Molecular mechanisms of peroxisome biogenesis in yeasts

Peroxisomes contain oxidases generating hydrogen peroxide, and catalase degrading this toxic compound. Another characteristic function of each eukaryotic peroxisome, from yeast to man, is fatty acid beta-oxidation. However, in peroxisomes a variety of other metabolic pathways are located. In fungi, peroxisomes contain enzymes involved in catabolism of unusual carbon and nitrogen sources (methanol, purines, D-amino acids, pipecolynic acid, sarcosine, glycolate, spermidine etc) as well as biosynthesis of lysine in yeasts and penicillin in mycelial fungi. Impairment of peroxisomal structure and functions causes many human disorders. The similar defects have been identified in yeast mutants defective in peroxisomal biogenesis. Peroxisomal biogenesis is actively studied during last two decades using uni- and multicellular model systems. It was observed that many aspects of peroxisomal biogenesis and proteins involved in this process display striking similarity between all eukaryotes, from yeasts to humans. Yeast is a convenient model system for this kind of research. Current review summarizes data on molecular events of peroxisomal biogenesis, functions of peroxine proteins, import of peroxisomal matrix and membrane proteins and on mechanisms of peroxisomedivision and inheritance.     

Mechanisms of autophagy and pexophagy in yeasts

Autophagy is a process of recycling of the intracellular constituents using vacuoles (lysosomes). General autophagy occurs due to involvement of highly conservative components found in all eukaryotes, from yeasts to higher plants and humans. Autophagy also could be a selective process and be involved in regulation of the cellular number of organelles, including that of peroxisomes. The process of specific autophagic peroxisome degradation is known as pexophagy. Yeasts appear to be convenient model for studying molecular mechanisms of pexophagy, and most known ATG genes (from the term AuTophaGy) were identified in yeast studies. This review examines characteristics of general autophagy, other types of autophagy as well as pexophagy, in particular, functions of Atg proteins in general autophagy and in macro- and micropexophagy. Special attention is given to mechanisms of phagophore assembly, the role of phosphatidylinositol-3-phosphate in pexophagy, the role of peroxines (proteins involved in peroxisome biogenesis) in pexophagy, as well as properties of Atg proteins specifically involved in micropexophagy.     

Novel peroxisome clustering mutants and peroxisome biogenesis mutants of Saccharomyces cerevisiae

The goal of this research is to identify and characterize the protein machinery that functions in the intracellular translocation and assembly of peroxisomal proteins in Saccharomyces cerevisiae. Several genes encoding proteins that are essential for this process have been identified previously by Kunau and collaborators, but the mutant collection was incomplete. We have devised a positive selection procedure that identifies new mutants lacking peroxisomes or peroxisomal function. Immunofluorescence procedures for yeast were simplified so that these mutants could be rapidly and efficiently screened for those in which peroxisome biogenesis is impaired. With these tools, we have identified four complementation groups of peroxisome biogenesis mutants, and one group that appears to express reduced amounts of peroxisomal proteins. Two of our mutants lack recognizable peroxisomes, although they might contain peroxisomal membrane ghosts like those found in Zellweger syndrome. Two are selectively defective in packaging peroxisomal proteins and moreover show striking intracellular clustering of the peroxisomes. The distribution of mutants among complementation groups implies that the collection of peroxisome biogenesis mutants is still incomplete. With the procedures described, it should prove straightforward to isolate mutants from additional complementation groups.     

PEX基因在过氧化物酶体形成及真菌致病性中的作用

过氧化物酶体(peroxisomes)是一类真核生物中普遍存在的细胞器,参与β-氧化、乙醛酸循环等多种重要的生化代谢。研究表明,过氧化物酶体在植物病原真菌侵染寄主过程中具有着举足轻重的作用。参与过氧化物酶体形成与增殖的基因,通常称为PEX基因。近年来,越来越多的PEX基因在植物病原真菌中得到鉴定,真菌过氧化物酶体的形成机制及其在植物病原真菌生长发育和致病过程中的作用越来越受到研究者的关注。本文围绕PEX 基因在过氧化物酶体形成中的作用、对过氧化物酶体相关生化代谢的影响,以及与植物病原真菌生长发育和致病性的关系进行了综述,以期为植物病原真菌致病机理研究和病害防控提供借鉴和参考。     

Proteins involved in microbody biogenesis and degradation in Aspergillus nidulans

Fungal microbodies (peroxisomes) are inducible organelles that proliferate in response to nutritional cues. Proteins involved in peroxisome biogenesis/proliferation are designated peroxins and are encoded by PEX genes. An autophagy-related process, termed pexophagy, is responsible for the selective removal of peroxisomes from the cell. Several genes involved in pexophagy are also required for autophagy and are collectively known as ATG genes. We have re-analysed the Aspergillus nidulans genome for the presence of PEX and ATG genes and have identified a number of previously missed genes. Also, we manually determined the correct intron positions in each identified gene. The data show that in A. nidulans and related fungi the basic set of genes involved in peroxisome biogenesis or degradation are conserved. However, both processes have features that more closely resemble organelle formation/degradation in mammals rather than yeast. Thus, filamentous fungi like A. nidulans are ideal model systems for peroxisome homeostasis in man.     

Role of the PAS1 gene of Pichia pastoris in peroxisome biogenesis

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.     

Peroxisome biogenesis occurs in an unsynchronized manner in close association with the endoplasmic reticulum in temperature-sensitive Yarrowia lipolytica Pex3p mutants

Pex3p is a peroxisomal integral membrane protein required early in peroxisome biogenesis, and Pex3p-deficient cells lack identifiable peroxisomes. Two temperature-sensitive pex3 mutant strains of the yeast Yarrowia lipolytica were made to investigate the role of Pex3p in the early stages of peroxisome biogenesis. In glucose medium at 16 degrees C, these mutants underwent de novo peroxisome biogenesis and exhibited early matrix protein sequestration into peroxisome-like structures found at the endoplasmic reticulum-rich periphery of cells or sometimes associated with nuclei. The de novo peroxisome biogenesis seemed unsynchronized, with peroxisomes occurring at different stages of development both within cells and between cells. Cells with peripheral nascent peroxisomes and cells with structures morphologically distinct from peroxisomes, such as semi/circular tubular structures that immunostained with antibodies to peroxisomal matrix proteins and to the endoplasmic reticulum-resident protein Kar2p, and that surrounded lipid droplets, were observed during up-regulation of peroxisome biogenesis in cells incubated in oleic acid medium at 16 degrees C. These structures were not detected in wild-type or Pex3p-deficient cells. Their role in peroxisome biogenesis remains unclear. Targeting of peroxisomal matrix proteins to these structures suggests that Pex3p directly or indirectly sequesters components of the peroxisome biogenesis machinery. Such a role is consistent with Pex3p overexpression producing cells with fewer, larger, and clustered peroxisomes.     

Pexophagy: The Selective Autophagy of Peroxisomes

Pichia pastoris and Hanseula polymorpha are methylotrophic yeasts capable of utilizing methanol, as a sole source of carbon and energy. Growth of these yeast species on methanol requires the synthesis of cytosolic and peroxisomal enzymes combined with the proliferation of peroxisomes. Peroxisomes are also abundantly present in the alkane-utilizing yeast Yarrowia lipolytica upon growth of cells on oleic acid. This feature has made these yeast species attractive model systems to dissect the molecular mechanisms controlling peroxisome biogenesis. We have found that upon glucose- or ethanol-induced catabolite inactivation of metabolically superfluous peroxisomes are rapidly and selectively degraded within the vacuole by a process called pexophagy, the selective removal of peroxisomes by autophagy-like processes. Utilizing several genetic screens, we have identified a number of genes that are essential for pexophagy. In this review, we will summarize our current knowledge of the molecular events of pexophagy.     

Pexophagy: the selective autophagy of peroxisomes

Pichia pastoris and Hansenula polymorpha are methylotrophic yeasts capable of utilizing methanol, as a sole source of carbon and energy. Growth of these yeast species on methanol requires the synthesis of cytosolic and peroxisomal enzymes combined with the proliferation of peroxisomes. Peroxisomes are also abundantly present in the alkane-utilizing yeast Yarrowia lipolytica upon growth of cells on oleic acid. This feature has made these yeast species attractive model systems to dissect the molecular mechanisms controlling peroxisome biogenesis. We have found that upon glucose- or ethanol-induced catabolite inactivation, metabolically superfluous peroxisomes are rapidly and selectively degraded within the vacuole by a process called pexophagy, the selective removal of peroxisomes by autophagy-like processes. Utilizing several genetic screens, we have identified a number of genes that are essential for pexophagy. In this review, we will summarize our current knowledge of the molecular events of pexophagy.     

Topogenesis of peroxisomal proteins

Molecular and biochemical analysis of the biogenesis of peroxisomes has made rapid progress in recent years. Research on the mechanism of targeting of peroxisomal proteins has revealed that many, but not all, peroxisomal proteins have a conserved tripeptide motif in their carboxy-terminal portions which is required for entry into peroxisomes; the topogenic signal mechanism thus differs in these instances from those employed in mitochondria and endoplasmic reticulum. Other factors involved in peroxisome biogenesis are also coming to light.     

Identification and functional characterization of Arabidopsis PEROXIN4 and the interacting protein PEROXIN22

Peroxins are genetically defined as proteins necessary for peroxisome biogenesis. By screening for reduced response to indole-3-butyric acid, which is metabolized to active auxin in peroxisomes, we isolated an Arabidopsis thaliana peroxin4 (pex4) mutant. This mutant displays sucrose-dependent seedling development and reduced lateral root production, characteristics of plant peroxisome malfunction. We used yeast two-hybrid analysis to determine that PEX4, an apparent ubiquitin-conjugating enzyme, interacts with a previously unidentified Arabidopsis protein, PEX22. A pex4 pex22 double mutant enhanced pex4 defects, confirming that PEX22 is a peroxin. Expression of both Arabidopsis genes together complemented yeast pex4 or pex22 mutant defects, whereas expression of either gene individually failed to rescue the corresponding yeast mutant. Therefore, it is likely that the Arabidopsis proteins can function similarly to the yeast PEX4-PEX22 complex, with PEX4 ubiquitinating substrates and PEX22 tethering PEX4 to the peroxisome. However, the severe sucrose dependence of the pex4 pex22 mutant is not accompanied by correspondingly strong defects in peroxisomal matrix protein import, suggesting that this peroxin pair may have novel plant targets in addition to those important in fungi. Isocitrate lyase is stabilized in pex4 pex22, indicating that PEX4 and PEX22 may be important during the remodeling of peroxisome matrix contents as glyoxysomes transition to leaf peroxisomes.     

Peroxisome biogenesis in the yeast Yarrowia lipolytica

Extensive perexisome proliferation during growth on oleic acid, combined with the availability of excellent genetic tools, makes the dimorphic yeast, Yarrowia lipolytica, a powerful model system to study the molecular mechanisms involved in peroxisome biogenesis. A combined genetic, biochemical, and morphological approach has revealed that the endoplasmic reticulum (ER) plays an essential role in the assembly of functional peroxisomes in this yeast. The trafficking of some membrane proteins to the peroxisomes occurs via the ER, results in their glyco-sylation in the ER lumen, does not involve transit through the Golgi, and requires the products of the SEC238, SRP54, PEX1, and PEX6 genes. The authors' data suggest a model for protein import into peroxisomes via two subpopulations of ER-derived vesicles that are distinct from secretory vesicles. A kinetic analysis of the trafficking of peroxisomal proteins in vivo has demonstrated that membrane and matrix proteins are initially targeted to multiple vesicular precursors that represent intermediates in the assembly pathway of peroxisomes. The authors have also recently identified a novel cytosolic chaperone, Pex20p, that assists in the oligomerization of thiolase in the cytosol and promotes its targeting to the peroxisome. These data provide the first evidence that a chaperone-assisted folding and oligomerization of thiolase in the cytosol is required for the import of this protein into the peroxisomal matrix.     

稻瘟病菌过氧化物酶体产生与降解的关键基因

过氧化物酶体(peroxisomes)是真核细胞中一类单层膜包被的细胞器,参与多种生化代谢.过氧化物酶体起源于内质网,过氧化物酶体形成相关的蛋白称为Peroxin,其编码基因通常写作PEX.细胞中过氧化物酶体的选择性消解称为过氧化物酶体自噬(pexophagy).参与细胞自噬(autophagy)的基因(ATG)大多参与过氧化物酶体自噬.近年来,丝状真菌中过氧化物酶体形成与降解机制的研究进展迅速,相关基因不断被鉴定.本文对相关研究进行了简要评述,并以稻瘟病菌为例,对丝状真菌基因组中可能的PEX和ATG基因进行了检索.发现稻瘟病菌中存在除PEX15,PEX17,PEX18,PEX21,PEX22,ATG19,ATG25,ATG30和ATG31之外的大多数PEX和ATG基因;同时,还存在多个丝状真菌特有的基因.说明过氧化物酶体的产生与消解在酵母、丝状真菌与哺乳动物之间相对保守,同时又各具特性.     

Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes

Peroxisomes play key roles in energy metabolism, cell signaling, and plant development. A better understanding of these important functions will be achieved with a more complete definition of the peroxisome proteome. The isolation of peroxisomes and their separation from mitochondria and other major membrane systems have been significant challenges in the Arabidopsis (Arabidopsis thaliana) model system. In this study, we present new data on the Arabidopsis peroxisome proteome obtained using two new technical advances that have not previously been applied to studies of plant peroxisomes. First, we followed density gradient centrifugation with free-flow electrophoresis to improve the separation of peroxisomes from mitochondria. Second, we used quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions. We provide evidence for peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analyses of Arabidopsis peroxisomes. Chimeric green fluorescent protein constructs of 35 proteins have been used to confirm their localization in peroxisomes or to identify endoplasmic reticulum contaminants. The distribution of many of these peroxisomal proteins between soluble, membrane-associated, and integral membrane locations has also been determined. This core peroxisomal proteome from nonphotosynthetic cultured cells contains a proportion of proteins that cannot be predicted to be peroxisomal due to the lack of recognizable peroxisomal targeting sequence 1 (PTS1) or PTS2 signals. Proteins identified are likely to be components in peroxisome biogenesis, beta-oxidation for fatty acid degradation and hormone biosynthesis, photorespiration, and metabolite transport. A considerable number of the proteins found in peroxisomes have no known function, and potential roles of these proteins in peroxisomal metabolism are discussed. This is aided by a metabolic network analysis that reveals a tight integration of functions and highlights specific metabolite nodes that most probably represent entry and exit metabolites that could require transport across the peroxisomal membrane.     

Pexophagy is responsible for 65% of cases of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBDs) is a group of diseases caused by mutations in one of the peroxins, proteins responsible for biogenesis of the peroxisomes. In recent years, it became clear that many peroxins (e.g., PEX3 and PEX14) play additional roles in peroxisome homeostasis (such as promoting autophagic degradation of peroxisomes or pexophagy), which are often opposite to their originally established functions in peroxisome formation and maintenance. Even more interesting, the peroxins that make up the peroxisomal AAA ATPase complex (AAA-complex) in yeast (Pex1, Pex6 and Pex15) or mammals (PEX1, PEX6, PEX26) are responsible for the downregulation of pexophagy. Moreover, this might be even their primary role in human: to prevent pexophagy by removing from the peroxisomal membrane the ubiquitinated peroxisomal matrix protein import receptor, Ub-PEX5, which is also a signal for the Ub-binding pexophagy receptor, NBR1. Remarkably, the peroxisomes rescued from pexophagy by autophagic inhibitors in PEX1^G843D (the most common PBD mutation) cells are able to import matrix proteins and improve their biochemical function suggesting that the AAA-complex per se is not essential for the protein import function in human. This paradigm-shifting discovery published in the current issue of Autophagy has raised hope for up to 65% of all PBD patients with various deficiencies in the AAA-complex. Recognizing PEX1, PEX6 and PEX26 as pexophagy suppressors will allow treating these patients with a new range of tools designed to target mammalian pexophagy.     

Localization of a portion of extranuclear ATM to peroxisomes

The gene mutated in the human genetic disorder ataxia-telangiectasia codes for a protein, ATM, the known functions of which include response to DNA damage, cell cycle control, and meiotic recombination. Consistent with these functions, ATM is predominantly present in the nucleus of proliferating cells; however, a significant proportion of the protein has also been detected outside the nucleus in cytoplasmic vesicles. To understand the possible role of extra-nuclear ATM, we initially investigated the nature of these vesicles. In this report we demonstrate that a portion of ATM co-localizes with catalase, that ATM is present in purified mouse peroxisomes, and that there are reduced levels of ATM in the post-mitochondrial membrane fraction of cells from a patient with a peroxisome biogenesis disorder. Furthermore the use of the yeast two-hybrid system demonstrated that ATM interacts directly with a protein involved in the import of proteins into the peroxisome matrix. Because peroxisomes are major sites of oxidative metabolism, we investigated catalase activity and lipid hydroperoxide levels in normal and A-T fibroblasts. Significantly decreased catalase activity and increased lipid peroxidation was observed in several A-T cell lines. The localization of ATM to peroxisomes may contribute to the pleiotropic nature of A-T.     

Environmental response of yeast peroxisomes

Nutritional changes can effect either the assembly or disassembly of yeast peroxisomes. In the past decade, insights regarding the molecular mechanisms of peroxisome assembly have been gained chiefly through the cloning of the PEX genes obtained by complementation of corresponding pex mutants in several yeast strains and Chinese hamster ovary cell lines. Depletion of these peroxins (proteins encoded by PEX genes) by deletion of the corresponding genes affects peroxisomal protein import biogenesis or proliferation. To complement these studies in the field, the authors undertook an investigation of the functions of a subset of Candida boidinii peroxisomal membrane proteins (PMPs), Pex11, Pmp47, and Pmp20, by analyzing strains of C. boidnii in which the genes encoding these proteins were deleted. The authors' studies show that Pex11p is involved in peroxisome proliferation; Pmp47 plays a role in the translocation, folding, or assembly of dihydroxyacetone synthase; and Pmp20 is probably involved in methanol metabolism. In contrast to the studies on peroxisome assembly, the molecular mechanisms of peroxisome degradation remain poorly understood. To shed light on this problem, the authors isolated Pichia pastoris mutants defective in peroxisome autopathy (pag mutants). A novel, double-fluorescence method used for the characterization of wild-type cells and of pag mutants enabled us to dissect the microautophagic degradation of peroxisomes into several distinct stages. These studies show that specific PAG gene products are involved in multiple steps of the process. Future cloning and characterization of the functions of PAG genes will reveal the molecular basis of peroxisome degradation.     

Peroxisomal Disorders: Clinical,Biochemical, and Molecular Aspects

Peroxisomes are subcellular organelles catalyzing a number of indispensable functions in cellular metabolism. The importance of peroxisomes in man is stressed by the existence of an expanding group of genetic diseases in which there is an impairment in one or more peroxisomal functions. Much has been learned in recent years about these functions and many of the enzymes involved have been characterized, purified and their cDNAs cloned. This has allowed resolution of the enzymatic and molecular basis of many of the single peroxisomal enzyme deficiencies. Similarly, the molecular basis of the peroxisome biogenesis disorders is also being resolved rapidly thanks to the successful use of CHO as well as yeast mutants. In this paper we will provide an overview of the peroxisomal disorders with particular emphasis on their clinical, biochemical and molecular characteristics.     

Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.