Small Peroxisomes Research Articles

Peroxisome development in yeast is associated with the formation of Pex3-dependent peroxisome-vacuole contact sites

Using electron and fluorescence microscopy techniques, we identified various physical contacts between peroxisomes and other cell organelles in the yeast Hansenula polymorpha.In exponential glucose-grown cells, which typically contain a single small peroxisome, contacts were only observed with the endoplasmic reticulum and the plasma membrane. Here we focus on a novel peroxisome-vacuole contact site that is formed when glucose-grown cells are shifted to methanol containing media, conditions that induce strong peroxisome development. At these conditions, the small peroxisomes rapidly increase in size, a phenomenon that is paralleled by the formation of distinct intimate contacts with the vacuole.Localization studies showed that the peroxin Pex3 accumulated in patches at the peroxisome-vacuole contact sites. In wild-type cells growing exponentially on medium containing glucose, peroxisome-vacuole contact sites were never observed. However, upon overproduction of Pex3 peroxisomes also associated to vacuoles at these growth conditions.Our observations strongly suggest a role for Pex3 in the formation of a novel peroxisome-vacuole contact site. This contact likely plays a role in membrane growth as it is formed solely at conditions of strong peroxisome expansion.

Effects of Sleeve Gastrectomy on Nonalcoholic Fatty Liver Disease in an Obese Rat Model.

Laparoscopic sleeve gastrectomy (SG) is an increasingly used bariatric surgery, which is reported to be effective for nonalcoholic fatty liver disease (NAFLD). Recently, activation of farnesoid X receptor (FXR), which is a nuclear receptor of bile acid (BA), was reported to contribute to the resolution of NAFLD. However, it is unclear whether SG has an effect on expression of FXR in the liver. We aimed to investigate the expression of FXR and its related factors in the liver after SG and to clarify the relationship between changes in FXR expression and NAFLD in an obese rat model. Thirty male Zucker fatty rats were divided into three groups: sham-operated (SO) control, pair-fed (PF) control, and SG. Eight weeks after the surgery, metabolic parameters, plasma levels of total BA and liver enzymes, liver triglyceride (TG) content, and mRNA expression of FXR and its related factors, such as small heterodimer partner (SHP) and peroxisome proliferator-activated receptor α (PPARα), were measured. Metabolic parameters in the SG group were significantly improved compared with the SO group. Liver enzymes and TG were significantly lower in the SG group than in the SO group. Plasma levels of BA were significantly higher in the SG group than in the SO and PF groups. mRNA expression of FXR, SHP, and PPARα in the liver was significantly higher in the SG group than in the SO group. These results suggest that the effects of SG on NAFLD should be associated with the expression of the FXR pathway in the liver in a Zucker fatty rat model.

Expression level of methanol-inducible peroxisomal proteins and peroxisome morphology are affected by oxygen conditions and mitochondrial respiratory pathway function in the methylotrophic yeastCandida boidinii

In the methylotrophic yeast, Candida boidinii, methanol-inducible peroxisomal proteins, for example alcohol oxidase (AOD), dihydroxyacetone synthase (DAS), and peroxisomal glutathione peroxidase (Pmp20), were induced only under aerobic conditions, while expression of PMP47 encoding peroxisomal integral membrane protein Pmp47 was independent of oxygen conditions. Expression of the methanol-inducible peroxisomal enzymes was repressed by inhibition of the mitochondrial respiratory chain. In the respiratory-deficient (ρ0) mutant strain, their induction was at very low levels despite the presence of oxygen, whereas the expression of PMP47 was unaffected. Taken together, these facts indicate that C. boidinii can sense oxygen conditions, and that mitochondrial respiratory function may have a profound effect on induction of methanol-inducible gene expression of peroxisomal proteins. Peroxisome morphology was also affected by oxygen conditions and respiratory function. Under hypoxic conditions or respiration-inhibited conditions, cells induced by methanol contained small peroxisomes, indicating that peroxisome biogenesis and the protein import machinery were not affected by oxygen conditions but that peroxisome morphology was dependent on induction of peroxisomal matrix proteins.

Adaptation of Hansenula polymorpha to methanol: a transcriptome analysis

BackgroundMethylotrophic yeast species (e.g. Hansenula polymorpha, Pichia pastoris) can grow on methanol as sole source of carbon and energy. These organisms are important cell factories for the production of recombinant proteins, but are also used in fundamental research as model organisms to study peroxisome biology. During exponential growth on glucose, cells of H. polymorpha typically contain a single, small peroxisome that is redundant for growth while on methanol multiple, enlarged peroxisomes are present. These organelles are crucial to support growth on methanol, as they contain key enzymes of methanol metabolism.In this study, changes in the transcriptional profiles during adaptation of H. polymorpha cells from glucose- to methanol-containing media were investigated using DNA-microarray analyses.ResultsTwo hours after the shift of cells from glucose to methanol nearly 20% (1184 genes) of the approximately 6000 annotated H. polymorpha genes were significantly upregulated with at least a two-fold differential expression. Highest upregulation (> 300-fold) was observed for the genes encoding the transcription factor Mpp1 and formate dehydrogenase, an enzyme of the methanol dissimilation pathway. Upregulated genes also included genes encoding other enzymes of methanol metabolism as well as of peroxisomal β-oxidation.A moderate increase in transcriptional levels (up to 4-fold) was observed for several PEX genes, which are involved in peroxisome biogenesis. Only PEX11 and PEX32 were higher upregulated. In addition, an increase was observed in expression of the several ATG genes, which encode proteins involved in autophagy and autophagy processes. The strongest upregulation was observed for ATG8 and ATG11.Approximately 20% (1246 genes) of the genes were downregulated. These included glycolytic genes as well as genes involved in transcription and translation.ConclusionTranscriptional profiling of H. polymorpha cells shifted from glucose to methanol showed the expected downregulation of glycolytic genes together with upregulation of the methanol utilisation pathway. This serves as a confirmation and validation of the array data obtained. Consistent with this, also various PEX genes were upregulated. The strong upregulation of ATG genes is possibly due to induction of autophagy processes related to remodeling of the cell architecture required to support growth on methanol. These processes may also be responsible for the enhanced peroxisomal β-oxidation, as autophagy leads to recycling of membrane lipids. The prominent downregulation of transcription and translation may be explained by the reduced growth rate on methanol (td glucose 1 h vs td methanol 4.5 h).

Peroxisome Size Provides Insights into the Function of Autophagy-related Proteins

Autophagy is a major pathway of intracellular degradation mediated by formation of autophagosomes. Recently, autophagy was implicated in the degradation of intracellular bacteria, whose size often exceeds the capacity of normal autophagosomes. However, the adaptations of the autophagic machinery for sequestration of large cargos were unknown. Here we developed a yeast model system to study the effect of cargo size on the requirement of autophagy-related (Atg) proteins. We controlled the size of peroxisomes before their turnover by pexophagy, the selective autophagy of peroxisomes, and found that peroxisome size determines the requirement of Atg11 and Atg26. Small peroxisomes can be degraded without these proteins. However, Atg26 becomes essential for degradation of medium peroxisomes. Additionally, the pexophagy-specific phagophore assembly site, organized by the dual interaction of Atg30 with functionally active Atg11 and Atg17, becomes essential for degradation of large peroxisomes. In contrast, Atg28 is partially required for all autophagy-related pathways independent of cargo size, suggesting it is a component of the core autophagic machinery. As a rule, the larger the cargo, the more cargo-specific Atg proteins become essential for its sequestration.

Transcriptional Down-regulation of Peroxisome Numbers Affects Selective Peroxisome Degradation in Hansenula polymorpha

We have isolated and characterized a novel transcription factor of Hansenula polymorpha that is involved in the regulation of peroxisomal protein levels. This protein, designated Mpp1p, belongs to the family of Zn(II)2Cys6 proteins. In cells deleted for the function of Mpp1p the levels of various proteins involved in peroxisome biogenesis (peroxins) and function (enzymes) are reduced compared with wild type or, in the case of the matrix protein dihydroxyacetone synthase, fully absent. Also, upon induction of mpp1 cells on methanol, the number of peroxisomes was strongly reduced relative to wild type cells and generally amounted to one organelle per cell. Remarkably, this single organelle was not susceptible to selective peroxisome degradation (pexophagy) and remained unaffected during exposure of methanol-induced cells to excess glucose conditions. We show that this mechanism is a general phenomenon in H. polymorpha in the case of cells that contain only a single peroxisome.

Pex11-related proteins in peroxisome dynamics: a role for the novel peroxin Pex27p in controlling peroxisome size and number in Saccharomyces cerevisiae.

Transcriptome profiling identified the gene PEX25 encoding Pex25p, a peroxisomal membrane peroxin required for the regulation of peroxisome size and maintenance in Saccharomyces cerevisiae. Pex25p is related to a protein of unknown function encoded by the open reading frame, YOR193w, of the S. cerevisiae genome. Yor193p is a peripheral peroxisomal membrane protein that exhibits high sequence similarity not only to Pex25p but also to the peroxisomal membrane peroxin Pex11p. Unlike Pex25p and Pex11p, Yor193p is constitutively expressed in wild-type cells grown in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. Cells deleted for the YOR193w gene show a few enlarged peroxisomes. Peroxisomes are greatly enlarged in cells harboring double deletions of the YOR193w and PEX25 genes, the YOR193w and PEX11 genes, and the PEX25 and PEX11 genes. Yeast two-hybrid analyses showed that Yor193p interacts with Pex25p and itself, Pex25p interacts with Yor193p and itself, and Pex11p interacts only with itself. Overexpression of YOR193w, PEX25, or PEX11 led to peroxisome proliferation and the formation of small peroxisomes. Our data suggest a role for Yor193p, renamed Pex27p, in controlling peroxisome size and number in S. cerevisiae.

The Dynamin-like GTPase DLP1 Is Essential for Peroxisome Division and Is Recruited to Peroxisomes in Part by PEX11

Peroxisome division involves the conserved PEX11 peroxisomal membrane proteins and in yeast has been shown to require Vps1p, a dynamin-like protein. We show here that DLP1, the human homolog of the yeast DNM1 and VPS1 genes, plays an important role in peroxisome division in human cells. Disruption of DLP1 function by either RNA interference or overexpressing dominant negative DLP1 mutants causes a dramatic reduction in peroxisome abundance, although overexpression of functional DLP1 has no effect on peroxisome abundance. Overexpression of PEX11 induces peroxisome division in a multistep process involving elongation of preexisting peroxisomes followed by their division. We find that DLP1 is dispensable for the first phase of this process but essential for the second. Furthermore, we show that DLP1 associates with peroxisomes and that PEX11 overexpression recruits DLP1 to peroxisome membranes. However, we were unable to detect physical interaction between PEX11 and DLP1, and the stoichiometry of PEX11 and peroxisome-associated DLP1 was far less than 1:1. Based on these and other aspects, we propose that DLP1 performs an essential but transient role in peroxisome division and that PEX11 promotes peroxisome division by recruiting DLP1 to peroxisome membranes through an indirect mechanism.

Stomach of Aplysia depilans (Mollusca, Opisthobranchia): a histochemical, ultrastructural, and cytochemical study.

We report the results of a morphological, histochemical, and cytochemical characterization of the Aplysia depilans stomach, an organ little studied in opisthobranchs. Very thin ciliated cells with microvilli on their apical surfaces are predominant in the epithelium lining the lumen of the stomach. Many lysosomes with a strong arylsulphatase activity were present in the apical regions of these cells that could also contain some lipid droplets and glycogen. Small peroxisomes were observed, usually around lipid droplets or mitochondria. Bottle-shaped secretory cells are very common in this epithelium and produce a secretion rich in proteins and acidic mucopolysaccharides. Most of the cytoplasm of these mucus-producing cells was filled with a very high number of granules and the nucleus is dislocated to the basal region. Cisternae of rough endoplasmic reticulum were abundant around the nucleus and several Golgi stacks were also present in this area. In spite of the variation in the electron density of the granules, only one type of secretory cell seems to be present in the stomach epithelium, since granules with very different electron densities were frequently found in the same cell. A few neurons were also found in the stomach epithelium of this species. Fibrocytes, muscle cells, nerves, and amebocytes were observed in the connective tissue of the stomach wall.

2P-0550 Possible mechanisms for the antiobesity effects of a novel compound OT-13540

Treatment of normolipidemic rats by alkylthiopropionic acid (CETTD), resulted in a dose- and time-dependent increase in total dihydroxyacetone phosphate acyltransferase (DHAPAT) activity, in extent comparable to that of 3-thiadicarboxylic acid (BCMTD) and alkylthioacetic acid (CMTTD). Thus, in CETTD- and CMTTD-treated rats, the specific DHAPAT activity increased in the microsomal, peroxisomal and mitochondrial fractions. In contrast, repeated administration of the peroxisome proliferator, BCMTD, decreased the specific DHAPAT activity both in the peroxisomal fraction and in purified peroxisomes. A three-fold increase in specific activity was, however, revealed in the mitochondrial fraction. Whether the variation of the DHAPAT activity in the mitochondrial and microsomal fractions among the feeding groups can be explained by increased number of enlarged and small peroxisomes sedimenting in the fractions, are to be considered. Subcellular fractionation studies confirmed previous findings that rat liver glycerophosphate acyltransferase (GPAT) was located both in mitochondria and the microsomal fraction. BCMTD was considerably more potent than CMTTD in stimulating the microsomal and mitochondrial GPAT activities. Administration of CETTD marginally affected the isoenzymes of GPAT. Diacylglycerol acyltransferase (DGAT) activity was increased by 35% in BCMTD and CMTTD treated rats, but by administration of CETTD the enzyme activity was decreased by more than 80%. The acyl-CoA cholesterol acyltransferase (ACAT) activity was marginally affected in animals treated with BCMTD, CMTTD and CETTD. Thus, the results indicate that the initial steps in the synthesis of triacylglycerols and ether glycerolipids as well as the last step in triacylglycerol synthesis could not be identified as mediating the fat accumulation or the lowering of triacylglycerol content in liver of CETTD, or BCMTD and CMTTD treated rats. On the other hand, CMTTD increased the palmitoyl-CoA oxidation in mitochondria, and CETTD considerably inhibited the activity. Therefore, it is conceivable that the development of fatty liver with CETTD is mostly due to inhibition of mitochondrial β-oxidation.

Differential induction of peroxisomal populations in subcellular fractions of rat liver

In rat liver, peroxisome proliferators induce profound changes in the number and protein composition of peroxisomes, which upon subcellular fractionation is reflected in heterogeneity in sedimentation properties of peroxisome populations. In this study we have investigated the time course of induction of the peroxisomal proteins catalase, acyl-CoA oxidase (ACO) and the 70 kDa peroxisomal membrane protein (PMP70) in different subcellular fractions. Rats were fed a di(2-ethylhexyl)phthalate (DEHP) containing diet for 8 days and livers were removed at different time-points, fractionated by differential centrifugation into nuclear, heavy and light mitochondrial, microsomal and soluble fractions, and organelle marker enzymes were measured. Catalase was enriched mainly in the light mitochondrial and soluble fractions, while ACO was enriched in the nuclear fraction (about 30%) and in the soluble fraction. PMP70 was found in all fractions except the soluble fraction. DEHP treatment induced ACO, catalase and PMP70 activity and immunoreactive protein, but the time course and extent of induction was markedly different in the various subcellular fractions. All three proteins were induced more rapidly in the nuclear fraction than in the light mitochondrial or microsomal fractions, with catalase and PMP70 being maximally induced in the nuclear fraction already at 2 days of treatment. Refeeding a normal diet quickly normalized most parameters. These results suggest that induction of a heavy peroxisomal compartment is an early event and that induction of ‘small peroxisomes’, containing PMP70 and ACO, is a late event. These data are compatible with a model where peroxisomes initially proliferate by growth of a heavy, possibly reticular-like, structure rather than formation of peroxisomes by division of pre-existing organelles into small peroxisomes that subsequently grow. The various peroxisome populations that can be separated by subcellular fractionation may represent peroxisomes at different stages of biogenesis.

Hepatic peroxisomes in isolated hyperpipecolic acidaemia: evidence supporting its classification as a single peroxisomal enzyme deficiency.

Hyperpipecolic acidaemia is still regarded as a peroxisomal assembly deficiency. The enzyme responsible for the accumulation of pipecolic acid is located in the peroxisomes in man. We studied the appearance and alterations of peroxisomes in liver biopsy material from three unrelated children suffering from isolated hyperpipecolic acidaemia, in which only the metabolism of pipecolic acid is disturbed, using light and electron microscopy after cytochemical staining for visualisation of peroxisomes. Morphometric results showed the presence of normal-sized to small peroxisomes, an increase in number and abnormally shaped organelles, suggesting enhancement of metabolic efficiency. In one case enlarged organelles were observed. Skin fibroblasts were studied in all patients: their peroxisomes appeared to be normal. The obvious presence of peroxisomes in isolated HPA indicates that this disorder should be classified as a single peroxisomal enzyme deficiency.

Presence of heterogeneous peroxisomal populations in the rat nervous tissue

Peroxisomes were purified from the nervous tissue of 14-day-old rats by means of a Nycodenz gradient. Peroxisomal enzymes exhibited different sedimentation patterns: dihydroxyacetone phosphate acyl-transferase equilibrates at 1.142 g/ml together with the first peak of catalase; palmitoyl-CoA oxidase and d-amino acid oxidase activities are mainly recovered at 1.154 g/ml; the second peak of catalase is found at 1.175 g/ml. Morphological and semi-quantitative analyses of immunogold-labelled peroxisomes reveal profound heterogeneity of the particles. Very small (=0.2 μm diameter), electron dense vesicles containing catalase or thiolase, but devoid of other tested enzymes, are preferentially found in the light region, together with larger (>0.2 <0.3 μm) and less electron dense palmitoyl-CoA oxidase-positive peroxisomes. At intermediate density (1.154 g/ml) peroxisomes of more uniform size (0.25–0.27 μm), containing palmitoyl-CoA oxidase or thiolase with or without catalase are preferentially found. This population extends toward the densest region of the gradient, where very large d-amino acid oxidase-containing peroxisomes are also found. In this region, smaller peroxisomes, often polymorphic, which are catalase- and thiolase-positive and d-amino acid oxidase/palmitoyl-CoA oxidase-negative, are also observed. The possibility that the heterogeneity of neural peroxisomes may reflect both cellular heterogeneity and ongoing peroxisomal biogenesis is discussed.

Purification of guinea pig small intestinal peroxisomes and the subcellular localization of glucose-6-phosphate dehydrogenase.

A method for the isolation of highly purified peroxisomes from guinea pig small intestine was developed. This two-stage process involved a rate-dependent banding of a light-mitochondria lambda-fraction followed by a density-dependent banding of the catalase enriched fractions obtained from the first step, using a horizontal rotor. Furthermore, the subcellular localization of glucose-6-phosphate dehydrogenase (NADP+-dependent) activity in guinea pig small intestine was examined. Analysis of density-gradient fractions indicated that approximately 3-4% of the cellular NADP+-dependent glucose-6-phosphate dehydrogenase activity is associated with peroxisomal fractions and that it is localized to the matrix of peroxisomes. It is therefore suggested that a peroxisomal source of NADPH may be utilized by enzyme systems that use NADPH specifically as a reductant.

Deviant Pex3p levels affect normal peroxisome formation in Hansenula polymorpha: a sharp increase of the protein level induces the proliferation of numerous, small protein-import competent peroxisomes.

Pex3p has been implicated in the biosynthesis of the peroxisomal membrane of the yeast Hansenula polymorpha. Here we show that in the initial stages of a sharp increase in Pex3p levels, induced in batch cultures of cells of a constructed H. polymorpha strain, which contained seven copies of PEX3 under control of the alcohol oxidase promoter (WT::PAOX.PEX3(7x)), strongly interfered with normal peroxisome proliferation. Ultrastructural studies demonstrated that in such cells numerous small peroxisomes had developed, which were absent in wild-type controls. These organelles, which contained typical peroxisomal matrix and membrane proteins (alcohol oxidase, catalase, Pex3p, Pex10p and Pex14p), showed a relatively low density (1.18 g cm-3) after sucrose gradient centrifugation of WT::PAOX.PEX3(7x) homogenates, compared to normal peroxisomes (1.23 g cm-3). We furthermore demonstrated that these early induced, small peroxisomes were protected against glucose-induced proteolytic degradation and did not fuse to form larger organelles. Remarkably, the induction of these small peroxisomes was paralleled by a partial defect in matrix protein import, reflected by the mislocalization of minor amounts of alcohol oxidase protein in the cytosol. However, when the cells were subsequently placed under conditions in which the synthesis of a new matrix enzyme (amine oxidase) was induced while simultaneously the excessive proliferation was repressed (by repression of the PAOX), amine oxidase protein was selectively incorporated into these organelles. This indicated that the small peroxisomes had regained a normal protein import capacity. Based on these results we argue that peroxisome proliferation and matrix protein import are coupled processes in H. polymorpha.

Biogenesis of peroxisomes in fetal liver.

Peroxisomes are single membrane-limited cell organelles that are involved in numerous metabolic functions. Peroxisomes do not contain DNA; the matrix and membrane proteins are encoded by the nuclear genome. It is assumed that new peroxisomes are formed by division of existing organelles. The present article gives an overview of microscopic studies and recent unpublished results dealing with peroxisome biogenesis in mammalian fetal liver and presents data on peroxisomes in oocytes. Cytochemical (catalase and D-aminoacid oxidase activity) and immunocytochemical data in rat and human liver (antigens of catalase, the three peroxisomal beta-oxidation enzymes, alanine: glyoxylate aminotransferase, peroxisomal membrane proteins with molecular weights of 42 and 70 kDa) indicate that during embryonic and fetal development the peroxisomal population undergoes a differentiation with respect to the composition of the matrix and to the size and number of the organelles. In the youngest stages, rare and small peroxisomes are present, into which the matrix components are imported in a sequential way. The import seems asynchronous in peroxisomes of the same hepatocyte. The size and number of the peroxisomes increase during liver development. In rat and human liver, no morphological or immunocytochemical evidence for an elaborate network of interconnected peroxisomes ("reticulum") was found. Instead, peroxisomes presented as individual organelles, which occasionally show membrane extensions. The importance of the metabolic functions of peroxisomes in human liver is emphasized by the peroxisomal disorders. In the liver of affected fetuses, the microscopic features associated with the defect can already be recognized; i.e., either catalase containing peroxisomes are absent and catalase is localized in the cytoplasm (in fetuses affected with Zellweger syndrome or with infantile Refsum disease) or peroxisomes are present but they are abnormally enlarged (e.g., a fetus affected with acyl-CoA oxidase deficiency). In the quail ovary, numerous peroxisomes are observed in the oocyte and in the granulosa cells during follicle maturation, but not in the full-grown egg. Thus, the mechanism of peroxisome inheritance remains unresolved.

Decrease in glycolate pathway enzyme activities in plastids and peroxisomes of the albostrians mutant of barley ( Hordeum vulgare L.)

The in-vitro activities of various enzymes involved in the glycolate pathway were analyzed in crude leaf extracts of the albostrians mutant of barley. This mutant line is characterized by the lack of plastid ribosomes in white leaf tissue, thus causing a complex modification of nuclear gene expression. In white leaf cells the activities of the plastidic enzymes phosphoglycolate phosphatase and glycerate kinase, as well as the activities of the peroxisomal enzymes glycolate oxidase, catalase, and hydroxypyruvate reductase, are drastically reduced by as much as 80–99% compared to green wild-type leaf tissue. The activity of plastidic NADPH-glyoxylate reductase, the total activity of serine hydroxymethyltransferase and the activity of mitochondrial fumarase was reduced by only 30–52%. The activities of the glyoxysomal enzymes isocitrate lyase and malate synthase were neither detectable in green nor white mutant leaf tissue. Electron microscopy showed fewer and smaller peroxisomes in white leaf tissue, accounting for at least part of the reduction of peroxisomal enzyme activities. Northern blot analysis for the catalase and glycolate oxidase transcripts revealed strongly reduced levels of steady-state mRNA accumulation. It is suggested that a putative plastidic signal(s) influences the expression of nuclear-encoded plastidic and peroxisomal enzymes of the glycolate pathway.

Contributions of the immunogold technique to investigation of the biology of peroxisomes.

The immunogold labeling technique has been extremely useful in investigation of the structure and function of peroxisomes. In this report a few examples of the application of this technique with significant implications in the field are briefly reviewed. The problem of extra-peroxisomal catalase, the subject of long controversy between the biochemists and cytochemists, was settled with the immunogold technique, which unequivocally revealed the presence of that enzyme not only in the cytoplasm, but also in the euchromatin region of nucleus, in addition to peroxisomes. On the other hand, lactate dehydrogenase, a typical cytoplasmic protein, has also been shown recently to be present in peroxisomes and to be involved in the reoxidation of NADH produced by the peroxisomal beta-oxidation system. The immunogold technique has revealed several distinct compartments in the matrix of mammalian peroxisomes: urate oxidase in the crystalline cores, alpha-hydroxy acid oxidase B in the marginal plates and D-amino acid oxidase in a non-crystalline condensed region of matrix. The specific alterations of peroxisomal proteins are reflected in their immunolabeling density with gold particles. Quantitation of gold-label by automatic image analysis has revealed that the induction of lipid beta-oxidation enzyme proteins by diverse hypolipidemic drugs is initiated and more pronounced in the pericentral regions of the liver lobule. Finally, immunogold labeling with an antibody to 70 kDa peroxisomal membrane protein has identified a novel class of small peroxisomes that initially incorporate radioactive amino acids more efficiently than regular peroxisomes and thus may represent early stages in the biogenesis of peroxisomes.

Pip2p: a transcriptional regulator of peroxisome proliferation in the yeast Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, peroxisomes are the exclusive site for the degradation of fatty acids. Upon growth with the fatty acid oleic acid as sole carbon source, not only are the enzymes of beta-oxidation and catalase A induced, but also the peroxisomal compartment as a whole increases in volume and the number of organelles per cell rises. We previously identified a cis-acting DNA sequence [oleate response element (ORE)] involved in induction of genes encoding peroxisomal proteins. The aim of our investigation was to test whether a single mechanism acting via the ORE coordinates the events necessary for the proliferation of an entire organelle. Here we report the cloning and characterization of the oleate-specific transcriptional activator protein Pip2p (pip: peroxisome induction pathway). Pip2p contains a typical Zn(2)-Cys(6) cluster domain and binds to OREs. A pip2 deletion strain is impaired in growth on oleate as sole carbon source and the induction of beta-oxidation enzymes is abolished. Moreover, only a few, small peroxisomes per cell can be detected. These results indicate that fatty acids activate Pip2p, which in turn activates the transcription of genes encoding beta-oxidation components and acts as the crucial activator of peroxisomes.

Regulation of Peroxisome Proliferator-Activated Receptor-α mRNA in Rat Liver

Chemical-induced peroxisome proliferation in rodent liver is postulated to occur via activation of members of the steroid hormone receptor superfamily, the peroxisome proliferator-activated receptors (PPARs). In the present study, the expression of the predominant liver subtype PPARα was examined and compared to that of acyl-CoA oxidase (ACO), a marker for peroxisome proliferation and a prototype for genes regulated via PPARs. Despite the induction of both mRNA speciesin vivoby the peroxisome proliferator perfluorodecanoic acid (PFDA), dose response and time course indicate PPARα and ACO are not controlled similarly. Messenger RNA levels for ACO increased rapidly in rat liver and declined over the subsequent 7 days following PFDA administration, while PPARα mRNA increased slower and remained elevated over this period. In addition, PPARα mRNA accumulation in PFDA-treated rats appears to be due primarily to hypophagia as pair feeding and complete caloric restriction result in a large increase in the concentration of this messenger RNA. Nuclear run-on experimentsin vivosuggest that, unlike ACO, PFDA as well as caloric restriction results in accumulation of PPARα mRNA which cannot be explained solely by transcriptional activation. These data indicate that PPARα mRNA accumulation has a very small peroxisome proliferator-dependent component and that other factors may be involved. A rat hepatoma cell line was examined to determine the direct effect of peroxisome proliferators on PPARα mRNA. PPARα and ACO mRNA levels were increased rapidly in the rat hepatoma cell line FaO after treatment with PFDA or the prototypical peroxisome proliferator Wy 14,643. In this cell line, PPARα mRNA levels are not affected by glucagon or insulin and in addition to peroxisome proliferators are induced in this cell line by oleic acid and dexamethasone. The latter treatment had the greatest effect on PPARα mRNA accumulation while having a minimal effect on ACO mRNA. Treatment of FaO cells with actinomycin D prior to Wy 14,643 abolished ACO and PPARα mRNA accumulation, demonstrating that there must be a transcriptional component of the peroxisome proliferator response. Therefore, although PPARα is responsive to peroxisome proliferators and direct effects are observed in cell culture, mRNA accumulationin vivois predominantly posttranscriptional, and endogenous regulators such as glucocorticoids may play critical roles in the tissue- and developmentally specific expression of this steroid hormone receptor.