzyme. This was shown by size comparison of the in vitro synthesized precursor and the 45 kDa subunit of the homodimeric glyoxysomal form. We isolated a full-length cDNA clone encoding the 48 539 Da precursor of thiolase. This plant protein displayed 40% and 47% identity with the precursor of fungal peroxisomal thiolase and human peroxisomal thiolase, respectively. Compared to bacterial thiolases, the precursor of the plant enzyme was distinguished by an N-terminal extension of 34 amino acid residues. This putative targeting sequence of cucumber thiolase shows similarities with the cleavable presequences of rat peroxisomal thiolase and plant peroxisomal malate dehydrogenase.     

Intraparticulate localization of some peroxisomal enzymes related to fatty acid beta-oxidation

Male Wistar rats were given a diet containing 0.05% (w/w) LK-903 (alpha-methyl-p-myristyroxycinnamic acid 1-monoglyceride) for 2 weeks. The activities of four hepatic peroxisomal enzymes involved in the fatty acyl-CoA beta-oxidizing system were determined. The activities of fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase were all increased about three times by administration of LK-903. The intraparticulate localizations of the four enzymes were then investigated by treatment of the purified peroxisomes with Triton X-100, by sonication, and by sucrose-density-gradient centrifugation after Triton X-100 treatment. The results suggest that thiolase is localized in the matrix of peroxisomes, that crotonase and beta-hydroxybutyryl-CoA dehydrogenase are located in the core, and that all or at least part of fatty acyl-CoA oxidase is associated with the core, though its association is weak.     

The NH2-terminal 14-16 amino acids of mitochondrial and bacterial thiolases can direct mature ornithine carbamoyltransferase into mitochondria

Unlike most mitochondrial matrix proteins, the mitochondrial 3-oxoacyl-CoA thiolase [EC 2.3.1.16] is synthesized with no cleavable presequence and possesses information for mitochondrial targeting and import in the mature protein. This mitochondrial thiolase is homologous with the mature portion of peroxisomal 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase [EC 2.3.1.9] of Zoogloea ramigera along the entire sequence. A hybrid gene encoding the NH2-terminal 16 residues (MALLRGVFIVAAKRTP) of the mitochondrial thiolase fused to the mature portion of rat ornithine carbamoyltransferase [EC 2.1.3.3] (lacking its own presequence) was transfected into COS cells, and subcellular localization of the fusion protein was analyzed. Cell fractionation and immunocytochemical analyses showed that the fusion protein was localized in the mitochondria. These results indicate that the NH2-terminal 16 residues of the mitochondrial thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. The fusion protein containing the NH2-terminal 14 residues (MSTPSIVIASARTA) of the bacterial thiolase was also localized in the mitochondria. On the other hand, the fusion protein containing the corresponding portion (MQASASDVVVVHGQRTP) of the peroxisomal thiolase appeared not to be localized to the mitochondria. These results show that the import signal of mitochondrial 3-oxoacyl-CoA thiolase originated from the NH2-terminal portion of the ancestral thiolase. The ancestral enzyme might have already possessed a mitochondrial import activity when mitochondria appeared first, or that it might have acquired the import activity during evolution by accumulation of point mutations in the NH2-terminal portion of the enzyme.     

Import of the peroxisomal targeting signal type 2 protein 3-ketoacyl-coenzyme a thiolase into glyoxysomes

Most peroxisomal matrix proteins possess a carboxy-terminal tripeptide targeting signal, termed peroxisomal targeting signal type 1 (PTS1), and follow a relatively well-characterized pathway of import into the organelle. The peroxisomal targeting signal type 2 (PTS2) pathway of peroxisomal matrix protein import is less well understood. In this study, we investigated the mechanisms of PTS2 protein binding and import using an optimized in vitro assay to reconstitute the transport events. The import of the PTS2 protein thiolase differed from PTS1 protein import in several ways. Thiolase import was slower than typical PTS1 protein import. Competition experiments with both PTS1 and PTS2 proteins revealed that PTS2 protein import was inhibited by addition of excess PTS2 protein, but it was enhanced by the addition of PTS1 proteins. Mature thiolase alone, lacking the PTS2 signal, was not imported into peroxisomes, confirming that the PTS2 signal is necessary for thiolase import. In competition experiments, mature thiolase did not affect the import of a PTS1 protein, but it did decrease the amount of radiolabeled full-length thiolase that was imported. This is consistent with a mechanism by which the mature protein competes with the full-length thiolase during assembly of an import complex at the surface of the membrane. Finally, the addition of zinc to PTS2 protein imports increased the level of thiolase bound and imported into the organelles.     

Properties of peroxisomal 3-ketoacyl-coA thiolase from rat liver

Peroxisomal 3-ketoacyl-CoA thiolase has a molecular weight of 89,000 and consists of 2 polypeptide chains of identical size. The enzyme has no interchain disulfide bonds and is reversibly dissociated to an inactive monomer in the cold. Mitochondrial 3-ketoacyl-CoA thiolase and acetoacetyl-CoA specific thiolase have molecular weights of 154,000 and 149,000, respectively. They each consist of 4 polypeptide chains of identical size. Peroxisomal thiolase and mitochondrial 3-ketoacyl-CoA thiolase operate by a ping-pong mechanism. The catalytic properties, including substrate specificity, of the peroxisomal enzyme were compared to those of mitochondrial 3-ketoacyl-CoA thiolase.     

Targeted disruption of the peroxisomal thiolase B gene in mouse: a new model to study disorders related to peroxisomal lipid metabolism

The peroxisomal beta-oxidation system consists of four steps catalysed by three enzymes: acyl-CoA oxidase, 3-hydroxyacyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (multifunctional enzyme) and thiolase. In humans, thiolase activity is encoded by one gene, whereas in rodents, three enzymes encoded by three distinct genes (i.e. thiolase A, thiolase B and SCP2/thiolase) catalyse the thiolase activity. So far, acyl-CoA oxidase- and multifunctional enzyme-deficient patients have been identified and knock-out mice for these genes have been produced. Conversely, no isolated thiolase-deficient patient has been found, and no thiolase (A or B)-deficient mice have been generated. Hence, to better understand the cause of isolated human thiolase deficiency, we disrupted the catalytic site of the mouse thiolase B by homologous recombination in order to analyse the phenotype of these thiolase B-deficient mice. Mice, made homozygous for the mutation, lack expression of thiolase B mRNA and are viable, fertile and healthy at birth. They exhibit no detectable phenotype defects and no compensation, rather a slight decrease in other peroxisomal thiolase (thiolase A and SCPx) mRNAs, was found.     

Pro-sterol carrier protein-2: role of the N-terminal presequence in structure, function, and peroxisomal targeting

Although the 20-amino acid presequence present in 15-kDa pro-sterol carrier protein-2 (pro-SCP-2, the precursor of the mature 13-kDa SCP-2) alters the function of SCP-2 in lipid metabolism, the molecular basis for this effect is unresolved. The presequence dramatically altered SCP-2 structure as determined by circular dichroism, mass spectroscopy, and antibody accessibility such that pro-SCP-2 had 3-fold less alpha-helix, 7-fold more beta-structure, 6-fold more reactive C terminus to carboxypeptidase A, 2-fold less binding of anti-SCP-2, and did not enhance sterol transfer from plasma membranes. These differences were not due to protein stability since (i) the same concentration of guanidine hydrochloride was required for 50% unfolding, and (ii) the ligand binding sites displayed the same high affinity (nanomolar K(d) values) in the order: cholesterol straight chain fatty acid > kinked chain fatty acid. Laser scanning confocal microscopy and double immunofluorescence demonstrated that pro-SCP-2 was more efficiently targeted to peroxisomes. Transfection of l-cells or McAR7777 hepatoma cells with cDNA encoding pro-SCP-2 resulted in 45% and 59% of SCP-2, respectively, colocalizing with the peroxisomal marker PMP70. In contrast, l-cells transfected with cDNA encoding SCP-2 exhibited 3-fold lower colocalization of SCP-2 with PMP70. In summary, the data suggest for the first time that the 20-amino acid presequence of pro-SCP-2 alters SCP-2 structure to facilitate peroxisomal targeting mediated by the C-terminal SKL peroxisomal targeting sequence.     

Zonal distribution of peroxisomal 3-oxoacyl-CoA thiolase mRNA in liver from rats treated with di-(2-ethylhexyl)phthalate

Treatment of rats with di-(2-ethylhexyl)phthalate leads to a dramatic increase in peroxisomal 3-oxoacyl-CoA thiolase RNA, the concentration being higher in the pericentral than in periportal hepatocytes. These findings indicate that the production of peroxisomal thiolase and the zonal distribution of the enzyme are regulated at a pretranslational level.     

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.     

Reinvestigation of peroxisomal 3-ketoacyl-CoA thiolase deficiency: identification of the true defect at the level of d-bifunctional protein

In this report, we reinvestigate the only patient ever reported with a deficiency of peroxisomal 3-ketoacyl-CoA thiolase (THIO). At the time when they were described, the abnormalities in this patient, which included accumulation of very-long-chain fatty acids and the bile-acid intermediate trihydroxycholestanoic acid, were believed to be the logical consequence of a deficiency of the peroxisomal beta-oxidation enzyme THIO. In light of the current knowledge of the peroxisomal beta-oxidation system, however, the reported biochemical aberrations can no longer be explained by a deficiency of this thiolase. In this study, we show that the true defect in this patient is at the level of d-bifunctional protein (DBP). Immunoblot analysis revealed the absence of DBP in postmortem brain of the patient, whereas THIO was normally present. In addition, we found that the patient had a homozygous deletion of part of exon 3 and intron 3 of the DBP gene, resulting in skipping of exon 3 at the cDNA level. Our findings imply that the group of single-peroxisomal beta-oxidation-enzyme deficiencies is limited to straight-chain acyl-CoA oxidase, DBP, and alpha-methylacyl-CoA racemase deficiency and that there is no longer evidence for the existence of THIO deficiency as a distinct clinical entity.