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.     

An Inventory of Peroxisomal Proteins and Pathways in Drosophila melanogaster

Peroxisomes are ubiquitous organelles housing a variety of essential biochemical pathways. Peroxisome dysfunction causes a spectrum of human diseases known as peroxisome biogenesis disorders (PBD). Although much is known regarding the mechanism of peroxisome biogenesis, it is still unclear how peroxisome dysfunction leads to the disease state. Several recent studies have shown that mutations in Drosophila peroxin genes cause phenotypes similar to those seen in humans with PBDs suggesting that Drosophila might be a useful system to model PBDs. We have analyzed the proteome of Drosophila to identify the proteins involved in peroxisomal biogenesis and homeostasis as well as metabolic enzymes that function within the organelle. The subcellular localization of five of these predicted peroxisomal proteins was confirmed. Similar to Caenorhabditis elegans, Drosophila appears to only utilize the peroxisome targeting signal type 1 system for matrix protein import. This work will further our understanding of peroxisomes in Drosophila and add to the usefulness of this emerging model system.     

Central role of peroxisomes in isoprenoid biosynthesis

Peroxisomes contain enzymes catalyzing a number of indispensable metabolic functions mainly related to lipid metabolism. The importance of peroxisomes in man is stressed by the existence of genetic disorders in which the biogenesis of the organelle is defective, leading to complex developmental and metabolic phenotypes. The purpose of this review is to emphasize some of the recent findings related to the localization of cholesterol biosynthetic enzymes in peroxisomes and to discuss the impairment of cholesterol biosynthesis in peroxisomal deficiency diseases.     

Molecular basis of peroxisomal biogenesis disorders caused by defects in peroxisomal matrix protein import

Peroxisomal biogenesis disorders (PBDs) represent a spectrum of autosomal recessive metabolic disorders that are collectively characterized by abnormal peroxisome assembly and impaired peroxisomal function. The importance of this ubiquitous organelle for human health is highlighted by the fact that PBDs are multisystemic disorders that often cause death in early infancy. Peroxisomes contribute to central metabolic pathways. Most enzymes in the peroxisomal matrix are linked to lipid metabolism and detoxification of reactive oxygen species. Proper assembly of peroxisomes and thus also import of their enzymes relies on specific peroxisomal biogenesis factors, so called peroxins with PEX being the gene acronym. To date, 13 PEX genes are known to cause PBDs when mutated. Studies of the cellular and molecular defects in cells derived from PBD patients have significantly contributed to the understanding of the functional role of the corresponding peroxins in peroxisome assembly. In this review, we discuss recent data derived from both human cell culture as well as model organisms like yeasts and present an overview on the molecular mechanism underlying peroxisomal biogenesis disorders with emphasis on disorders caused by defects in the peroxisomal matrix protein import machinery. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.     

The evolutionary origin of peroxisomes: an ER-peroxisome connection

The peroxisome is an essential eukaryotic organelle, crucial for lipid metabolism and free radical detoxification, development, differentiation, and morphogenesis from yeasts to humans. Loss of peroxisomes invariably leads to fatal peroxisome biogenesis disorders in man. The evolutionary origin of peroxisomes remains unsolved; proposals for either a symbiogenetic or cellular membrane invagination event are unconclusive. To address this question, we have probed with a peroxisomal proteome, an "ensemble" of 19 representative eukaryotic complete genomes. Molecular phylogenetic and sequence comparison tools allowed us to identify four proteins as peroxisomal markers for unequivocal in silico peroxisome detection. We have then detected the Apicomplexa phylum as the first group of organisms devoid of peroxisomes, in the presence of mitochondria. Finally, we deliver evidence against a prokaryotic ancestor of peroxisomes: (1) the peroxisomal membrane is composed of purely eukaryotic bricks and is thus useful to trace the eukaryotes in their evolutionary paths and (2) the peroxisomal matrix protein import system shares mechanistic similarities with the endoplasmic reticulum/proteasome degradation process, indicating a common evolutionary history.     

The peroxisome: still a mysterious organelle

More than half a century of research on peroxisomes has revealed unique features of this ubiquitous subcellular organelle, which have often been in disagreement with existing dogmas in cell biology. About 50 peroxisomal enzymes have so far been identified, which contribute to several crucial metabolic processes such as β-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, and render peroxisomes indispensable for human health and development. It became obvious that peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. However, many aspects of peroxisome biology are still mysterious. This review addresses recent exciting discoveries on the biogenesis, formation and degradation of peroxisomes, on peroxisomal dynamics and division, as well as on the interaction and cross talk of peroxisomes with other subcellular compartments. Furthermore, recent advances on the role of peroxisomes in medicine and in the identification of novel peroxisomal proteins are discussed.     

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.     

The Proteome of Human Liver Peroxisomes: Identification of Five New Peroxisomal Constituents by a Label-Free Quantitative Proteomics Survey

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.     

Peroxisome senescence in human fibroblasts

The molecular mechanisms of peroxisome biogenesis have begun to emerge; in contrast, relatively little is known about how the organelle functions as cells age. In this report, we characterize age-related changes in peroxisomes of human cells. We show that aging compromises peroxisomal targeting signal 1 (PTS1) protein import, affecting in particular the critical antioxidant enzyme catalase. The number and appearance of peroxisomes are altered in these cells, and the organelles accumulate the PTS1-import receptor, Pex5p, on their membranes. Concomitantly, cells produce increasing amounts of the toxic metabolite hydrogen peroxide, and we present evidence that this increased load of reactive oxygen species may further reduce peroxisomal protein import and exacerbate the effects of aging.     

Peroxisome biogenesis disorders: Molecular basis for impaired peroxisomal membrane assembly: In metabolic functions and biogenesis of peroxisomes in health and disease

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS). Gene defects of peroxins required for both membrane assembly and matrix protein import are identified: ten mammalian pathogenic peroxins for ten complementation groups of PBDs, are required for matrix protein import; three, Pex3p, Pex16p and Pex19p, are shown to be essential for peroxisome membrane assembly and responsible for the most severe ZS in PBDs of three complementation groups 12, 9, and 14, respectively. Patients with severe ZS with defects of PEX3, PEX16, and PEX19 tend to carry severe mutation such as nonsense mutations, frameshifts and deletions. With respect to the function of these three peroxins in membrane biogenesis, two distinct pathways have been proposed for the import of peroxisomal membrane proteins in mammalian cells: a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex16p-dependent class II pathway. In class II pathway, Pex19p also forms a soluble complex with newly synthesized Pex3p as the chaperone for Pex3p in the cytosol and directly translocates it to peroxisomes. Pex16p functions as the peroxisomal membrane receptor that is specific to the Pex3p-Pex19p complexes. A model for the import of peroxisomal membrane proteins is suggested, providing new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p. Another model suggests that in Saccharomyces cerevisiae peroxisomes likely emerge from the endoplasmic reticulum. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of peroxisomes in Health and Disease.     

The surprising complexity of peroxisome biogenesis

Peroxisomes are small organelles with a single boundary membrane. All of their matrix proteins are nuclear-encoded, synthesized on free ribosomes in the cytosol, and post-translationally transported into the organelle. This may sound familiar, but in fact, peroxisome biogenesis is proving to be surprisingly unique. First, there are several classes of plant peroxisomes, each specialized for a different metabolic function and sequestering specific matrix enzymes. Second, although the mechanisms of peroxisomal protein import are conserved between the classes, multiple pathways of protein targeting and translocation have been defined. At least two different types of targeting signals direct proteins to the peroxisome matrix. The most common peroxisomal targeting signal is a tripeptide limited to the carboxyl terminus of the protein. Some peroxisomal proteins possess an amino-terminal signal which may be cleaved after import. Each targeting signal interacts with a different cytosolic receptor; other cytosolic factors or chaperones may also form a complex with the peroxisomal protein before it docks on the membrane. Peroxisomes have the unusual capacity to import proteins that are fully folded or assembled into oligomers. Although at least 20 proteins (mostly peroxins) are required for peroxisome biogenesis, the role of only a few of these have been determined. Future efforts will be directed towards an understanding of how these proteins interact and contribute to the complex process of protein import into peroxisomes.     

The biogenesis protein PEX14 is an optimal marker for the identification and localization of peroxisomes in different cell types, tissues, and species in morphological studies

Catalase and ABCD3 are frequently used as markers for the localization of peroxisomes in morphological experiments. Their abundance, however, is highly dependent on metabolic demands, reducing the validity of analyses of peroxisomal abundance and distribution based solely on these proteins. We therefore attempted to find a protein which can be used as an optimal marker for peroxisomes in a variety of species, tissues, cell types and also experimental designs, independently of peroxisomal metabolism. We found that the biogenesis protein peroxin 14 (PEX14) is present in comparable amounts in the membranes of every peroxisome and is optimally suited for immunoblotting, immunohistochemistry, immunofluorescence, and immunoelectron microscopy. Using antibodies against PEX14, we could visualize peroxisomes with almost undetectable catalase content in various mammalian tissue sections (submandibular and adrenal gland, kidney, testis, ovary, brain, and pancreas from mouse, cat, baboon, and human) and cell cultures (primary cells and cell lines). Peroxisome labeling with catalase often showed a similar tissue distribution to the mitochondrial enzyme mitochondrial superoxide dismutase (both responsible for the degradation of reactive oxygen species), whereas ABCD3 exhibited a distinct labeling only in cells involved in lipid metabolism. We increased the sensitivity of our methods by using QuantumDots?, which have higher emission yields compared to classic fluorochromes and are unsusceptible to photobleaching, thereby allowing more exact quantification without artificial mistakes due to heterogeneity of individual peroxisomes. We conclude that PEX14 is indeed the best marker for labeling of peroxisomes in a variety of tissues and cell types in a consistent fashion for comparative morphometry.     

Molecular insights into peroxisome homeostasis and peroxisome biogenesis disorders

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

Peroxisome metabolism and cellular aging

The essential role of peroxisomes in fatty acid oxidation, anaplerotic metabolism, and hydrogen peroxide turnover is well established. Recent findings suggest that these and other related biochemical processes governed by the organelle may also play a critical role in regulating cellular aging. The goal of this review is to summarize and integrate into a model the evidence that peroxisome metabolism actually helps define the replicative and chronological age of a eukaryotic cell. In this model, peroxisomal reactive oxygen species (ROS) are seen as altering organelle biogenesis and function, and eliciting changes in the dynamic communication networks that exist between peroxisomes and other cellular compartments. At low levels, peroxisomal ROS activate an anti-aging program in the cell; at concentrations beyond a specific threshold, a pro-aging course is triggered.     

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

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

Removal of Pex3p is an important initial stage in selective peroxisome degradation in Hansenula polymorpha

Selective degradation of peroxisomes (macropexophagy) in Hansenula polymorpha involves the sequestration of individual organelles to be degraded by membranes prior to the fusion of this compartment with the vacuole and subsequent degradation of the whole organelle by vacuolar hydrolases. Here we show that Pex3p, a peroxisomal membrane protein essential for peroxisome biogenesis, escapes this autophagic process. Upon induction of macropexophagy, Pex3p is removed from the organelle tagged for degradation prior to its sequestration. Our data indicate that Pex3p degradation is essential to allow the initiation of the organellar degradation process. Also, in a specific peroxisome degradation-deficient (pdd) mutant in which sequestration still occurs but the vacuolar fusion event is disturbed, the turnover of Pex3p is still observed. Taken together, our data suggest that degradation of Pex3p is part of the initial degradation machinery of individual peroxisomes.     

Evaluation of the role of the endoplasmic reticulum-Golgi transit in the biogenesis of peroxisomal membrane proteins in wild type and peroxisome biogenesis mutant CHO cells

Peroxisomes are thought to be formed by division of pre-existing peroxisomes after the import of newly synthesized proteins. However, it has been recently suggested that the endoplasmic reticulum (ER) provides an alternative de novo mechanism for peroxisome biogenesis in some cells. To test a possible role of the ER-Golgi transit in peroxisome biogenesis in mammalian cells, we evaluated the biogenesis of three peroxisomal membrane proteins (PMPs): ALDRP (adrenoleukodystrophy related protein), PMP70 and Pex3p in CHO cells. We constructed chimeric genes encoding these PMPs and green fluorescent protein (GFP), and transiently transfected them to wild type and mutant CHO cells, in which normal peroxisomes were replaced by peroxisomal membrane ghosts. The expressed proteins were targeted to peroxisomes and peroxisomal ghosts correctly in the presence or absence of Brefeldin A (BFA), a drug known to block the ER-Golgi transit. Furthermore, low temperature did not disturb the targeting of Pex3p-GFP to peroxisomes. We also constructed two chimeric proteins of PMPs containing an ER retention signal "DEKKMP": GFP-ALDRP-DEKKMP and myc- Pex3p-DEKKMP. These proteins were mostly targeted to peroxisomes. No colocalization with an ER maker was found. These results suggest that the classical ER-Golgi pathway does not play a major role in the biogenesis of mammalian PMPs.     

Peroxisome biogenesis: where Arf and coatomer might be involved

The present review summarizes recent observations on binding of Arf and COPI coat to isolated rat liver peroxisomes. The general structural and functional features of both Arf and coatomer were considered along with the requirements and dependencies of peroxisomal Arf and coatomer recruitment. Studies on the expression of mammalian Pex11 proteins, mainly Pex11alpha and Pex11beta, intimately related to the process of peroxisome proliferation, revealed a sequence of individual steps including organelle elongation/tubulation, formation of membrane and matrix protein patches segregating distinct proteins from each other, development of membrane constrictions and final membrane fission. Based on the similarities of the processes leading to cargo selection and concentration on Golgi membranes on the one hand and to the formation of peroxisomal protein patches on the other hand, an implication of Arf and COPI in distinct processes of peroxisomal proliferation is hypothesized. Alternatively, peroxisomal Arf/COPI might facilitate the formation of COPI-coated peroxisomal vesicles functioning in cargo transport and retrieval from peroxisomes to the ER. Recent observations suggesting transport of Pex3 and Pex19 during early steps of peroxisome biogenesis from the ER to peroxisomes inevitably propose such a retrieval mechanism, provided the ER to peroxisome pathway is based on transporting vesicles.     

Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane

We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.