Topographical localization of peroxisomal acyl-CoA ligases: differential localization of palmitoyl-CoA and lignoceroyl-CoA ligases

We found that peroxisomal lignoceroyl-CoA ligase, like palmitoyl-CoA ligase, is present in the peroxisomal membrane whereas the peroxisomal beta-oxidation enzyme system is localized in the matrix. To further define the role of peroxisomal acyl-CoA ligases (membrane component) in providing acyl-CoA for peroxisomal beta-oxidation, we examined the transverse topographical localization of enzymatic sites of palmitoyl-CoA and lignoceroyl-CoA ligases in the peroxisomal membranes. The disruption of peroxisomes by various techniques resulted in the release of a "latent" pool of lignoceroyl-CoA ligase activity while palmitoyl-CoA ligase activity remained the same. Proteolytic enzyme treatment inhibited palmitoyl-CoA ligase activity in intact peroxisomes but had no effect on lignoceroyl-CoA ligase activity. Lignoceroyl-CoA ligase activity was inhibited only if peroxisomes were disrupted with detergent before trypsin treatment. Antibodies to palmitoyl-CoA ligase and to peroxisomal membrane proteins (PMP) inhibited palmitoyl-CoA ligase in intact peroxisomes, and no pool of "latent" activity appeared when antibody-treated peroxisomes were disrupted with detergent. On the other hand, disruption of PMP antibody-treated peroxisomes with detergent resulted in the appearance of a "latent" pool of lignoceroyl-CoA ligase activity. These results demonstrate that the enzymatic site of palmitoyl-CoA ligase is on the cytoplasmic surface whereas that for lignoceroyl-CoA ligase is on the luminal surface of peroxisomal membranes. This implies that palmitoyl-CoA is synthesized on the cytoplasmic surface and is then transferred to the matrix through the peroxisomal membrane for beta-oxidation in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro insertion of the 22-kD peroxisomal membrane protein into isolated rat liver peroxisomes

The membrane insertion of the 22-kD integral peroxisomal membrane protein (PMP 22) was studied in a system in which peroxisomes isolated from rat liver were incubated with the [35S]methionine-labeled in vitro translation product of PMP 22 mRNA. Membrane insertion of PMP 22 was demonstrated by protease treatment of peroxisomes in the absence and presence of detergent. Approximately 35% of total in vitro translated PMP 22 became protease resistant after a 1-h incubation at 26 degrees C. Import was dependent on time and temperature, did not require ATP or GTP and was not inhibited by N-ethylmaleimide treatment of neither the soluble components of the translation mixture nor of the isolated peroxisomes. In contrast to these results it was recently shown that the import of the peroxisomal marker, firefly luciferase, into peroxisomes of permeabilized cells was dependent on ATP hydrolysis and was blocked by N-ethylmaleimide pretreatment of the cytosol-depleted cells (Rapp et al., 1993; Wendland and Subramani, 1993). Therefore, the present data suggest that insertion of PMP 22 into the peroxisomal membrane and translocation of firefly luciferase into peroxisomes follow distinct mechanisms. At low temperature binding of PMP 22 to the peroxisomal membrane was not influenced whereas insertion was strongly inhibited. Pretreatment of peroxisomes with subtilisin reduced binding to a low level and completely abolished insertion. Therefore it is suggested that binding is prerequisite to insertion and that insertion may be mediated by a proteinaceous receptor.     

Localization of nitric-oxide synthase in plant peroxisomes

The presence of nitric-oxide synthase (NOS) in peroxisomes from leaves of pea plants (Pisum sativum L.) was studied. Plant organelles were purified by differential and sucrose density gradient centrifugation. In purified intact peroxisomes a Ca(2+)-dependent NOS activity of 5.61 nmol of L-[(3)H]citrulline mg(-1) protein min(-1) was measured while no activity was detected in mitochondria. The peroxisomal NOS activity was clearly inhibited (60-90%) by different well characterized inhibitors of mammalian NO synthases. The immunoblot analysis of peroxisomes with a polyclonal antibody against the C terminus region of murine iNOS revealed an immunoreactive protein of 130 kDa. Electron microscopy immunogold-labeling confirmed the subcellular localization of NOS in the matrix of peroxisomes as well as in chloroplasts. The presence of NOS in peroxisomes suggests that these oxidative organelles are a cellular source of nitric oxide (NO) and implies new roles for peroxisomes in the cellular signal transduction mechanisms.     

Suramin prevents import of acyl-CoA oxidase into rat liver peroxisomes.

The inhibitory effect of suramin on the import of [35S]acyl-CoA oxidase into purified rat liver peroxisomes was investigated in vitro. The import of acyl-CoA oxidase was inhibited completely by 10 microM suramin, whilst the latency of catalase remained unchanged. The important value decreased 60% by pretreatment of peroxisomes with 10 microM suramin, but it did not decrease by pretreatment of translation products. Polysulfonate compounds which have two clusters of negative charges, such as Cibacron blue F3GA and Trypan blue, as well as suramin, inhibited the import, whilst mono- and disulfonate compounds did not.     

Peroxisomes in disease.

Cytochemical, biochemical and morphological changes in peroxisomes have been described in human metabolic disorders, in experimental models of disease and in response to drugs and toxins. These include the cerebrohepatorenal syndromes, in which peroxisomes can not be detected and mitochondrial respiration is inhibited, atherosclerosis, alcoholic cardiomyopathy, and tolerance to oxygen toxicity. Although information on the role of peroxisomes in disease is limited, increased awareness of their widespread distribution and the availability of an improved cytochemical procedure for staining peroxisomes in human specimens should provide new insights into their function.     

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.     

Ultrastructural localization of L-alpha-hydroxy acid oxidase in rat liver perioxisomes.

The localization of L-alpha-hydroxy acid oxidase in rat liver peroxisomes was studied using slight modifications of the Shnitka and Talibi (1971) method. Best results were obtained with formaldehyde fixation and incubation with glycolate as substrate. Following incubation the copper ferrocyanide reaction product was amplified with 3,3'-diamino-benzidine according to Hanker et al. (1972a,b). Dense reaction product was visible in hepatocyte peroxisomes by light and electron microscopy. Some diffusion of enzyme and/or reaction product into the adjacent cytoplasm occurred around the peroxisomes. Apparent non-specific deposits occurred on the plasmalemma, in the nucleus, and occasionally over mitochondria. Glutaraldehyde fixation severely inhibited enzymatic activity, and the enzyme showed less activity toward L-lactate and DL-alpha-hydroxybutyrate.     

Translocation of acyl-CoA oxidase into peroxisomes requires ATP hydrolysis but not a membrane potential

An efficient system for the import of newly synthesized proteins into highly purified rat liver peroxisomes was reconstituted in vitro. 35S- Labeled acyl-CoA oxidase (AOx) was incorporated into peroxisomes in a proteinase K-resistant fashion. This import was specific (did not occur with mitochondria) and was dependent on temperature, time, and peroxisome concentration. Under optimal conditions approximately 30% of [35S]AOx became proteinase resistant. The import of AOx into peroxisomes could be dissociated into two steps: (a) binding occurred at 0 degrees C in the absence of ATP; (b) translocation occurred only at 26 degrees C and required the hydrolysis of ATP. GTP would not substitute for ATP and translocation was not inhibited by carbonylcyanide-m-chlorophenylhydrazone, valinomycin, or other ionophores.     

Conclusive evidence that very-long-chain fatty acids are oxidized exclusively in peroxisomes in human skin fibroblasts

We have investigated the contribution of peroxisomes and mitochondria to the beta-oxidation of palmitate (C16:0) and cerotate (C26:0) in intact human skin fibroblasts. The oxidation of both fatty acids was found to be inhibited by rotenone plus antimycin and cyanide, respectively, although to a different extent. When 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) was used to specifically block carnitine palmitoyltransferase I, it was found that palmitate beta-oxidation was inhibited almost completely whereas cerotate beta-oxidation was not affected. Since carnitine palmitoyltransferase is essential for the oxidation of fatty acids in mitochondria this result provides conclusive evidence that oxidation of very-long-chain fatty acids is initiated in peroxisomes and not in mitochondria.     

Peroxisomal fatty acyl-coenzyme A oxidation in chicken liver

The activities of antimycin A-insensitive palmitoyl-CoA oxidation and of palmitoyl-CoA oxidase in peroxisomes from chicken liver were similar to those of rat liver. Catalase and d-amino acid oxidase activities in peroxisomes from chicken liver were lower than those of rat liver and urate oxidase was not detected. Carnitine acetyltransferase and palmitoyltransferase levels in chicken liver were 18- and 2-fold higher, respectively, than those of rat liver. Peroxisomal palmitoyl-CoA oxidation of chicken liver was inhibited by cyanide, in contrast to that of rat liver, although it was insensitive to antimycin A. Subcellular distribution of this enzyme was similar to that of rat liver; i.e., it was located only in the peroxisomes. The fatty acyl-CoA oxidase had a higher affinity toward medium- to long-chain fatty acyl-CoAs (C₈ to C₁₆) than shorter-chain analogs. The fatty acyl-CoA dehydrogenase had a broad affinity toward fatty acyl-CoAs (C₄ to C₁₈). Carnitine acetyltransferase was distributed equally in both peroxisomes and mitochondria. Carnitine palmitoyltransferase was distributed in the proportion of 20 and 80% in peroxisomes and mitochondria, respectively.     

Effect of catalase-specific inhibitor 3-amino-1,2,4-triazole on yeast peroxisomal catalase in vivo

3-Amino-1,2,4-triazole (3-AT) is known as an inhibitor of catalase to whose active center it specifically and covalently binds. Subcellular fractionation and immunoelectronmicroscopic observation of the yeast Candida tropicalis revealed that, in 3-AT-treated cells in which the 3-AT was added to the n-alkane medium from the beginning of cultivation, catalase transported into peroxisomes was inactivated and was present as insoluble aggregated forms in the organelle. The aggregation of catalase in peroxisomes occurred only in these 3-AT-treated cells and not in cells in which 3-AT was added at the late exponential growth phase. Furthermore, 3-AT did not affect the transportation of catalase into peroxisomes. The appearance of aggregation only in cells to which 3-AT was added from the beginning of cultivation suggests that, in the process of catalase transportation into yeast peroxisomes, some conformational change may take place and that correct folding may be inhibited by the binding of 3-AT to the active center of catalase. Accordingly, 3-AT will be an interesting compound for investigation of the transport machinery of the peroxisomal tetrameric catalase.     

Long-chain-acyl-CoA synthetase and very-long-chain-acyl-CoA synthetase activities in peroxisomes and microsomes from rat liver. An enzymological study

We have investigated the palmitic acid (C16:0) and cerotic acid (C26:0) activating activities in rat-liver microsomes and peroxisomes. The activation of the two fatty acids showed similar dependencies on ATP and coenzyme A, reflected in about equal apparent Km values both in microsomes and peroxisomes. In microsomes and peroxisomes similar apparent Km values for palmitic acid were found (15 microM and 22.8 microM, respectively), whereas apparent Km values for cerotic acid were 8.4 microM and 1.0 microM in microsomes and peroxisomes, respectively. The activation of cerotic acid was found to be inhibited to a progressively greater extent by increasing concentrations of 1-pyrenedecanoic acid (P10) as compared to the activation of palmitic acid, both in microsomes and peroxisomes. The inhibition by P10 of palmitic acid activation and cerotic acid activation was non-competitive in both organelles. From the observation that P10 activation is not affected by palmitic acid and cerotic acid, we conclude that P10 is activated by a distinct enzyme. Furthermore, our results are in accordance with earlier suggestions that activation of cerotic acid is brought about by an enzyme distinct from the palmitoyl-CoA synthetase.     

Selective inhibition of hepatic peroxisomal fatty acid beta-oxidation by enoximone.

Although beta-oxidation of fatty acids occurs in both peroxisomes and mitochondria, beta-oxidizing enzymes in these organelles have distinct differences in their specifity and sensitivity to inhibitors. In this study, the effects of the phosphodiesterase inhibitor enoximone on hepatic peroxisomal and mitochondrial beta-oxidation were investigated. In liver homogenates from control rats, cyanide-insensitive peroxisomal beta-oxidation of palmitoyl-CoA was inhibited progressively by increasing concentrations of enoximone. Similar results were obtained in liver homogenates from rats pretreated with the known peroxisomal proliferator diethylhexylphthalate. In contrast, mitochondrial beta-oxidation of palmitoyl-CoA was not inhibited by enoximone. These data show that enoximone selectively inhibits basal as well as induced peroxisomal, but not mitochondrial, beta-oxidation of the CoA thioester of long-chain fatty acids. The availability of specific inhibitors of peroxisomal beta-oxidation should prove useful in elucidating regulatory mechanisms operative in this pathway in normal as well as in proliferated peroxisomes.     

Liver mitochondria, confirmed as intact by complete suppression of succinate uptake and oxidation, possess a carnitine palmitoyltransferase I that is totally inhibited by malonyl CoA.

Succinate dehydrogenase activity in mitochondria, which were isolated by centrifuging partially purified mitochondria through 1. 315 M sucrose, was completely suppressed when [14C]succinate uptake was abolished by prior incubation of the mitochondria with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and valinomycin. The conclusion that these mitochondria were intact was confirmed by the fact that, when these mitochondria were broken by a freeze-thaw cycle followed by sonication, such inhibition was totally abolished. The yield of mitochondria, microsomes, and peroxisomes from the initial homogenate was 17.8, <0.1, and 0%, respectively, indicating that the mitochondria were not only intact but also essentially free of contamination from microsomes and peroxisomes. The overt form of carnitine palmitoyltransferase (CPT I) in these intact and pure mitochondria was totally inhibited by malonyl CoA, indicating that previous reports of incomplete inhibition in mitochondrial preparations resulted from interference from CPT activity in the inner mitochondrial membrane (CPT II), microsomes, or peroxisomes.     

Peroxisomal chain-shortening of prostaglandin F2 alpha

We have recently reported that prostaglandin F2 alpha can be chain-shortened by isolated rat liver peroxisomes. In the present study it is further established by cell fractionation experiments that the enzymes involved in this reaction are localized to peroxisomes. Under the conditions employed, the highest activity was found in the light mitochondrial fraction. Further fractionation of the light mitochondrial fraction by sucrose density gradient centrifugation showed that the prostaglandin oxidation activity comigrated with peroxisomal marker enzymes. Di(2-ethylhexyl)phthalate treatment resulted in a tenfold increased capacity for the conversion of prostaglandin F2 alpha into tetranorprostaglandin F1 alpha. The reaction was not inhibited by KCN. The reaction was further characterized with respect to cofactor requirements. The prostaglandin oxidation was found to be completely dependent on NAD, CoA, ATP, Mg2+ and was stimulated by FAD. Incubation of prostaglandin E2 with peroxisomes resulted in conversion into several products. After alkaline hydrolysis, one of these was identified as tetranorprostaglandin B1.     

Induction of tubular peroxisomes by UV irradiation and reactive oxygen species in HepG2 cells.

Peroxisomes in the human hepatoblastoma cell line HepG2 exhibit a high degree of plasticity. Whereas in confluent cultures they appear as small (0.1-0.3 micrometer) spherical particles, they undergo dramatic changes, forming elongated tubules measuring up to 5 micrometer on separation of cells and cultivation at low density. We recently showed that several growth factors, including nerve growth factor (NGF), induce the formation of tubular peroxisomes and that this induction is sensitive to K 252b, a specific tyrosine kinase inhibitor, suggesting the involvement of this signal transduction pathway. Because tyrosine kinase is also involved in signal transduction via the reactive oxygen species (ROS), we have analyzed in this study the effects of UV irradiation, H(2)O(2), and oxygen on tubulation of peroxisomes. UVC irradiation induced a significant increase in formation of tubular peroxisomes (40-50% of cells) and this effect was dose-dependently inhibited by pretreatment with N-acetyl cysteine, confirming the involvement of ROS in the UV effect. Furthermore, H(2)O(2) also directly induced the tubulation of peroxisomes, although to a lesser extent. Finally, cultivation under hypoxic conditions (1.5% O(2)) drastically reduced the inducing effect of fetal calf serum on tubulation of peroxisomes, suggesting the involvement of oxygen-mediated signaling. Taken together, our observations indicate that ROS induce the tubulation of peroxisomes in HepG2 cells. Because peroxisomes harbor most of the enzymes for catabolism of ROS, the tubulation and expansion of the peroxisome compartment could have a cell rescue function against the destructive effects of ROS.     

Peroxisome degradation in mammals

This review summarizes the historical aspects of the study of peroxisome degradation in mammalian cells. Peroxisomes have diverse metabolic roles in response to environmental changes and are degraded in a preferential manner, by comparison with cytosolic proteins. This review introduces three hypotheses on the degradation mechanisms: (a) the action of the peroxisome-specific Lon protease; (b) the membrane disruption effect of 15-lipoxygenase; and (c) autophagy that sequesters and degrades the organelles by lysosomal enzymes. Among these hypotheses, autophagy is now recognized as the most important mechanism for excess peroxisome degradation. One of the most striking characteristics of peroxisomes is that they are markedly proliferated in the liver by the administration of hypolipidemic drugs and industrial plasticizers. The effects of these substances were fully reversed after withdrawal of the substances, and most of the excess peroxisomes were selectively degraded and recovered to a normal number and size. Autophagic degradation of peroxisomes has been examined using this characteristic phenomenon. Excessive peroxisome degradation that occurs after cessation of hypolipidemic drugs has been extensively investigated biochemically and morphologically. The evidence shows that the degradation of excess peroxisomes and peroxisomal enzymes is inhibited by 3-methyladenine (3-MA), a specific inhibitor of autophagy. Furthermore, in liver-specific autophagy-deficient mice, rapid removal of peroxisomes was exclusively impaired, and degradation of peroxisomal enzymes was not detected. Thus, the significant contribution of autophagic machinery to peroxisomal degradation in mammals was confirmed. However, the important question of the mechanism for the selective recognition of peroxisomes by autophagosomes remains to be fully elucidated.     

Adrenoleukodystrophy: impaired oxidation of fatty acids due to peroxisomal lignoceroyl-CoA ligase deficiency

Very long chain fatty acids (lignoceric acid) are oxidized in peroxisomes and pathognomonic amounts of these fatty acids accumulate in X-adrenoleukodystrophy (X-ALD) due to a defect in their oxidation. However, in cellular homogenates from X-ALD cells, lignoceric acid is oxidized at a rate of 38% of control cells. Therefore, to identify the source of this residual activity we raised antibody to palmitoyl-CoA ligase and examined its effect on the activation and oxidation of palmitic and lignoceric acids in isolated peroxisomes from control and X-ALD fibroblasts. The normalization of peroxisomal lignoceric acid oxidation in the presence of exogenously added acyl-CoA ligases and along with the complete inhibition of activation and oxidation of palmitic and lignoceric acids in peroxisomes from X-ALD by antibody to palmitoyl-CoA ligase provides direct evidence that lignoceroyl-CoA ligase is deficient in X-ALD and demonstrates that the residual activity for the oxidation of lignoceric acid was derived from the activation of lignoceric acid by peroxisomal palmitoyl-CoA ligase. This antibody inhibited the activation and oxidation of palmitic acid but had little effect on these activities for lignoceric acid in peroxisomes from control cells. Furthermore, these data provide evidence that peroxisomal palmitoyl-CoA and lignoceroyl-CoA ligases are two different enzymes.     

Cellular oxidation of lignoceric acid is regulated by the subcellular localization of lignoceroyl-CoA ligases

The acyl-CoA ligases convert free fatty acids to acyl-CoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum. Because their activity is obligatory for fatty acid metabolism, it is important to identify their substrate specificities and subcellular distributions to further understand the cellular regulation of these pathways. To define the role of the enzymes and organelles involved in the metabolism of very long chain (VLC) fatty acids, we studied human genetic cell mutants impaired for the metabolism of these molecules. Fibroblast cell lines were derived from patients with X-linked adrenoleukodystrophy (X-ALD) and Zellweger's cerebro-hepato-renal syndrome (CHRS). While peroxisomes are present and morphologically normal in X-ALD, they are either greatly reduced in number or absent in CHRS. Palmitoyl-CoA ligase is known to be present in mitochondria, peroxisomes, and endoplasmic reticulum (microsomes). We found enzyme-dependent formation of lignoceroyl-CoA in these same organelles (specific activities were 0.32 +/- 0.12, 0.86 +/- 0.12, and 0.78 +/- 0.07 nmol/h per mg protein, respectively). However, lignoceroyl-CoA synthesis was inhibited by an antibody to palmitoyl-CoA ligase in isolated mitochondria while it was not inhibited in peroxisomes or endoplasmic reticulum (ER). This suggests that palmitoyl-CoA ligase and lignoceroyl-CoA are different enzymes and that mitochondria lack lignoceroyl-CoA ligase. This conclusion is further supported by data showing that oxidation of lignoceric acid was found almost exclusively in peroxisomes (0.17 nmol/h per mg protein) but was largely absent from mitochondria and the finding that monolayers of CHRS fibroblasts lacking peroxisomes showed a pronounced deficiency in lignoceric acid oxidation in situ (1.8% of control). In spite of the observation that lignoceroyl-CoA ligase activity is present on the cytoplasmic surface of ER, our data indicate that lignoceroyl-CoA synthesized by ER is not available for oxidation in mitochondria. This organelle plays no physiological role in the beta-oxidation of VLC fatty acids. Furthermore, the normal peroxisomal oxidation of lignoceroyl-CoA but deficient oxidation of lignoceric acid in X-ALD cells indicates that cellular VLC fatty acid oxidation is dependent on peroxisomal lignoceroyl-CoA ligase. These studies allow us to propose a model for the subcellular localization of various acyl-CoA ligases and to describe how these enzymes control cellular fatty acid metabolism.     

The Arabidopsis pex12 and pex13 mutants are defective in both PTS1- and PTS2-dependent protein transport to peroxisomes

Peroxisome biogenesis requires various complex processes including organelle division, enlargement and protein transport. We have been studying a number of Arabidopsis apm mutants that display aberrant peroxisome morphology. Two of these mutants, apm2 and apm4, showed green fluorescent protein fluorescence in the cytosol as well as in peroxisomes, indicating a decrease of efficiency of peroxisome targeting signal 1 (PTS1)-dependent protein transport to peroxisomes. Interestingly, both mutants were defective in PTS2-dependent protein transport. Plant growth was more inhibited in apm4 than apm2 mutants, apparently because protein transport was more severely decreased in apm4 than in apm2 mutants. APM2 and APM4 were found to encode proteins homologous to the peroxins PEX13 and PEX12, respectively, which are thought to be involved in transporting matrix proteins into peroxisomes in yeasts and mammals. We show that APM2/PEX13 and APM4/PEX12 are localized on peroxisomal membranes, and that APM2/PEX13 interacts with PEX7, a cytosolic PTS2 receptor. Additionally, a PTS1 receptor, PEX5, was found to stall on peroxisomal membranes in both mutants, suggesting that PEX12 and PEX13 are components that are involved in protein transport on peroxisomal membranes in higher plants. Proteins homologous to PEX12 and PEX13 have previously been found in Arabidopsis but it is not known whether they are involved in protein transport to peroxisomes. Our findings reveal that APM2/PEX13 and APM4/PEX12 are responsible for matrix protein import to peroxisomes in planta.