Involvement of glutathione peroxidase 1 in growth and peroxisome formation in Saccharomyces cerevisiae in oleic acid medium

Saccharomyces cerevisiae is able to use some fatty acids, such as oleic acid, as a sole source of carbon. β-oxidation, which occurs in a single membrane-enveloped organelle or peroxisome, is responsible for the assimilation of fatty acids. In S. cerevisiae, β-oxidation occurs only in peroxisomes, and H(2)O(2) is generated during this fatty acid-metabolizing pathway. S. cerevisiae has three GPX genes (GPX1, GPX2, and GPX3) encoding atypical 2-Cys peroxiredoxins. Here we show that expression of GPX1 was induced in medium containing oleic acid as a carbon source in an Msn2/Msn4-dependent manner. We found that Gpx1 was located in the peroxisomal matrix. The peroxisomal Gpx1 showed peroxidase activity using thioredoxin or glutathione as a reducing power. Peroxisome biogenesis was induced when cells were cultured with oleic acid. Peroxisome biogenesis was impaired in gpx1? cells, and subsequently, the growth of gpx1? cells was lowered in oleic acid-containing medium. Gpx1 contains six cysteine residues. Of the cysteine-substituted mutants of Gpx1, Gpx1(C36S) was not able to restore growth and peroxisome formation in oleic acid-containing medium, therefore, redox regulation of Gpx1 seems to be involved in the mechanism of peroxisome formation.     

Oleate β-oxidation in yeast involves thioesterase but not Yor180c protein that is not a dienoyl-CoA isomerase

The β-oxidation of oleic acid in Saccharomyces cerevisiae (S. cerevisiae) was studied by comparing the growth of wild-type cells on oleic acid or palmitic acid with the growth of mutants that either had a deletion in the YOR180c (DCI1) gene reported to encode Δ^3,5,Δ^2,4-dienoyl-CoA isomerase (dienoyl-CoA isomerase) or in the PTE1 gene encoding peroxisomal thioesterase 1. Growth of wild-type cells was indistinguishable from that of YOR180c mutant cells on either palmitic acid or oleic acid, whereas the PTE1 mutant grew slower and to a lower density on oleic acid but not on palmitic acid. The identification of 3,5-tetradecadienoic acid in the medium of wild-type cells but not in the medium of the PTE1 mutant proves the operation of the thioesterase-dependent pathway of oleate β-oxidation in S. cerevisiae. Dienoyl-CoA isomerase activity was very low in wild-type cells, fourfold higher in the YOR180c mutant, and not associated with purified Yor180c protein. These observations support the conclusion that the YOR180c gene does not encode dienoyl-CoA isomerase.     

Glutathione peroxidase 2 in Saccharomyces cerevisiae is distributed in mitochondria and involved in sporulation

Gpx2, one of three glutathione peroxidase homologs (Gpx1, Gpx2, and Gpx3) in Saccharomyces cerevisiae, is an atypical 2-Cys peroxiredoxin that prefers to use thioredoxin as a reducing agent in vitro. Despite Gpx2 being an antioxidant, no obvious phenotype of gpx2Δ mutant cells in terms of oxidative stress has yet been found. To gain a clue as to Gpx2’s physiological function in vivo, here we identify its intracellular distribution. Gpx2 was found to exist in the cytoplasm and mitochondria. In mitochondria, Gpx2 was associated with the outer membrane of the cytoplasmic-side, as well as the inner membrane of the matrix-side. The redox state of the mitochondrial Gpx2 was regulated by Trx1 and Trx2 (cytoplasmic thioredoxin), and by Trx3 (mitochondrial matrix thioredoxin). In addition, we found that the disruption of GPX2 reduced the sporulation efficiency of diploid cells.     

Synthesis of Polyhydroxyalkanoate in the Peroxisome of Saccharomyces cerevisiae by Using Intermediates of Fatty Acid β-Oxidation

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the β-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.     

Very-long-chain polyunsaturated fatty acids accumulate in phosphatidylcholine of fibroblasts from patients with Zellweger syndrome and acyl-CoA oxidase1 deficiency

Peroxisomes are subcellular organelles that function in multiple anabolic and catabolic processes, including β-oxidation of very-long-chain fatty acids (VLCFA) and biosynthesis of ether phospholipids. Peroxisomal disorders caused by defects in peroxisome biogenesis or peroxisomal β-oxidation manifest as severe neural disorders of the central nervous system. Abnormal peroxisomal metabolism is thought to be responsible for the clinical symptoms of these diseases, but their molecular pathogenesis remains to be elucidated. We performed lipidomic analysis to identify aberrant metabolites in fibroblasts from patients with Zellweger syndrome (ZS), acyl-CoA oxidase1 (AOx) deficiency, D-bifunctional protein (D-BP) and X-linked adrenoleukodystrophy (X-ALD), as well as in peroxisome-deficient Chinese hamster ovary cell mutants. In cells deficient in peroxisomal biogenesis, plasmenylethanolamine was remarkably reduced and phosphatidylethanolamine was increased. Marked accumulation of very-long-chain saturated fatty acid and monounsaturated fatty acids in phosphatidylcholine was observed in all mutant cells. Very-long-chain polyunsaturated fatty acid (VLC-PUFA) levels were significantly elevated, whilst phospholipids containing docosahexaenoic acid (DHA, C22:6n-3) were reduced in fibroblasts from patients with ZS, AOx deficiency, and D-BP deficiency, but not in fibroblasts from an X-ALD patient. Because patients with AOx deficiency suffer from more severe symptoms than those with X-ALD, accumulation of VLC-PUFA and/or reduction of DHA may be associated with the severity of peroxisomal diseases.     

The Arabidopsis Peroxisomal ABC Transporter,Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2Δ Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2Δ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2Δ mutant, and restored β-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.     

Pex18p and Pex21p,a Novel Pair of Related Peroxins Essential for Peroxisomal Targeting by the PTS2 Pathway

We have identified ScPex18p and ScPex21p, two novel S. cerevisiae peroxins required for protein targeting via the PTS2 branch of peroxisomal biogenesis. Targeting by this pathway is known to involve the interaction of oligopeptide PTS2 signals with Pex7p, the PTS2 receptor. Pex7p function is conserved between yeasts and humans, with defects in the human protein causing rhizomelic chondrodysplasia punctata (RCDP), a severe, lethal peroxisome biogenesis disorder characterized by aberrant targeting of several PTS2 peroxisomal proteins, but uncertainty remains about the subcellular localization of this receptor. Previously, we have reported that ScPex7p resides predominantly in the peroxisomal matrix, suggesting that it may function as a highly unusual intraorganellar import receptor, and the data presented in this paper identify Pex18p and Pex21p as key components in the targeting of Pex7p to peroxisomes. They each interact specifically with Pex7p both in two-hybrid analyses and in vitro. In cells lacking both Pex18p and Pex21p, Pex7p remains cytosolic and PTS2 targeting is completely abolished. Pex18p and Pex21p are weakly homologous to each other and display partial functional redundancy, indicating that they constitute a two-member peroxin family specifically required for Pex7p and PTS2 targeting.     

Engineering the Monomer Composition of Polyhydroxyalkanoates Synthesized in Saccharomyces cerevisiae

Polyhydroxyalkanoates (PHAs) have received considerable interest as renewable-resource-based, biodegradable, and biocompatible plastics with a wide range of potential applications. We have engineered the synthesis of PHA polymers composed of monomers ranging from 4 to 14 carbon atoms in either the cytosol or the peroxisome of Saccharomyces cerevisiae by harnessing intermediates of fatty acid metabolism. Cytosolic PHA production was supported by establishing in the cytosol critical β-oxidation chemistries which are found natively in peroxisomes. This platform was utilized to supply medium-chain (C₆ to C₁₄) PHA precursors from both fatty acid degradation and synthesis to a cytosolically expressed medium-chain-length (mcl) polymerase from Pseudomonas oleovorans. Synthesis of short-chain-length PHAs (scl-PHAs) was established in the peroxisome of a wild-type yeast strain by targeting the Ralstonia eutropha scl polymerase to the peroxisome. This strain, harboring a peroxisomally targeted scl-PHA synthase, accumulated PHA up to approximately 7% of its cell dry weight. These results indicate (i) that S. cerevisiae expressing a cytosolic mcl-PHA polymerase or a peroxisomal scl-PHA synthase can use the 3-hydroxyacyl coenzyme A intermediates from fatty acid metabolism to synthesize PHAs and (ii) that fatty acid degradation is also possible in the cytosol as β-oxidation might not be confined only to the peroxisomes. Polymers of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers can be controlled by feeding the appropriate substrates. This ability should permit the rational design and synthesis of polymers with desired material properties.     

Role of Pex11p in Lipid Homeostasis in Yarrowia lipolytica

Peroxisomes are essential organelles in the cells of most eukaryotes, from yeasts to mammals. Their role in β-oxidation is particularly essential in yeasts; for example, in Saccharomyces cerevisiae, fatty acid oxidation takes place solely in peroxisomes. In this species, peroxisome biogenesis occurs when lipids are present in the culture medium, and it involves the Pex11p protein family: ScPex11p, ScPex25p, ScPex27p, and ScPex34p. Yarrowia lipolytica has three Pex11p homologues, which are YALI0C04092p (YlPex11p), YALI0C04565p (YlPex11C), and YALI0D25498p (Pex11/25p). We found that these genes are regulated by oleic acid, and as has been observed in other organisms, YlPEX11 deletion generated giant peroxisomes when mutant yeast were grown in oleic acid medium. Moreover, ΔYlpex11 was unable to grow on fatty acid medium and showed extreme dose-dependent sensitivity to oleic acid. Indeed, when the strain was grown in minimum medium with 0.5% glucose and 3% oleic acid, lipid body lysis and cell death were observed. Cell death and lipid body lysis may be partially explained by an imbalance in the expression of the genes involved in lipid storage, namely, DGA1, DGA2, and LRO1, as well as that of TGL4, which is involved in lipid remobilization. TGL4 deletion and DGA2 overexpression resulted in decreased oleic acid sensitivity and delayed cell death of ΔYlpex11, which probably stemmed from the release of free fatty acids into the cytoplasm. All these results show that YlPex11p plays an important role in lipid homeostasis in Y. lipolytica.     

Molecular insights into peroxisome homeostasis and peroxisome biogenesis disorders

Peroxisomes are single-membrane organelles essential for cell metabolism including the β-oxidation of fatty acids, synthesis of etherlipid plasmalogens, and redox homeostasis. Investigations into peroxisome biogenesis and the human peroxisome biogenesis disorders (PBDs) have identified 14 PEX genes encoding peroxins involved in peroxisome biogenesis and the mutation of PEX genes is responsible for the PBDs. Many recent findings have further advanced our understanding of the biology, physiology, and consequences of a functional deficit of peroxisomes. In this Review, we discuss cell defense mechanisms that counteract oxidative stress by 1) a proapoptotic Bcl-2 factor BAK-mediated release to the cytosol of H₂O₂-degrading catalase from peroxisomes and 2) peroxisomal import suppression of catalase by Ser232-phosphorylation of Pex14, a docking protein for the Pex5–PTS1 complex. With respect to peroxisome division, the important issue of how the energy-rich GTP is produced and supplied for the division process was recently addressed by the discovery of a nucleoside diphosphate kinase-like protein, termed DYNAMO1 in a lower eukaryote, which has a mammalian homologue NME3. In regard to the mechanisms underlying the pathogenesis of PBDs, a new PBD model mouse defective in Pex14 manifests a dysregulated brain-derived neurotrophic factor (BDNF)-TrkB pathway, an important signaling pathway for cerebellar morphogenesis. Communications between peroxisomes and other organelles are also addressed.     

Arabidopsis ABERRANT PEROXISOME MORPHOLOGY9 is a peroxin that recruits the PEX1-PEX6 complex to peroxisomes

Peroxisomes have pivotal roles in several metabolic processes, such as the detoxification of H₂O₂ and β-oxidation of fatty acids, and their functions are tightly regulated by multiple factors involved in peroxisome biogenesis, including protein transport. This study describes the isolation of an embryonic lethal Arabidopsis thaliana mutant, aberrant peroxisome morphology9 (apem9), which is compromised in protein transport into peroxisomes. The APEM9 gene was found to encode an unknown protein. Compared with apem9 having the nucleotide substitution, the knockdown mutants showed severe defects in peroxisomal functions and plant growth. We showed that expression of APEM9 altered PEROXIN6 (PEX6) subcellular localization from the cytosol to peroxisomes. In addition, we showed that PEX1 and PEX6 comprise a heterooligomer and that this complex was recruited to peroxisomal membranes via protein–protein interactions of APEM9 with PEX6. These findings show that APEM9 functions as an anchoring protein, similar to Pex26 in mammals and Pex15p in yeast. Interestingly, however, the identities of amino acids among these anchoring proteins are quite low. These results indicate that although the association of the PEX1-PEX6 complex with peroxisomal membranes is essential for peroxisomal functions, the protein that anchors this complex evolved uniquely in plants.     

Induction of Peroxisomes by Butyrate-Producing Probiotics

We previously found that peroxisomal biogenesis factor 11a (Pex11a) deficiency is associated with a reduction in peroxisome abundance and impaired fatty acid metabolism in hepatocytes, and results in steatosis. In the present study, we investigated whether butyrate induces Pex11a expression and peroxisome proliferation, and studied its effect on lipid metabolism. C57BL/6 mice fed standard chow or a high-fat diet (HFD) were treated with tributyrin, 4-phelybutyrate acid (4-PBA), or the butyrate-producing probiotics (Clostridium butyricum MIYAIRI 588 [CBM]) plus inulin (dietary fiber), and the body weight, white adipose tissue, serum triglycerides, mRNA expression, and peroxisome abundance were evaluated. Tributyrin or 4-PBA treatment significantly decreased body weight and increased hepatic mRNA expression of peroxisome proliferator-activated receptor-α (PPARα) and Pex11a. In addition, 4-PBA treatment increased peroxisome abundance and the expression of genes involved in peroxisomal fatty acid β-oxidation (acyl-coenzyme A oxidase 1 and hydroxysteroid [17-beta] dehydrogenase 4). CBM and inulin administration reduced adipose tissue mass and serum triglycerides, induced Pex11a, acyl-coenzyme A oxidase 1, and hydroxysteroid (17-beta) dehydrogenase 4 genes, and increased peroxisome abundance in mice fed standard chow or an HFD. In conclusion, elevation of butyrate availability (directly through administration of butyrate or indirectly via administration of butyrate-producing probiotics plus fiber) induces PPARα and Pex11a and the genes involved in peroxisomal fatty acid β-oxidation, increases peroxisome abundance, and improves lipid metabolism. These results may provide a new therapeutic strategy against hyperlipidemia and obesity.     

Repercussion of a deficiency in mitochondrial ß-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ß-oxidation cycle in Aspergillus nidulans

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ß-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ß-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ß-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ß-oxidation cycle can handle both long and short-chain intermediates. While the ?foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ß-oxidation is actually enhanced in its absence. While no change in PHA was observed in the ?scdA?echA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in ?scdA?echA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ß-oxidation cycle when the mitochondrial ß-oxidation pathway is defective.     

Peroxisome biogenesis and the role of protein import

Peroxisomes are metabolic organelles with enzymatic content that are found in virtually all cells and are involved in β-oxidation of fatty acids, hydrogen peroxide-based respiration and defence against oxidative stress. The steps of their biogenesis involves "peroxins", proteins encoded by PEX genes. Peroxins are involved in three key stages of peroxisome development: (1) import of peroxisomal membrane proteins; (2) import of peroxisomal matrix proteins and (3) peroxisome proliferation. Of these three areas, peroxisomal matrix-protein import is by far the best understood and accounts for most of the available published data on peroxisome biogenesis. Defects in peroxisome biogenesis result in peroxisome biogenesis disorders (PBDs), which although rare, have no known cure to-date. This review explores current understanding of each key area in peroxisome biogenesis, paying particular attention to the role of protein import.     

CmPEX6, a Gene Involved in Peroxisome Biogenesis,Is Essential for Parasitism and Conidiation by the Sclerotial Parasite Coniothyrium minitans

Coniothyrium minitans is a sclerotial parasite of the plant-pathogenic fungus Sclerotinia sclerotiorum, and conidial production and parasitism are two important aspects for commercialization of this biological control agent. To understand the mechanism of conidiation and parasitism at the molecular level, we constructed a transfer DNA (tDNA) insertional library with the wild-type strain ZS-1. A conidiation-deficient mutant, ZS-1TN22803, was uncovered through screening of this library. This mutant could produce pycnidia on potato dextrose agar (PDA), but most were immature and did not bear conidia. Moreover, this mutant lost the ability to parasitize or rot the sclerotia of S. sclerotiorum. Analysis of the tDNA flanking sequences revealed that a peroxisome biogenesis factor 6 (PEX6) homolog of Saccharomyces cerevisiae, named CmPEX6, was disrupted by the tDNA insertion in this mutant. Targeted gene replacement and gene complementation tests confirmed that a null mutation of CmPEX6 was responsible for the phenotype of ZS-1TN22803. Further analysis showed that both ZS-1TN22803 and the targeted replacement mutants could not grow on PDA medium containing oleic acid, and they produced much less nitric oxide (NO) and hydrogen peroxide (H₂O₂) than wild-type strain ZS-1. The conidiation of ZS-1TN22803 was partially restored by adding acetyl-CoA or glyoxylic acid to the growth media. Our results suggest that fatty acid β-oxidation, reactive oxygen and nitrogen species, and possibly other unknown pathways in peroxisomes are involved in conidiation and parasitism by C. minitans.