Structural and metabolic requirements for activators of the peroxisome proliferator-activated receptor

Fatty acids have recently been demonstrated to activate peroxisome proliferator-activated receptors (PPARs) but specific structural requirements of fatty acids to produce this response have not yet been determined. Importantly, it has hitherto not been possible to show specific binding of these compounds to PPAR. To test whether a common PPAR binding metabolite might be formed, we tested the effects of long-chain omega-3 polyunsaturated fatty acids, differentially beta-oxidizable fatty acids and inhibitors of fatty acid metabolism. We determined the activation of a reporter gene by a chimaeric receptor encompassing the DNA binding domain of the glucocorticoid receptor and the ligand binding domain of PPAR. The omega-3 unsaturated fatty acids were slightly more potent PPAR activators in vitro than saturated fatty acids. The peroxisomal proliferation-inducing, non-beta-oxidizable, tetradecylthioacetic acid activated PPAR to the same extent as the strong peroxisomal proliferator WY 14,643, whereas the homologous beta-oxidizable tetradecylthiopropionic acid was only as potent as a non-substituted fatty acid. Cyclooxygenase inhibitors, radical scavengers or cytochrome P450 inhibitors did not affect activation of PPAR. In conclusion, beta-oxidation is apparently not required for the formation of the PPAR-activating molecule and this moiety might be a fatty acid, its ester with CoA, or a further derivative of the activated fatty acid prior to beta-oxidation of the acyl-CoA ester. These data should aid understanding of signal transduction via PPAR and the identification of a receptor ligand.     

The Saccharomyces cerevisiae FAT1 gene encodes an acyl-CoA synthetase that is required for maintenance of very long chain fatty acid levels

The Saccharomyces cerevisiae FAT1 gene appears to encode an acyl-CoA synthetase that is involved in the regulation of very long chain (C20-C26) fatty acids. Fat1p, has homology to a rat peroxisomal very long chain fatty acyl-CoA synthetase. Very long chain acyl-CoA synthetase activity is reduced in strains containing a disrupted FAT1 gene and is increased when FAT1 is expressed in insect cells under control of a baculovirus promoter. Fat1p accounts for approximately 90% of the C24-specific acyl-CoA synthetase activity in glucose-grown cells and approximately 66% of the total activity in cells grown under peroxisomal induction conditions. Localization of functional Fat1p:green fluorescent protein gene fusions and subcellular fractionation of C24 acyl-CoA synthetase activities indicate that the majority of Fat1p is located in internal cellular locations. Disruption of the FAT1 gene results in the accumulation of very long chain fatty acids in the sphingolipid and phospholipid fractions. This includes a 10-fold increase in C24 acids and a 6-fold increase in C22 acids. These abnormal accumulations are further increased by perturbation of very long chain fatty acid synthesis. Overexpression of Elo2p, a component of the fatty acid elongation system, in fat1Delta cells causes C20-C26 levels to rise to approximately 20% of the total fatty acids. These data suggest that Fat1p is involved in the maintenance of cellular very long chain fatty acid levels, apparently by facilitating beta-oxidation of excess intermediate length (C20-C24) species. Although fat1Delta cells were reported to grow poorly in oleic acid-supplemented medium when fatty acid synthase activity is inactivated by cerulenin, fatty acid import is not significantly affected in cells containing disrupted alleles of FAT1 and FAS2 (a subunit of fatty acid synthase). These results suggest that the primary cause of the growth-defective phenotype is a failure to metabolize the incorporated fatty acid rather than a defect in fatty acid transport. Certain fatty acyl-CoA synthetase activities, however, do appear to be essential for bulk fatty acid transport in Saccharomyces. Simultaneous disruption of FAA1 and FAA4, which encode long chain (C14-C18) fatty acyl-CoA synthetases, effectively blocks the import of long chain saturated and unsaturated fatty acids.     

Role of hepatic fatty acid:coenzyme A ligases in the metabolism of xenobiotic carboxylic acids

1. Formation of acyl-coenzymes (Co)A occurs as an obligatory step in the metabolism of a variety of endogenous substrates, including fatty acids. The reaction is catalysed by ATP-dependent acid:CoA ligases (EC 6.2.1.1-2.1.3; AMP forming), classified on the basis of their ability to conjugate saturated fatty acids of differing chain lengths, short (C2-C4), medium (C4-C12) and long (C10-C22). The enzymes are located in various cell compartments (cytosol, smooth endoplasmic reticulum, mitochondria and peroxisomes) and exhibit wide tissue distribution, with highest activity associated with liver and adipose tissue. 2. Formation of acyl-CoA is not unique to endogenous substrates, but also occurs as an obligatory step in the metabolism of some xenobiotic carboxylic acids. The mitochondrial medium-chain CoA ligase is principally associated with metabolism via amino acid conjugation and activates substrates such as benzoic and salicylic acids. Although amino acid conjugation was previously considered an a priori route of metabolism for xenobiotic-CoA, it is now recognized that these highly reactive and potentially toxic intermediates function as alternative substrates in pathways of intermediary metabolism, particularly those associated with lipid biosyntheses. 3. In addition to a role in fatty acid metabolism, the hepatic microsomal and peroxisomal long-chain-CoA-ligases have been implicated in the formation of the acyl-CoA thioesters of a variety of hypolipidaemic and peroxisome proliferating agents (e.g. clofibric acid) and of the R(-)-enantiomers of the commonly used 2-arylpropionic acid non-steroidal anti-inflammatory drugs (e.g. ibuprofen). In vitro kinetic studies using rat hepatic microsomes and peroxisomes have alluded to the possibility of xenobiotic-CoA ligase multiplicity. Although cDNA encoding a long-chain ligase have been isolated from rat and human liver, there is currently no molecular evidence of multiple isoforms. The gene has been localized to chromosome 4 and homology searches have revealed a significant similarity with enzymes of the luciferase family. 4. Increasing recognition that formation of a CoA conjugate increases chemical reactivity of xenobiotic carboxylic acids has led to an awareness that the relative activity, substrate specificity and intracellular location of the xenobiotic-CoA ligases may explain differences in toxicity. 5. Continued characterization of the human xenobiotic-CoA ligases in terms of substrate/inhibitor profiles and regulation, will allow a greater understanding of the role of these enzymes in the metabolism of carboxylic acids.     

Expression of putative fatty acid transporter genes are regulated by peroxisome proliferator-activated receptor alpha and gamma activators in a tissue- and inducer-specific manner

Regulation of gene expression of three putative long-chain fatty acid transport proteins, fatty acid translocase (FAT), mitochondrial aspartate aminotransferase (mAspAT), and fatty acid transport protein (FATP), by drugs that activate peroxisome proliferator-activated receptor (PPAR) alpha and gamma were studied using normal and obese mice and rat hepatoma cells. FAT mRNA was induced in liver and intestine of normal mice and in hepatoma cells to various extents only by PPARalpha-activating drugs. FATP mRNA was similarly induced in liver, but to a lesser extent in intestine. The induction time course in the liver was slower for FAT and FATP mRNA than that of an mRNA encoding a peroxisomal enzyme. An obligatory role of PPARalpha in hepatic FAT and FATP induction was demonstrated, since an increase in these mRNAs was not observed in PPARalpha-null mice. Levels of mAspAT mRNA were higher in liver and intestine of mice treated with peroxisome proliferators, while levels in hepatoma cells were similar regardless of treatment. In white adipose tissue of KKAy obese mice, thiazolidinedione PPARgamma activators (pioglitazone and troglitazone) induced FAT and FATP more efficiently than the PPARalpha activator, clofibrate. This effect was absent in brown adipose tissue. Under the same conditions, levels of mAspAT mRNA did not change significantly in these tissues. In conclusion, tissue-specific expression of FAT and FATP genes involves both PPARalpha and -gamma. Our data suggest that among the three putative long-chain fatty acid transporters, FAT and FATP appear to have physiological roles. Thus, peroxisome proliferators not only influence the metabolism of intracellular fatty acids but also cellular uptake, which is likely to be an important regulatory step in lipid homeostasis.     

A new colorimetric method for the determination of free fatty acids with acyl-CoA synthetase and acyl-CoA oxidase

A rapid and sensitive spectrophotometric assay for free fatty acids using acyl-CoA synthetase and acyl-CoA oxidase is described. It is sensitive to as low as 5 nmol of free fatty acids, and the standard curve is linear up to 100 nmol. The assay consists of the measurement of H2O2 produced from free fatty acids by acyl-CoA synthetase and acyl-CoA oxidase. The quantity of H2O2 is determined by the absorbance at 550 nm in the presence of catalase and 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (AHMT). This method shows a broad specificity to long-chain fatty acids and the recoveries of added fatty acids (C12-C18) are more than 90%. The presence or absence of serum components or Escherichia coli cell-free extracts has no significant effect on the recovery of added palmitic acid.     

New insights in peroxisomal beta-oxidation. Implications for human peroxisomal disorders

In mammals including man, peroxisomes play a pivotal role in the breakdown of various carboxylates via beta-oxidation. Physiological substrates include very long chain fatty acids (e.g. lignoceric acid), medium and long chain dicarboxylic acids, certain polyunsaturated fatty acids, 2-methylbranched isoprenoid-derived fatty acids (e.g. pristanic acid), prostanoids (prostaglandins, leukotrienes thromboxanes), and bile acid intermediates (di- and trihydroxycoprostanic acid). Substrate spectrum and specificity studies of the four different beta-oxidation steps in rat and man indicate that these carboxylates, in contrast to previous belief, are degraded by separate systems composed of different enzymes. Bile acid intermediates are degraded in hepatic peroxisomes via 2-methylacyl-CoA racemase, trihydroxycoprostanoyl-CoA oxidase (in rat) or branched acyl-CoA oxidase (in man), D-specific multifunctional protein 2 (MFP 2) and sterol carrier protein X/thiolase. beta-oxidation of pristanic acid can occur in all tissues and relies on the action of 2-methylacyl-CoA racemase (for the 2R-isomer), pristanoyl-CoA oxidase (in rat) or branched chain acyl-CoA oxidase (in man), D-specific multifunctional protein 2 (MFP 2) and sterol carrier protein X/thiolase. The enzymes catalyzing the breakdown of straight chain fatty acids are palmitoyl-CoA oxidase, L-specific multifunctional protein 1 (MFP 1) and the dimeric thiolase. These enzymes are present in all tissues and are identical to those initially characterized in hepatic peroxisomes. Due to the presence of peroxisome targeting signals in all the above mentioned proteins, they are localised in the cytosolic or absent (due to proteolysis) in tissues of patients with a generalized peroxisome deficiency (e.g. Zellweger syndrome). In addition to these lethal inherited disorders that are caused by defects in the biogenesis of peroxisomes, a growing number of patients with peroxisomal beta-oxidation deficiencies have been described. The implications of the presence of separate beta-oxidation systems for the latter disorders is quite profound and calls, in many cases, for a reevaluation of the diagnosis of such patients.     

Steatohepatitis, spontaneous peroxisome proliferation and liver tumors in mice lacking peroxisomal fatty acyl-CoA oxidase. Implications for peroxisome proliferator-activated receptor alpha natural ligand metabolism

Peroxisomal beta-oxidation system consists of four consecutive reactions to preferentially metabolize very long chain fatty acids. The first step of this system, catalyzed by acyl-CoA oxidase (AOX), converts fatty acyl-CoA to 2-trans-enoyl-CoA. Herein, we show that mice deficient in AOX exhibit steatohepatitis, increased hepatic H2O2 levels, and hepatocellular regeneration, leading to a complete reversal of fatty change by 6 to 8 months of age. The liver of AOX-/- mice with regenerated hepatocytes displays profound generalized spontaneous peroxisome proliferation and increased mRNA levels of genes that are regulated by peroxisome proliferator-activated receptor alpha (PPARalpha). Hepatic adenomas and carcinomas develop in AOX-/- mice by 15 months of age due to sustained activation of PPARalpha. These observations implicate acyl-CoA and other putative substrates for AOX, as biological ligands for PPARalpha; thus, a normal AOX gene is indispensable for the physiological regulation of PPARalpha.     

A new peroxisomal beta-oxidation disorder in twin neonates: defective oxidation of both cerotic and pristanic acids

Twin brothers were born with clinical symptoms indicating that they were suffering from Zellweger syndrome. However, instead of a generalized peroxisomal dysfunction, only very long-chain fatty acids and the pristanic acid/phytanic acid ratio were elevated in plasma and decreased oxidation of very long-chain fatty acids and pristanic acid was the only impairment found in fibroblasts. The other peroxisomal parameters tested were normal, including normal oxidation of phytanic acid and normal activity of dihydroxyacetonephosphate acyltransferase in fibroblasts as well as normal plasma bile acids. Although the biochemical results point to a defect in peroxisomal beta-oxidation, the isolated finding of impaired oxidation of very long-chain fatty acids and pristanic acid has to our knowledge not been reported previously and is difficult to explain by a deficiency of a known peroxisomal beta-oxidation enzyme.     

In vitro inhibition of carnitine acyltransferase activity in mitochondria from rat and mouse liver by a diethylhexylphthalate metabolite

The effects of mono(2-ethyl-5-oxohexyl)phthalate [ME(O)HP], a di(2-ethylhexyl)phthalate (DEHP) metabolite and a potent peroxisomal inducer, on the mitochondrial beta-oxidation were investigated. In isolated rat hepatocytes, ME(O)HP inhibited long chain fatty acid oxidation and had no effect on the ketogenesis of short chain fatty acids, suggesting that the inhibition occurred at the site of carnitine-dependent transport across the mitochondrial inner membrane. In rat liver mitochondria, ME(O)HP inhibited carnitine acyltransferase I (CAT I; EC 2.3.1.21) competitively with the substrates palmitoyl-CoA and octanoyl-CoA. An analogous treatment of mouse mitochondria produced a similar competitive inhibition of palmitoyl-CoA transport whereas ME(O)HP exposure with guinea pig and human liver mitochondria revealed little or no effect. The addition of clofibric acid, nafenopin or methylclofenopate revealed no direct effects upon CAT I activity. Inhibition of transferase activity by ME(O)HP was reversed in mitochondria which had been solubilized with octyl glucoside to expose the latent form of carnitine acyltransferase (CAT II), suggesting that the inhibition was specific for CAT I. Our results demonstrate that in vitro ME(O)HP inhibits fatty acid oxidation in rat liver at the site of transport across the mitochondrial inner membrane with a marked species difference and support the idea that induction of peroxisome proliferation could be due to an initial biochemical lesion of the fatty acid metabolism.     

Partial purification and properties of the fatty acid elongation systems in the outer and inner membranes of beef liver mitochondria

Purified outer membrane of beef liver mitochondria was found to elongate medium chain fatty acyl-CoA primer by the incorporation of [1-14C]acetyl-CoA. This enzymic activity, extracted by Triton X-100, was purified 8-fold by ammonium sulfate fractionation followed by chromatography on a Sephadex column. Purified inner membrane, when processed through an identical purification procedure, yielded a second enzyme system which incorporated [1-14C]acetyl-CoA into long chain fatty acids in the presence of medium chain fatty acyl-CoA primer. This enzyme preparation was about four times as active as the preparation from the outer membrane, and used NADH as the reductant for the synthesis. The molecular weights of the inner and the outer membrane enzyme systems, estimated by gel filtration as well as sucrose density gradient centrifugation, were approx. 57 000 and 126 000, respectively. The partially purified outer membrane enzyme system required NADH and a medium chain acyl-CoA primer for the incorporation of [1-14C]acetyl-CoA into long chain fatty acids. KNC stimulated the reaction. NADPH could substitute for NADH only to a limited extent. Malonyl-CoA was ineffective as a substrate in this reaction. The optimum pH of the reaction was 7.2-7.6 in 0.1 M potassium phosphate buffer. Dithiothreitol, beta-mercaptoethanol, N-ethylmaleimide and high concentrations of ATP and acyl-CoA primer inhibited the reaction. The specificity for the acyl-CoA primer in the reaction was very broad. All the primers tested, C8 to C16, incorporated acetyl-CoA significantly. However, maximum incorporation was observed with dodecanoyl-CoA. Decanoyl-CoA was the best primer for the enzyme system isolated from the inner membrane. About 42% of the radioactivity in the fatty acids synthesized by the outer membrane enzyme system, from myristoyl-CoA and [1-C14]acetyl-CoA, was in palmitic acid. Of the remaining activity, 41% was in stearic acid and about 38% in longer-chain acids. Hence, the elongation of the primer fatty acid by one C2 unit appeared to be the predominant process in this synthesis. In the elongation of myristoyl-C0A by the inner membrane enzyme system, palmitic acid which constituted nearly 78% of the fatty acids synthesized, was the primary product.     

IDP3 encodes a peroxisomal NADP-dependent isocitrate dehydrogenase required for the beta-oxidation of unsaturated fatty acids

In Saccharomyces cerevisiae the metabolic degradation of saturated fatty acids is exclusively confined to peroxisomes. In addition to a functional beta-oxidation system, the degradation of unsaturated fatty acids requires auxiliary enzymes, including a Delta2, Delta3-enoyl-CoA isomerase and an NADPH-dependent 2,4-dienoyl-CoA reductase. We found both enzymes to be present in yeast peroxisomes. The impermeability of the peroxisomal membrane for pyrimidine nucleotides led to the question of how the NADPH needed by the reductase is regenerated in the peroxisomal lumen. We report the identification and functional analysis of the IDP3 gene product, which is a yeast peroxisomal NADP-dependent isocitrate dehydrogenase. The newly identified peroxisomal protein is homologous to the mitochondrial Idp1p and cytosolic Idp2p, which both are yeast NADP-dependent isocitrate dehydrogenases. Yeast cells lacking Idp3p grow normally on saturated fatty acids, but growth is impaired on unsaturated fatty acids, indicating that the peroxisomal Idp3p is involved in their metabolic utilization. The data presented are consistent with the assumption that peroxisomes of S. cerevisiae contain the enzyme equipment needed for the degradation of unsaturated fatty acids, including an NADP-dependent isocitrate dehydrogenase, a putative constituent of a peroxisomal NADPH-regenerating redox system.     

Localization of nervonic acid beta-oxidation in human and rodent peroxisomes: impaired oxidation in Zellweger syndrome and X-linked adrenoleukodystrophy

Studies with purified subcellular organelles from rat liver indicate that nervonic acid (C24:1) is beta-oxidized preferentially in peroxisomes. Lack of effect by etomoxir, inhibitor of mitochondrial beta-oxidation, on beta-oxidation of lignoceric acid (C24:0), a peroxisomal function, and that of nervonic acid (24:1) compared to the inhibition of palmitic acid (16:0) oxidation, a mitochondrial function, supports the conclusion that nervonic acid is oxidized in peroxisomes. Moreover, the oxidation of nervonic and lignoceric acids was deficient in fibroblasts from patients with defects in peroxisomal beta-oxidation [Zellweger syndrome (ZS) and X-linked adrenoleukodystrophy (X-ALD)]. Similar to lignoceric acid, the activation and beta-oxidation of nervonic acid was deficient in peroxisomes isolated from X-ALD fibroblasts. Transfection of X-ALD fibroblasts with human cDNA encoding for ALDP (X-ALD gene product) restored the oxidation of both nervonic and lignoceric acids, demonstrating that the same molecular defect may be responsible for the abnormality in the oxidation of nervonic as well as lignoceric acid. Moreover, immunoprecipitation of activities for acyl-CoA ligase for both lignoceric acid and nervonic acid indicate that saturated and monoenoic very long chain (VLC) fatty acids may be activated by the same enzyme. These results clearly demonstrate that similar to saturated VLC fatty acids (e.g., lignoceric acid), VLC monounsaturated fatty acids (e.g., nervonic acid) are oxidized preferentially in peroxisomes and that this activity is impaired in X-ALD. In view of the fact that the oxidation of unsaturated VLC fatty acids is defective in X-ALD patients, the efficacy of dietary monoene therapy, "Lorenzo's oil," in X-ALD needs to be evaluated.     

Targeted deletion of the PEX2 peroxisome assembly gene in mice provides a model for Zellweger syndrome, a human neuronal migration disorder

Zellweger syndrome is a peroxisomal biogenesis disorder that results in abnormal neuronal migration in the central nervous system and severe neurologic dysfunction. The pathogenesis of the multiple severe anomalies associated with the disorders of peroxisome biogenesis remains unknown. To study the relationship between lack of peroxisomal function and organ dysfunction, the PEX2 peroxisome assembly gene (formerly peroxisome assembly factor-1) was disrupted by gene targeting. Homozygous PEX2-deficient mice survive in utero but die several hours after birth. The mutant animals do not feed and are hypoactive and markedly hypotonic. The PEX2-deficient mice lack normal peroxisomes but do assemble empty peroxisome membrane ghosts. They display abnormal peroxisomal biochemical parameters, including accumulations of very long chain fatty acids in plasma and deficient erythrocyte plasmalogens. Abnormal lipid storage is evident in the adrenal cortex, with characteristic lamellar-lipid inclusions. In the central nervous system of newborn mutant mice there is disordered lamination in the cerebral cortex and an increased cell density in the underlying white matter, indicating an abnormality of neuronal migration. These findings demonstrate that mice with a PEX2 gene deletion have a peroxisomal disorder and provide an important model to study the role of peroxisomal function in the pathogenesis of this human disease.     

Early modulation of genes encoding peroxisomal and mitochondrial beta-oxidation enzymes by 3-thia fatty acids

The aim of the present study was to elucidate the effects of a single dose of 3-thia fatty acids (tetradecylthioacetic acid and 3-thiadicarboxylic acid) over a 24-hr study period on the expression of genes related to peroxisomal and mitochondrial beta-oxidation in liver of rats. The plasma triglyceride level decreased at 2-4 hr, 4-8 hr, and 8-24 hr, respectively, after a single dose of 150, 300, or 500 mg of 3-thia fatty acids/kg body weight. Four to eight hours after administration of 3-thia fatty acids, a several-fold-induced gene expression of peroxisomal multifunctional protein, fatty acyl-CoA oxidase (EC 1.3.3.6), fatty acid binding protein, and 2,4-dienoyl-CoA reductase (EC 1.3.1.43) resulted, concomitant with increased activity of 2,4-dienoyl-CoA reductase and fatty acyl-CoA oxidase. The expression of carnitine palmitoyltransferase-I and carnitine palmitoyltransferase-II increased at 2 and 4 hr, respectively, although at a smaller scale. In cultured hepatocytes, 3-thia fatty acids stimulated fatty acid oxidation after 4 hr, and this was both L-carnitine- and L-aminocarnitine-sensitive. The hepatic content of eicosapentaenoic acid and docosahexaenoic acid decreased throughout the study period. In contrast, the hepatic content of oleic acid tended to increase after 24 hr and was significantly increased after repeated administration of 3-thia fatty acids. Similarly, the expression of delta9-desaturase was unchanged during the 24-hr study, but increased after feeding for 5 days. To conclude, carnitine palmitoyltransferase-I expression seemed to be induced earlier than 2,4-dienoyl-CoA reductase and fatty acid binding protein, and not later than the peroxisomal fatty acyl-CoA oxidase. The expression of delta9-desaturase showed a more delayed response.     

Clofibrate-inducible, 28-kDa peroxisomal integral membrane protein is encoded by PEX11

We cloned a human PEX11 cDNA by expressed sequence tag homology search using yeast Candida boidinii PEX11, followed by screening of human liver cDNA library. PEX11 encoded a peroxisomal protein Pex11p comprising 247 amino acids, with two transmembrane segments and a dilysine motif at the C-terminus. Pex11p comigrated in SDS-PAGE with a 28-kDa peroxisomal integral membrane protein (PMP28) isolated from the liver of clofibrate-treated rats and was crossreactive to anti-PMP28 antibody, thereby indicating PEX11 to encode PMP28. Pex11p exposes both N- and C-terminal parts to the cytosol. PEX11 was not responsible for ten complementation groups of human peroxisome deficiency disorders.     

Peroxisome synthesis in the absence of preexisting peroxisomes

Zellweger syndrome and related diseases are caused by defective import of peroxisomal matrix proteins. In all previously reported Zellweger syndrome cell lines the defect could be assigned to the matrix protein import pathway since peroxisome membranes were present, and import of integral peroxisomal membrane proteins was normal. However, we report here a Zellweger syndrome patient (PBD061) with an unusual cellular phenotype, an inability to import peroxisomal membrane proteins. We also identified human PEX16, a novel integral peroxisomal membrane protein, and found that PBD061 had inactivating mutations in the PEX16 gene. Previous studies have suggested that peroxisomes arise from preexisting peroxisomes but we find that expression of PEX16 restores the formation of new peroxisomes in PBD061 cells. Peroxisome synthesis and peroxisomal membrane protein import could be detected within 2-3 h of PEX16 injection and was followed by matrix protein import. These results demonstrate that peroxisomes do not necessarily arise from division of preexisting peroxisomes. We propose that peroxisomes may form by either of two pathways: one that involves PEX11-mediated division of preexisting peroxisomes, and another that involves PEX16-mediated formation of peroxisomes in the absence of preexisting peroxisomes.     

Saccharomyces cerevisiae contains four fatty acid activation (FAA) genes: an assessment of their role in regulating protein N-myristoylation and cellular lipid metabolism

Saccharomyces cerevisiae has been used as a model for studying the regulation of protein N-myristoylation. MyristoylCoA:protein N-myristoyl-transferase (Nmt1p), is essential for vegetative growth and uses myristoylCoA as its substrate. MyristoylCoA is produced by the fatty acid synthetase (Fas) complex and by cellular acylCoA synthetases. We have recently isolated three unlinked Fatty Acid Activation (FAA) genes encoding long chain acylCoA synthetases and have now recovered a fourth by genetic complementation. When Fas is active and NMT1 cells are grown on media containing a fermentable carbon source, none of the FAA genes is required for vegetative growth. When Fas is inactivated by a specific inhibitor (cerulenin), NMT1 cells are not viable unless the media is supplemented with long chain fatty acids. Supplementation of cellular myristoylCoA pools through activation of imported myristate (C14:0) is predominantly a function of Faa1p, although Faa4p contributes to this process. Cells with nmt181p need larger pools of myristoylCoA because of the mutant enzyme's reduced affinity for this substrate. Faa1p and Faa4p are required for maintaining the viability of nmt1-181 strains even when Fas is active. Overexpression of Faa2p can rescue nmt1-181 cells due to activation of an endogenous pool of C14:0. This pool appears to be derived in part from membrane phospholipids since overexpression of Plb1p, a nonessential lysophospholipase/phospholipase B, suppresses the temperature-sensitive growth arrest and C14:0 auxotrophy produced by nmt1-181. None of the four known FAAs is exclusively responsible for targeting imported fatty acids to peroxisomal beta-oxidation pathways. Introduction of a peroxisomal assembly mutation, pas1 delta, into isogenic NMT1 and nmt1-181 strains with wild type FAA alleles revealed that when Fas is inhibited, peroxisomes contribute to myristoylCoA pools used by Nmt1p. When Fas is active, a fraction of cellular myristoylCoA is targeted to peroxisomes. A NMT1 strain with deletions of all four FAAs is still viable at 30 degrees C on media containing myristate, palmitate, or oleate as the sole carbon source--indicating that S. cerevisiae contains at least one other FAA which directs fatty acids to beta-oxidation pathways.     

Nucleotide triphosphates are required for the transport of glycolate oxidase into peroxisomes

All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and posttranslationally targeted to the organelle. We have used an in vitro assay to reconstitute protein import into pumpkin (Cucurbita pepo) glyoxysomes, a class of peroxisome found in the cotyledons of oilseed plants, to study the mechanisms involved in protein transport across peroxisome membranes. Results indicate that ATP hydrolysis is required for protein import into peroxisomes; nonhydrolyzable analogs of ATP could not substitute for this requirement. Nucleotide competition studies suggest that there may be a nucleotide binding site on a component of the translocation machinery. Peroxisomal protein import also was supported by GTP hydrolysis. Nonhydrolyzable analogs of GTP did not substitute in this process. Experiments to determine the cation specificity of the nucleotide requirement show that the Mg2+ salt was preferred over other divalent and monovalent cations. The role of a putative protonmotive force across the peroxisomal membrane was also examined. Although low concentrations of ionophores had no effect on protein import, relatively high concentrations of all ionophores tested consistently reduced the level of protein import by approximately 50%. This result suggests that a protonmotive force is not absolutely required for peroxisomal protein import.     

Endocytosis in rat intrahepatic bile duct epithelial cells of horseradish peroxidase injected into the common bile duct or the portal vein

This article reviews the role of the peroxisome in cellular signalling, with particular emphasis on the unique contributions of this organelle to the complex regulatory inter-relationships of cellular processes within the mammalian organism. Among the topics covered are the close alignments between the signalling systems governing peroxisome proliferation and those of the steroid hormone/thyroid hormone/vitamin D nuclear-receptor superfamily; the regulation of the permeability of the peroxisomal membrane; the involvements of lysophosphatidic acid as an intra- and inter-cellular messenger; the special role of the phosphatidylcholine cycle and its derivative messengers in relation to peroxisomal metabolism; peroxisomal contributions to the regulation of oxygen free radical levels in tissues and the significance of these radicals as second messengers; the evidence of peroxisomal influences on inter-cellular signalling from metabolic turnover studies; modifications of the regulatory significance of fatty acids by the peroxisome; the commonalities in metabolic relationships between the peroxisome and other cellular organelles; and regulatory shuttles associated with peroxisomal function. It is concluded that the peroxisome displays several significant interconnections with the cellular-signalling apparatus, that it is capable of imprinting a characteristic influence on the regulatory network in the cell, and that the contributions of this organelle deserve greater consideration in future investigations of cell-signalling phenomena.