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1.  The peroxin Pex34p functions with the Pex11 family of peroxisomal divisional proteins to regulate the peroxisome population in yeast 
Molecular Biology of the Cell  2011;22(10):1727-1738.
Pex34p is a novel peroxisomal protein involved in controlling peroxisome abundance in Saccharomyces cerevisiae. Pex34p acts to control peroxisome numbers both alone and in cooperation with the Pex11 protein family of peroxisome divisional proteins.
Peroxisomes are ubiquitous organelles involved in diverse metabolic processes, most notably the metabolism of lipids and the detoxification of reactive oxygen species. Peroxisomes are highly dynamic and change in size and number in response to both intra- and extracellular cues. In the yeast Saccharomyces cerevisiae, peroxisome growth and division are controlled by both the differential import of soluble matrix proteins and a specialized divisional machinery that includes peroxisome-specific factors, such as members of the Pex11 protein family, and general organelle divisional factors, such as the dynamin-related protein Vps1p. Global yeast two-hybrid analyses have demonstrated interactions between the product of the S. cerevisiae gene of unknown function, YCL056c, and Pex proteins involved in peroxisome biogenesis. Here we show that the protein encoded by YCL056c, renamed Pex34p, is a peroxisomal integral membrane protein that acts independently and also in concert with the Pex11 protein family members Pex11p, Pex25p, and Pex27p to control the peroxisome populations of cells under conditions of both peroxisome proliferation and constitutive peroxisome division. Yeast two-hybrid analysis showed that Pex34p interacts physically with itself and with Pex11p, Pex25p, and Pex27p but not with Vps1p. Pex34p can act as a positive effector of peroxisome division as its overexpression leads to increased numbers of peroxisomes in wild type and pex34Δ cells. Pex34p requires the Pex11 family proteins to promote peroxisome division. Our discovery of Pex34p as a protein involved in the already complex control of peroxisome populations emphasizes the necessity of cells to strictly regulate their peroxisome populations to be able to respond appropriately to changing environmental conditions.
PMCID: PMC3093324  PMID: 21441307
2.  Arginine improves peroxisome functioning in cells from patients with a mild peroxisome biogenesis disorder 
Zellweger spectrum disorders (ZSDs) are multisystem genetic disorders caused by a lack of functional peroxisomes, due to mutations in one of the PEX genes, encoding proteins involved in peroxisome biogenesis. The phenotypic spectrum of ZSDs ranges from an early lethal form to much milder presentations. In cultured skin fibroblasts from mildly affected patients, peroxisome biogenesis can be partially impaired which results in a mosaic catalase immunofluorescence pattern. This peroxisomal mosaicism has been described for specific missense mutations in various PEX genes. In cell lines displaying peroxisomal mosaicism, peroxisome biogenesis can be improved when these are cultured at 30°C. This suggests that these missense mutations affect the folding and/or stability of the encoded protein. We have studied if the function of mutant PEX1, PEX6 and PEX12 can be improved by promoting protein folding using the chemical chaperone arginine.
Fibroblasts from three PEX1 patients, one PEX6 and one PEX12 patient were cultured in the presence of different concentrations of arginine. To determine the effect on peroxisome biogenesis we studied the following parameters: number of peroxisome-positive cells, levels of PEX1 protein and processed thiolase, and the capacity to β-oxidize very long chain fatty acids and pristanic acid.
Peroxisome biogenesis and function in fibroblasts with mild missense mutations in PEX1, 6 and 12 can be improved by arginine.
Arginine may be an interesting compound to promote peroxisome function in patients with a mild peroxisome biogenesis disorder.
PMCID: PMC3844471  PMID: 24016303
Peroxisome biogenesis disorder; Zellweger spectrum disorder; Misfolded protein; Peroxisomal mosaicism; Arginine; Therapy
3.  Role of the PAS1 gene of Pichia pastoris in peroxisome biogenesis 
The Journal of Cell Biology  1994;127(5):1259-1273.
Several groups have reported the cloning and sequencing of genes involved in the biogenesis of yeast peroxisomes. Yeast strains bearing mutations in these genes are unable to grow on carbon sources whose metabolism requires peroxisomes, and these strains lack morphologically normal peroxisomes. We report the cloning of Pichia pastoris PAS1, the homologue (based on a high level of protein sequence similarity) of the Saccharomyces cerevisiae PAS1. We also describe the creation and characterization of P. pastoris pas1 strains. Electron microscopy on the P. pastoris pas1 cells revealed that they lack morphologically normal peroxisomes, and instead contain membrane-bound structures that appear to be small, mutant peroxisomes, or "peroxisome ghosts." These "ghosts" proliferated in response to induction on peroxisome-requiring carbon sources (oleic acid and methanol), and they were distributed to daughter cells. Biochemical analysis of cell lysates revealed that peroxisomal proteins are induced normally in pas1 cells. Peroxisome ghosts from pas1 cells were purified on sucrose gradients, and biochemical analysis showed that these ghosts, while lacking several peroxisomal proteins, did import varying amounts of several other peroxisomal proteins. The existence of detectable peroxisome ghosts in P. pastoris pas1 cells, and their ability to import some proteins, stands in contrast with the results reported by Erdmann et al. (1991) for the S. cerevisiae pas1 mutant, in which they were unable to detect peroxisome-like structures. We discuss the role of PAS1 in peroxisome biogenesis in light of the new information regarding peroxisome ghosts in pas1 cells.
PMCID: PMC2120248  PMID: 7962088
4.  A Subtle Interplay Between Three Pex11 Proteins Shapes De Novo Formation and Fission of Peroxisomes 
Traffic (Copenhagen, Denmark)  2011;13(1):157-167.
The organization of eukaryotic cells into membrane-bound compartments must be faithfully sustained for survival of the cell. A subtle equilibrium exists between the degradation and the proliferation of organelles. Commonly, proliferation is initiated by a membrane remodeling process. Here, we dissect the function of proteins driving organelle proliferation in the particular case of peroxisomes. These organelles are formed either through a growth and division process from existing peroxisomes or de novo from the endoplasmic reticulum (ER). Among the proteins involved in the biogenesis of peroxisomes, peroxins, members of the Pex11 protein family participate in peroxisomal membrane alterations. In the yeast Saccharomyces cerevisiae, the Pex11 family consists of three proteins, Pex11p, Pex25p and Pex27p. Here we demonstrate that yeast mutants lacking peroxisomes require the presence of Pex25p to regenerate this organelle de novo. We also provide evidence showing that Pex27p inhibits peroxisomal function and illustrate that Pex25p initiates elongation of the peroxisomal membrane. Our data establish that although structurally conserved each of the three Pex11 protein family members plays a distinct role. While ScPex11p promotes the proliferation of peroxisomes already present in the cell, ScPex25p initiates remodeling at the peroxisomal membrane and ScPex27p acts to counter this activity. In addition, we reveal that ScPex25p acts in concert with Pex3p in the initiation of de novo peroxisome biogenesis from the ER.
PMCID: PMC3245845  PMID: 21951626
fatty acid consumption; inheritance assay; membrane elongation; membrane proteins; organelle biogenesis; peroxisomes; PEX11; PEX25; PEX27; proliferation
5.  The Role of the Endoplasmic Reticulum in Peroxisome Biogenesis 
Peroxisomes are essential cellular organelles involved in lipid metabolism. Patients affected by severe peroxisome biogenesis disorders rarely survive their first year. Genetic screens in several model organisms have identified more than 30 PEX genes that are required for the formation of functional peroxisomes. Despite significant work on the PEX genes, the biogenic origin of peroxisomes remains controversial. For at least two decades, the prevailing model postulated that peroxisomes propagate by growth and fission of preexisting peroxisomes. In this review, we focus on the recent evidence supporting a new, semiautonomous model of peroxisomal biogenesis. According to this model, peroxisomal membrane proteins (PMPs) traffic from the endoplasmic reticulum (ER) to the peroxisome by a vesicular budding, targeting, and fusion process while peroxisomal matrix proteins are imported into the organelle by an autonomous, posttranslational mechanism. We highlight the contradictory conclusions reached to answer the question of how PMPs are inserted into the ER. We then review what we know and what still remains to be elucidated about the mechanism of PMP exit from the ER and the contribution of preperoxisomal vesicles to mature peroxisomes. Finally, we discuss discrepancies in our understanding of de novo peroxisome biogenesis in wild-type cells. We anticipate that resolving these key issues will lead to a more complete picture of peroxisome biogenesis.
Recent evidence supports a semiautonomous model of peroxisomal biogenesis. In this model, peroxisomal matrix proteins are imported directly from the cytosol, whereas peroxisomal membrane proteins come from the ER.
PMCID: PMC3632059  PMID: 23637287
6.  Peroxisome proliferation in Arabidopsis 
Plant Signaling & Behavior  2008;3(9):671-673.
Peroxisomes are subcellular organelles with multiple functions mediated by their plasticity and dynamic behavior in plants. Changes in their shape, size, number and enzyme content occur in response to developmental and metabolic cues as well as environmental conditions. The number of peroxisomes per cell is thus mainly determined by its capacity to proliferate. In mammals, peroxisome proliferators such as the hypolipidemic drug clofibrate are perceived by the Peroxisome Proliferator-Activated Receptors (PPARs) nuclear receptors. Therein, activated transcription of the peroxisome biogenesis PEX11 genes and the recruitment of dynamin-related proteins lead to peroxisome proliferation. We recently reported that Arabidopsis thaliana, despite of lacking a PPAR homolog protein, activated the proliferation of peroxisomes in response to clofibrate. Concomitantly, clofibrate activated the expression of wound-responsive genes through the jasmonic acid signaling master regulator COI1 F-box protein. Besides, wounding activated the expression of the peroxisome biogenesis-related PEX1 and PEX14 genes, but not of PEX11 or DRP3A, which analogously to mammals, code for the main regulators of peroxisome proliferation in Arabidopsis. Thus, wounding did not activate peroxisome proliferation. Noteworthy, jasmonic acid-treated plants contained fewer but larger peroxisomes. Despite of the cross-talk between clofibrate- and wound-induced signaling, the proliferation of peroxisomes and the wound-activated defense remained uncoupled.
PMCID: PMC2634552  PMID: 19704821
peroxisome proliferation; clofibrate; wound-related signaling; arabidopsis
7.  A Drosophila model for the Zellweger spectrum of peroxisome biogenesis disorders 
Disease Models & Mechanisms  2011;4(5):659-672.
Human peroxisome biogenesis disorders are lethal genetic diseases in which abnormal peroxisome assembly compromises overall peroxisome and cellular function. Peroxisomes are ubiquitous membrane-bound organelles involved in several important biochemical processes, notably lipid metabolism and the use of reactive oxygen species for detoxification. Using cultured cells, we systematically characterized the peroxisome assembly phenotypes associated with dsRNA-mediated knockdown of 14 predicted Drosophila homologs of PEX genes (encoding peroxins; required for peroxisome assembly and linked to peroxisome biogenesis disorders), and confirmed that at least 13 of them are required for normal peroxisome assembly. We also demonstrate the relevance of Drosophila as a genetic model for the early developmental defects associated with the human peroxisome biogenesis disorders. Mutation of the PEX1 gene is the most common cause of peroxisome biogenesis disorders and is one of the causes of the most severe form of the disease, Zellweger syndrome. Inherited mutations in Drosophila Pex1 correlate with reproducible defects during early development. Notably, Pex1 mutant larvae exhibit abnormalities that are analogous to those exhibited by Zellweger syndrome patients, including developmental delay, poor feeding, severe structural abnormalities in the peripheral and central nervous systems, and early death. Finally, microarray analysis defined several clusters of genes whose expression varied significantly between wild-type and mutant larvae, implicating peroxisomal function in neuronal development, innate immunity, lipid and protein metabolism, gamete formation, and meiosis.
PMCID: PMC3180231  PMID: 21669930
8.  Isolation of peroxisome assembly mutants from Saccharomyces cerevisiae with different morphologies using a novel positive selection procedure 
The Journal of Cell Biology  1992;119(1):153-162.
We have developed a positive selection system for the isolation of Saccharomyces cerevisiae mutants with disturbed peroxisomal functions. The selection is based on the lethality of hydrogen peroxide (H2O2) that is produced in wild type cells during the peroxisomal beta- oxidation of fatty acids. In total, 17 mutants having a general impairment of peroxisome biogenesis were isolated, as revealed by their inability to grow on oleic acid as the sole carbon source and their aberrant cell fractionation pattern of peroxisomal enzymes. The mutants were shown to have monogenetic defects and to fall into 12 complementation groups. Representative members of each complementation group were morphologically examined by immunocytochemistry using EM. In one mutant the induction and morphology of peroxisomes is normal but import of thiolase is abrogated, while in another the morphology differs from the wild type: stacked peroxisomal membranes are present that are able to import thiolase but not catalase. These mutants suggest the existence of multiple components involved in peroxisomal protein import. Some mutants show the phenotype characteristic of glucose-repressed cells, an indication for the interruption of a signal transduction pathway resulting in organelle proliferation. In the remaining mutants morphologically detectable peroxisomes are absent: this phenotype is also known from fibroblasts of patients suffering from Zellweger syndrome, a disorder resulting from impairment of peroxisomes.
PMCID: PMC2289622  PMID: 1356111
9.  The Proteome of Human Liver Peroxisomes: Identification of Five New Peroxisomal Constituents by a Label-Free Quantitative Proteomics Survey 
PLoS ONE  2013;8(2):e57395.
The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD+ becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.
PMCID: PMC3583843  PMID: 23460848
10.  Mechanisms of organelle biogenesis govern stochastic fluctuations in organelle abundance 
eLife  2014;3:e02678.
Fluctuations in organelle abundance can profoundly limit the precision of cell biological processes from secretion to metabolism. We modeled the dynamics of organelle biogenesis and predicted that organelle abundance fluctuations depend strongly on the specific mechanisms that increase or decrease the number of a given organelle. Our model exactly predicts the size of experimentally measured Golgi apparatus and vacuole abundance fluctuations, suggesting that cells tolerate the maximum level of variability generated by the Golgi and vacuole biogenesis pathways. We observe large increases in peroxisome abundance fluctuations when cells are transferred from glucose-rich to fatty acid-rich environments. These increased fluctuations are significantly diminished in mutants lacking peroxisome fission factors, leading us to infer that peroxisome biogenesis switches from de novo synthesis to primarily fission. Our work provides a general framework for exploring stochastic organelle biogenesis and using fluctuations to quantitatively unravel the biophysical pathways that control the abundance of subcellular structures.
eLife digest
Any cell that has a nucleus also contains various other organelles, such as the mitochondria that generate energy inside the cells. Like the nucleus, most of these organelles are enclosed within a membrane. Unlike the nucleus, however, there can be two or more copies of other types of organelles in a healthy cell.
How do the numbers of the different organelles in a cell change? The copy number for a given organelle can be increased in two ways: by the synthesis of new organelles, or the fission of an existing organelle to form two new organelles. Conversely, the number of organelles can also be decreased in two ways: an organelle can decay, or two organelles can fuse to form one new organelle. The steady state for a given organelle results from a balance of these creative and destructive processes.
Researchers have thought for some time that cells are able to count how many organelles of a given type they contain. It was also thought that cells have some control over this number, but it was not known how precisely cells could control the number of organelles they contained. It was also not known how this level of precision was influenced by the different processes responsible for making new organelles.
To address these issues, Mukherji and O'Shea have developed a stochastic model that treats the processes of organelle creation and destruction as if they were simple chemical reactions. A tool from statistical physics, known as the fluctuation-dissipation theorem, was then used to analyze this model and derive an equation that predicts how the fluctuations in organelle number depend on the rates of the processes that govern organelle number.
Mukherji and O'Shea used this model to make predictions about various organelles in the budding yeast S. cerevisiae. For two of these—vacuoles and the Golgi apparatus—the processes that lead to an increase or decrease in the number of organelles are well understood. In both cases the model accurately predicted the level of fluctuations measured in experiments. Moreover, Mukherji and O'Shea found that cells exhibited the maximum predicted level of fluctuations that could be generated by the processes that either increased or decreased the number of each organelle.
The model was also able to shed light on a long-running debate over the cellular origins of an organelle called the peroxisome. This organelle—which is involved in breaking down fatty acids and other compounds—has been studied much less than the Golgi apparatus and vacuoles, but there is compelling evidence that new peroxisomes are created by de novo synthesis and by the fission of existing peroxisomes.
Mukherji and O'Shea found that fluctuations in the number of peroxisomes suggest that the production of new peroxisomes is dominated by fission when the yeast cells are grown in a medium that is rich in oleic acid: peroxisomes are metabolically active and proliferate rapidly in such a medium. In a glucose-rich medium, on the other hand, most new peroxisomes are produced by de novo synthesis. The case of the peroxisome thus highlights the possibility of extending this mathematical framework to explain the creation and destruction of organelles and other subcellular structures in a range of organisms and environments.
PMCID: PMC4046565  PMID: 24916159
systems biology; organelles; mathematical modeling; fluctuations; cell biology; S. cerevisiae
11.  PEX11β induces peroxisomal gene expression and alters peroxisome number during early Xenopus laevis development 
Peroxisomes are organelles whose roles in fatty acid metabolism and reactive oxygen species elimination have contributed much attention in understanding their origin and biogenesis. Many studies have shown that de novo peroxisome biogenesis is an important regulatory process, while yeast studies suggest that total peroxisome numbers are in part regulated by proteins such as Pex11, which can facilitate the division of existing peroxisomes. Although de novo biogenesis and divisions are likely important mechanisms, the regulation of peroxisome numbers during embryonic development is poorly understood. Peroxisome number and function are particularly crucial in oviparous animals such as frogs where large embryonic yolk and fatty acid stores must be quickly metabolized, and resulting reactive oxygen species eliminated. Here we elucidate the role of Pex11β in regulating peroxisomal gene expression and number in Xenopus laevis embryogenesis.
Microinjecting haemagglutinin (HA) tagged Pex11β in early embryos resulted in increased RNA levels for peroxisome related genes PMP70 and catalase at developmental stages 10 and 20, versus uninjected embryos. Catalase and PMP70 proteins were found in punctate structures at stage 20 in control embryos, whereas the injection of ectopic HA-Pex11β induced their earlier localization in punctate structures at stage 10. Furthermore, the peroxisomal marker GFP-SKL, which was found localized as peroxisome-like structures at stage 20, was similarly found at stage 10 when co-microinjected with HA-Pex11β.
Overexpressed Pex11β altered peroxisomal gene levels and induced the early formation of peroxisomes-like structures during development, both of which demonstrate that Pex11β may be a key regulator of peroxisome number in early Xenopus embryos.
PMCID: PMC3095563  PMID: 21526995
12.  Genome-Wide Localization Study of Yeast Pex11 Identifies Peroxisome–Mitochondria Interactions through the ERMES Complex 
Journal of Molecular Biology  2015;427(11):2072-2087.
Pex11 is a peroxin that regulates the number of peroxisomes in eukaryotic cells. Recently, it was found that a mutation in one of the three mammalian paralogs, PEX11β, results in a neurological disorder. The molecular function of Pex11, however, is not known. Saccharomyces cerevisiae Pex11 has been shown to recruit to peroxisomes the mitochondrial fission machinery, thus enabling proliferation of peroxisomes. This process is essential for efficient fatty acid β-oxidation. In this study, we used high-content microscopy on a genome-wide scale to determine the subcellular localization pattern of yeast Pex11 in all non-essential gene deletion mutants, as well as in temperature-sensitive essential gene mutants. Pex11 localization and morphology of peroxisomes was profoundly affected by mutations in 104 different genes that were functionally classified. A group of genes encompassing MDM10, MDM12 and MDM34 that encode the mitochondrial and cytosolic components of the ERMES complex was analyzed in greater detail. Deletion of these genes caused a specifically altered Pex11 localization pattern, whereas deletion of MMM1, the gene encoding the fourth, endoplasmic-reticulum-associated component of the complex, did not result in an altered Pex11 localization or peroxisome morphology phenotype. Moreover, we found that Pex11 and Mdm34 physically interact and that Pex11 plays a role in establishing the contact sites between peroxisomes and mitochondria through the ERMES complex. Based on these results, we propose that the mitochondrial/cytosolic components of the ERMES complex establish a direct interaction between mitochondria and peroxisomes through Pex11.
Graphical abstract
•Molecular function of Pex11, a protein with roles in metabolism and disease, is unknown.•Genome-wide screening determined subcellular localization of Pex11-GFP in yeast.•Mutants defective in components of the ERMES complex show altered Pex11 localization.•Pex11 physically interacts with the ERMES complex component Mdm34.•ERMES complex and Pex11 mediate interaction between mitochondria and peroxisomes.
PMCID: PMC4429955  PMID: 25769804
PPAR, peroxisome proliferator-activated receptor; ER, endoplasmic reticulum; GFP, green fluorescent protein; MYTH, membrane yeast two-hybrid; BiFC, bimolecular fluorescence complementation; high-content microscopy; computational image analysis; lipid metabolism; peroxisomal disorders; organelle juxtaposition
13.  Tysnd1 Deficiency in Mice Interferes with the Peroxisomal Localization of PTS2 Enzymes, Causing Lipid Metabolic Abnormalities and Male Infertility 
PLoS Genetics  2013;9(2):e1003286.
Peroxisomes are subcellular organelles involved in lipid metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids, among others. Peroxisome matrix proteins are synthesized in the cytoplasm. Targeting signals (PTS or peroxisomal targeting signal) at the C-terminus (PTS1) or N-terminus (PTS2) of peroxisomal matrix proteins mediate their import into the organelle. In the case of PTS2-containing proteins, the PTS2 signal is cleaved from the protein when transported into peroxisomes. The functional mechanism of PTS2 processing, however, is poorly understood. Previously we identified Tysnd1 (Trypsin domain containing 1) and biochemically characterized it as a peroxisomal cysteine endopeptidase that directly processes PTS2-containing prethiolase Acaa1 and PTS1-containing Acox1, Hsd17b4, and ScpX. The latter three enzymes are crucial components of the very-long-chain fatty acids β-oxidation pathway. To clarify the in vivo functions and physiological role of Tysnd1, we analyzed the phenotype of Tysnd1−/− mice. Male Tysnd1−/− mice are infertile, and the epididymal sperms lack the acrosomal cap. These phenotypic features are most likely the result of changes in the molecular species composition of choline and ethanolamine plasmalogens. Tysnd1−/− mice also developed liver dysfunctions when the phytanic acid precursor phytol was orally administered. Phyh and Agps are known PTS2-containing proteins, but were identified as novel Tysnd1 substrates. Loss of Tysnd1 interferes with the peroxisomal localization of Acaa1, Phyh, and Agps, which might cause the mild Zellweger syndrome spectrum-resembling phenotypes. Our data established that peroxisomal processing protease Tysnd1 is necessary to mediate the physiological functions of PTS2-containing substrates.
Author Summary
Peroxisomes are subcellular organelles that are present in almost all eukaryotic cells. The syllables “per-oxi” reflect the oxidative functions of these single-membrane-bound organelles in various metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids. In an earlier study we identified a protease named Tysnd1 that is specifically located in the peroxisomes and processes the enzymes catalyzing the peroxisomal β-oxidation of very-long-chain fatty acids. In this study, we identified two novel Tysnd1 substrates, Agps and Phyh, which are involved in plasmalogen synthesis and phytanic acid metabolism, respectively. To further investigate the in vivo function of Tysnd1, we analyzed Tysnd1 knock-out mice. Mice that lack Tysnd1 showed reduced peroxisomal β-oxidation activity and an altered plasmalogen composition, as well as an abnormal phytanic acid metabolism. Male infertility is one of the major phenotypic manifestations of Tysnd1 deficiency. Our data support the idea that Tysnd1 affects the localization and activity of some of its substrates inside peroxisomes. Altogether, our Tysnd1-deficient mouse model expands the current peroxisome biology knowledge with regard to the molecular pathogenic mechanisms that may be relevant to some patients with Zellweger syndrome spectrum disorders.
PMCID: PMC3573110  PMID: 23459139
14.  How Peroxisomes Affect Aflatoxin Biosynthesis in Aspergillus Flavus 
PLoS ONE  2012;7(10):e48097.
In filamentous fungi, peroxisomes are crucial for the primary metabolism and play a pivotal role in the formation of some secondary metabolites. Further, peroxisomes are important site for fatty acids β-oxidation, the formation of reactive oxygen species and for their scavenging through a complex of antioxidant activities. Oxidative stress is involved in different metabolic events in all organisms and it occurs during oxidative processes within the cell, including peroxisomal β-oxidation of fatty acids. In Aspergillus flavus, an unbalance towards an hyper-oxidant status into the cell is a prerequisite for the onset of aflatoxin biosynthesis. In our preliminary results, the use of bezafibrate, inducer of both peroxisomal β-oxidation and peroxisome proliferation in mammals, significantly enhanced the expression of pex11 and foxA and stimulated aflatoxin synthesis in A. flavus. This suggests the existence of a correlation among peroxisome proliferation, fatty acids β-oxidation and aflatoxin biosynthesis. To investigate this correlation, A. flavus was transformed with a vector containing P33, a gene from Cymbidium ringspot virus able to induce peroxisome proliferation, under the control of the promoter of the Cu,Zn-sod gene of A. flavus. This transcriptional control closely relates the onset of the antioxidant response to ROS increase, with the proliferation of peroxisomes in A. flavus. The AfP33 transformant strain show an up-regulation of lipid metabolism and an higher content of both intracellular ROS and some oxylipins. The combined presence of a higher amount of substrates (fatty acids-derived), an hyper-oxidant cell environment and of hormone-like signals (oxylipins) enhances the synthesis of aflatoxins in the AfP33 strain. The results obtained demonstrated a close link between peroxisome metabolism and aflatoxin synthesis.
PMCID: PMC3477134  PMID: 23094106
15.  Pmp27 promotes peroxisomal proliferation 
The Journal of Cell Biology  1995;129(2):345-355.
Peroxisomes perform many essential functions in eukaryotic cells. The weight of evidence indicates that these organelles divide by budding from preexisting peroxisomes. This process is not understood at the molecular level. Peroxisomal proliferation can be induced in Saccharomyces cerevisiae by oleate. This growth substrate is metabolized by peroxisomal enzymes. We have identified a protein, Pmp27, that promotes peroxisomal proliferation. This protein, previously termed Pmp24, was purified from peroxisomal membranes, and the corresponding gene, PMP27, was isolated and sequenced. Pmp27 shares sequence similarity with the Pmp30 family in Candida boidinii. Pmp27 is a hydrophobic peroxisomal membrane protein but it can be extracted by high pH, suggesting that it does not fully span the bilayer. Its expression is regulated by oleate. The function of Pmp27 was probed by observing the phenotype of strains in which the protein was eliminated by gene disruption or overproduced by expression from a multicopy plasmid. The strain containing the disruption (3B) was able to grow on all carbon sources tested, including oleate, although growth on oleate, glycerol, and acetate was slower than wild type. Strain 3B contained peroxisomes with all of the enzymes of beta-oxidation. However, in addition to the presence of a few modestly sized peroxisomes seen in a typical thin section of a cell growing on oleate-containing medium, cells of strain 3B also contained one or two very large peroxisomes. In contrast, cells in a strain in which Pmp27 was overexpressed contained an increased number of normal-sized peroxisomes. We suggest that Pmp27 promotes peroxisomal proliferation by participating in peroxisomal elongation or fission.
PMCID: PMC2199913  PMID: 7721939
16.  Peroxisomes and sexual development in fungi 
Peroxisomes are versatile and dynamic organelles that are essential for the development of most eukaryotic organisms. In fungi, many developmental processes, such as sexual development, require the activity of peroxisomes. Sexual reproduction in fungi involves the formation of meiotic-derived sexual spores, often takes place inside multicellular fruiting bodies and requires precise coordination between the differentiation of multiple cell types and the progression of karyogamy and meiosis. Different peroxisomal functions contribute to the orchestration of this complex developmental process. Peroxisomes are required to sustain the formation of fruiting bodies and the maturation and germination of sexual spores. They facilitate the mobilization of reserve compounds via fatty acid β-oxidation and the glyoxylate cycle, allowing the generation of energy and biosynthetic precursors. Additionally, peroxisomes are implicated in the progression of meiotic development. During meiotic development in Podospora anserina, there is a precise modulation of peroxisome assembly and dynamics. This modulation includes changes in peroxisome size, number and localization, and involves a differential activity of the protein-machinery that drives the import of proteins into peroxisomes. Furthermore, karyogamy, entry into meiosis and sorting of meiotic-derived nuclei into sexual spores all require the activity of peroxisomes. These processes rely on different peroxisomal functions and likely depend on different pathways for peroxisome assembly. Indeed, emerging studies support the existence of distinct import channels for peroxisomal proteins that contribute to different developmental stages.
PMCID: PMC3764329  PMID: 24046747
peroxisomes; peroxins; fungi; sexual development; meiosis; organelle biogenesis; cell differentiation
17.  Genome-wide analysis of signaling networks regulating fatty acid–induced gene expression and organelle biogenesis 
The Journal of Cell Biology  2008;181(2):281-292.
Reversible phosphorylation is the most common posttranslational modification used in the regulation of cellular processes. This study of phosphatases and kinases required for peroxisome biogenesis is the first genome-wide analysis of phosphorylation events controlling organelle biogenesis. We evaluate signaling molecule deletion strains of the yeast Saccharomyces cerevisiae for presence of a green fluorescent protein chimera of peroxisomal thiolase, formation of peroxisomes, and peroxisome functionality. We find that distinct signaling networks involving glucose-mediated gene repression, derepression, oleate-mediated induction, and peroxisome formation promote stages of the biogenesis pathway. Additionally, separate classes of signaling proteins are responsible for the regulation of peroxisome number and size. These signaling networks specify the requirements of early and late events of peroxisome biogenesis. Among the numerous signaling proteins involved, Pho85p is exceptional, with functional involvements in both gene expression and peroxisome formation. Our study represents the first global study of signaling networks regulating the biogenesis of an organelle.
PMCID: PMC2315675  PMID: 18426976
18.  Cloning and characterization of PAS5: a gene required for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris 
The Journal of Cell Biology  1993;123(3):535-548.
The biogenesis and maintenance of cellular organelles is of fundamental importance in all eukaryotic cells. One such organelle is the peroxisome. The establishment of a genetic system to study peroxisome biogenesis in the methylotrophic yeast Pichia pastoris has yielded many different complementation groups of peroxisomal assembly (pas) or peroxisome-deficient (per) mutants. Each appears to be deficient in functional peroxisomes. One of these mutants, pas5, has been characterized, complemented, and the gene sequenced. Ultrastructural studies show that normal peroxisomes are not present in pas5, but aberrant peroxisomal structures resembling "membranous ghosts" are frequently observed. The "peroxisome ghosts" appear to be induced and segregated to daughter cells normally. Biochemical fractionation analysis of organelles of the pas5 mutant reveals that peroxisomal matrix enzymes are induced normally but are found mostly in the cytosol. However, purification of peroxisome ghosts from the mutant shows that small amounts (< 5%) of matrix enzymes are imported. The PAS5 gene was cloned and found to encode a 127-kD protein, which contains a 200-amino acid-long region of homology with PAS1, NEM- sensitive factor (NSF), and other related ATPases. Weak homology to a yeast myosin was also observed. The gene is not essential for growth on glucose but is essential for growth on oleic acid and methanol. The role of PAS5 in peroxisome biogenesis is discussed.
PMCID: PMC2200126  PMID: 8227124
19.  Peroxisome Function Regulates Growth on Glucose in the Basidiomycete Fungus Cryptococcus neoformans▿  
Eukaryotic Cell  2006;6(1):60-72.
The function of the peroxisomes was examined in the pathogenic basidiomycete Cryptococcus neoformans. Recent studies reveal the glyoxylate pathway is required for virulence of diverse microbial pathogens of plants and animals. One exception is C. neoformans, in which isocitrate lyase (encoded by ICL1) was previously shown not to be required for virulence, and here this was extended to exclude also a role for malate synthase (encoded by MLS1). The role of peroxisomes, in which the glyoxylate pathway enzymes are localized in many organisms, was examined by mutation of two genes (PEX1 and PEX6) encoding AAA (ATPases associated with various cellular activities)-type proteins required for peroxisome formation. The pex1 and pex6 deletion mutants were unable to localize the fluorescent DsRED-SKL protein to peroxisomal punctate structures, in contrast to wild-type cells. pex1 and pex6 single mutants and a pex1 pex6 double mutant exhibit identical phenotypes, including abolished growth on fatty acids but no growth difference on acetate. Because both icl1 and mls1 mutants are unable to grow on acetate as the sole carbon source, these findings demonstrate that the glyoxylate pathway can function efficiently outside the peroxisome in C. neoformans. The pex1 mutant exhibits wild-type virulence in a murine inhalation model and in an insect host, demonstrating that peroxisomes are not required for virulence under these conditions. An unusual phenotype of the pex1 and pex6 mutants was that they grew poorly with glucose as the carbon source, but nearly wild type with galactose, which suggested impaired hexokinase function and that C. neoformans peroxisomes might function analogously to the glycosomes of the trypanosomid parasites. Deletion of the hexokinase HXK2 gene reduced growth in the presence of glucose and suppressed the growth defect of the pex1 mutant on glucose. The hexokinase 2 protein of C. neoformans contains a predicted peroxisome targeting signal (type 2) motif; however, Hxk2 fused to fluorescent proteins was not localized to peroxisomes. Thus, we hypothesize that glucose or glycolytic metabolites are utilized in the peroxisome by an as yet unidentified enzyme or regulate a pathway required by the fungus in the absence of peroxisomes.
PMCID: PMC1800366  PMID: 17041184
20.  Conserved Function of Pex11p and the Novel Pex25p and Pex27p in Peroxisome Biogenesis 
Molecular Biology of the Cell  2003;14(10):4316-4328.
We describe the isolation and characterization of a homologous pair of proteins, Pex25p (YPL112c) and Pex27p (YOR193w), whose C-termini are similar to the entire Pex11p. All three proteins localize to the peroxisomal membrane and are likely to form homo-oligomers. Deletion of any of the three genes resulted in enlarged peroxisomes as revealed by fluorescence and electron microscopy. The partial growth defect on fatty acids of a pex25Δ mutant was not exacerbated by the additional deletion of PEX27; however, when PEX11 was deleted on top of that, growth was abolished on all fatty acids. Moreover, a severe peroxisomal protein import defect was observed in the pex11Δpex25Δpex27Δ triple mutant strain. This import defect was also observed when cells were grown on ethanol-containing medium, where peroxisomes are not required, suggesting that the function of the proteins in peroxisome biogenesis exceeds their role in proliferation. When Pex25p was overexpressed in the triple mutant strain, growth on oleic acid was completely restored and a massive proliferation of laminar membranes and peroxisomes was observed. Our data demonstrate that Pex11p, Pex25p, and Pex27p build a family of proteins whose members are required for peroxisome biogenesis and play a role in the regulation of peroxisome size and number.
PMCID: PMC207022  PMID: 14517338
21.  Mutants of the Yeast Yarrowia lipolytica Defective in Protein Exit from the Endoplasmic Reticulum Are Also Defective in Peroxisome Biogenesis 
Molecular and Cellular Biology  1998;18(5):2789-2803.
Mutations in the SEC238 and SRP54 genes of the yeast Yarrowia lipolytica not only cause temperature-sensitive defects in the exit of the precursor form of alkaline extracellular protease and of other secretory proteins from the endoplasmic reticulum and in protein secretion but also lead to temperature-sensitive growth in oleic acid-containing medium, the metabolism of which requires the assembly of functionally intact peroxisomes. The sec238A and srp54KO mutations at the restrictive temperature significantly reduce the size and number of peroxisomes, affect the import of peroxisomal matrix and membrane proteins into the organelle, and significantly delay, but do not prevent, the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX1 and PEX6 genes, which encode members of the AAA family of N-ethylmaleimide-sensitive fusion protein-like ATPases, not only affect the exit of precursor forms of secretory proteins from the endoplasmic reticulum but also prevent the exit of the peroxisomal membrane proteins Pex2p and Pex16p from the endoplasmic reticulum and cause the accumulation of an extensive network of endoplasmic reticulum membranes. None of the peroxisomal matrix proteins tested associated with the endoplasmic reticulum in sec238A, srp54KO, pex1-1, and pex6KO mutant cells. Our data provide evidence that the endoplasmic reticulum is required for peroxisome biogenesis and suggest that in Y. lipolytica, the trafficking of some membrane proteins, but not matrix proteins, to the peroxisome occurs via the endoplasmic reticulum, results in their glycosylation within the lumen of the endoplasmic reticulum, does not involve transport through the Golgi, and requires the products encoded by the SEC238, SRP54, PEX1, and PEX6 genes.
PMCID: PMC110658  PMID: 9566898
22.  Peroxisomal Peripheral Membrane Protein YlInp1p Is Required for Peroxisome Inheritance and Influences the Dimorphic Transition in the Yeast Yarrowia lipolytica▿ † 
Eukaryotic Cell  2007;6(9):1528-1537.
Eukaryotic cells have evolved molecular mechanisms to ensure the faithful inheritance of organelles by daughter cells in order to maintain the benefits afforded by the compartmentalization of biochemical functions. Little is known about the inheritance of peroxisomes, organelles of lipid metabolism. We have analyzed peroxisome dynamics and inheritance in the dimorphic yeast Yarrowia lipolytica. Most peroxisomes are anchored at the periphery of cells of Y. lipolytica. In vivo video microscopy showed that at cell division, approximately half of the anchored peroxisomes in the mother cell are dislodged individually from their static positions and transported to the bud. Peroxisome motility is dependent on the actin cytoskeleton. YlInp1p is a peripheral peroxisomal membrane protein that affects the partitioning of peroxisomes between mother cell and bud in Y. lipolytica. In cells lacking YlInp1p, most peroxisomes were transferred to the bud, with only a few remaining in the mother cell, while in cells overexpressing YlInp1p, peroxisomes were preferentially retained in the mother cell, resulting in buds nearly devoid of peroxisomes. Our results are consistent with a role for YlInp1p in anchoring peroxisomes in cells. YlInp1p has a role in the dimorphic transition in Y. lipolytica, as cells lacking the YlINP1 gene more readily convert from the yeast to the mycelial form in oleic acid-containing medium, the metabolism of which requires peroxisomal activity, than does the wild-type strain. This study reports the first analysis of organelle inheritance in a true dimorphic yeast and identifies the first protein required for peroxisome inheritance in Y. lipolytica.
PMCID: PMC2043367  PMID: 17644654
23.  PEX5 and Ubiquitin Dynamics on Mammalian Peroxisome Membranes 
PLoS Computational Biology  2014;10(1):e1003426.
Peroxisomes are membrane-bound organelles within eukaryotic cells that post-translationally import folded proteins into their matrix. Matrix protein import requires a shuttle receptor protein, usually PEX5, that cycles through docking with the peroxisomal membrane, ubiquitination, and export back into the cytosol followed by deubiquitination. Matrix proteins associate with PEX5 in the cytosol and are translocated into the peroxisome lumen during the PEX5 cycle. This cargo translocation step is not well understood, and its energetics remain controversial. We use stochastic computational models to explore different ways the AAA ATPase driven removal of PEX5 may couple with cargo translocation in peroxisomal importers of mammalian cells. The first model considered is uncoupled, in which translocation is spontaneous, and does not immediately depend on PEX5 removal. The second is directly coupled, in which cargo translocation only occurs when its PEX5 is removed from the peroxisomal membrane. The third, novel, model is cooperatively coupled and requires two PEX5 on a given importomer for cargo translocation — one PEX5 with associated cargo and one with ubiquitin. We measure both the PEX5 and the ubiquitin levels on the peroxisomes as we vary the matrix protein cargo addition rate into the cytosol. We find that both uncoupled and directly coupled translocation behave identically with respect to PEX5 and ubiquitin, and the peroxisomal ubiquitin signal increases as the matrix protein traffic increases. In contrast, cooperatively coupled translocation behaves dramatically differently, with a ubiquitin signal that decreases with increasing matrix protein traffic. Recent work has shown that ubiquitin on mammalian peroxisome membranes can lead to selective degradation by autophagy, or ‘pexophagy.’ Therefore, the high ubiquitin level for low matrix cargo traffic with cooperatively coupled protein translocation could be used as a disuse signal to mediate pexophagy. This mechanism may be one way that cells could regulate peroxisome numbers.
Author Summary
Peroxisomes are small organelles that must continually import matrix proteins to contribute to cholesterol and bile acid synthesis, among other important functions. Cargo matrix proteins are shuttled to the peroxisomal membrane, but the only source of energy that has been identified to translocate the cargo into the peroxisome is consumed during the removal of the shuttle protein. Ubiquitin is used to recycle peroxisomal shuttle proteins, but is more generally used in cells to signal degradation of damaged or unneeded cellular components. How shuttle removal and cargo translocation are coupled energetically has been difficult to determine directly, so we investigate how different models of coupling would affect the measurable levels of ubiquitin on mammalian peroxisomes. We find that for the simplest models of coupling, ubiquitin levels decrease as cargo levels decrease. Conversely, for a novel cooperative model of coupling we find that ubiquitin levels increase as cargo levels decrease. This effect could allow the cell to degrade peroxisomes when they are not used, or to avoid degrading peroxisomes as cargo levels increase. Regardless of which model is found to be right, we have shown that ubiquitination levels of peroxisomes should respond to the changing traffic of matrix proteins into peroxisomes.
PMCID: PMC3894153  PMID: 24453954
24.  Expanding functional repertoires of fungal peroxisomes: contribution to growth and survival processes 
It has long been regarded that the primary function of fungal peroxisomes is limited to the β-oxidation of fatty acids, as mutants lacking peroxisomal function fail to grow in minimal medium containing fatty acids as the sole carbon source. However, studies in filamentous fungi have revealed that peroxisomes have diverse functional repertoires. This review describes the essential roles of peroxisomes in the growth and survival processes of filamentous fungi. One such survival mechanism involves the Woronin body, a Pezizomycotina-specific organelle that plugs the septal pore upon hyphal lysis to prevent excessive cytoplasmic loss. A number of reports have demonstrated that Woronin bodies are derived from peroxisomes. Specifically, the Woronin body protein Hex1 is targeted to peroxisomes by peroxisomal targeting sequence 1 (PTS1) and forms a self-assembled structure that buds from peroxisomes to form the Woronin body. Peroxisomal deficiency reduces the ability of filamentous fungi to prevent excessive cytoplasmic loss upon hyphal lysis, indicating that peroxisomes contribute to the survival of these multicellular organisms. Peroxisomes were also recently found to play a vital role in the biosynthesis of biotin, which is an essential cofactor for various carboxylation and decarboxylation reactions. In biotin-prototrophic fungi, peroxisome-deficient mutants exhibit growth defects when grown on glucose as a carbon source due to biotin auxotrophy. The biotin biosynthetic enzyme BioF (7-keto-8-aminopelargonic acid synthase) contains a PTS1 motif that is required for both peroxisomal targeting and biotin biosynthesis. In plants, the BioF protein contains a conserved PTS1 motif and is also localized in peroxisomes. These findings indicate that the involvement of peroxisomes in biotin biosynthesis is evolutionarily conserved between fungi and plants, and that peroxisomes play a key role in fungal growth.
PMCID: PMC3713238  PMID: 23882222
peroxisome; fungi; Woronin body; biotin; mitochondria
25.  Regulation of Peroxisome dynamics 
Current opinion in cell biology  2009;21(1):119-126.
Peroxisomes are single-membraned organelles ubiquitous to eukaryotic cells that house metabolic reactions that generate and destroy harmful oxidative intermediates. They are dynamic structures whose morphology, abundance, composition and function depend on the cell type and environment. Perhaps due to the potentially damaging and protective metabolic roles of peroxisomes and their dynamic presence in the cell, peroxisome biogenesis is emerging as a process that involves complex underlying mechanisms of regulated formation and maintenance. There are roughly 30 known peroxins, proteins involved in peroxisome biogenesis, many of which have been conserved from yeast to mammals (Table I). This review focuses on the biogenesis of peroxisomes with an emphasis on the regulation of peroxisome formation and the import of peroxisomal matrix proteins in the model organism Saccharomyces cerevisiae.
PMCID: PMC2681484  PMID: 19188056

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