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The whole-genome sequence of strain K-4, isolated from grass silage in Thailand, which constitutes a chromosome and two plasmids, is 2,914,933 bp long, has a GC content of 37.5%, and contains 2,734 predicted protein-coding genes. Average nucleotide identity based on BLAST+ (ANIb) and digital DNA-DNA hybridization (dDDH) values indicated that the strain K-4 was closely related to Enterococcus faecalis .

Ethanolamine-containing alkenyl ether glycerophospholipids, plasmalogens, are major cell membrane components of mammalian cells that activate membrane protein receptors such as ion transporters and G-protein coupled receptors. However, the mechanism by which plasmalogens modulate receptor function is unknown. Here, we found that exogenously added plasmalogens activate transient receptor potential cation channel subfamily C member 4 (TRPC4) to increase Ca

Summary: Reactive oxygen species (ROS) play critical roles in metabolism and disease, yet a comprehensive analysis of the cellular response to oxidative stress is lacking. To systematically identify regulators of oxidative stress, we conducted genome-wide Cas9/CRISPR and shRNA screens. This revealed a detailed picture of diverse pathways that control oxidative stress response, ranging from the TCA cycle and DNA repair machineries to iron transport, trafficking, and metabolism. Paradoxically, disrupting the pentose phosphate pathway (PPP) at the level of phosphogluconate dehydrogenase (PGD) protects cells against ROS. This dramatically alters metabolites in the PPP, consistent with rewiring of upper glycolysis to promote antioxidant production. In addition, disruption of peroxisomal import unexpectedly increases resistance to oxidative stress by altering the localization of catalase. Together, these studies provide insights into the roles of peroxisomal matrix import and the PPP in redox biology and represent a rich resource for understanding the cellular response to oxidative stress. : Despite its importance in metabolism and disease, a comprehensive analysis of the cellular response to oxidative stress is lacking. Here, Dubreuil et al. use genome-wide screens to identify cellular regulators of oxidative stress. They investigate paradoxical mechanisms by which disruption of the pentose phosphate and peroxisomal import pathways protect cells. Keywords: CRISPR, genome-wide screen, shRNA, pentose phosphate pathway, peroxisomal import pathway, catalase, phosphogluconate dehydrogenase, oxidative stress, reactive oxygen species

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 H

Peroxisomes are presented in all eukaryotic cells and play essential roles in many of lipid metabolic pathways, including β-oxidation of fatty acids and synthesis of ether-linked glycerophospholipids, such as plasmalogens. Impaired peroxisome biogenesis, including defects of membrane assembly, import of peroxisomal matrix proteins, and division of peroxisome, causes peroxisome biogenesis disorders (PBDs). Fourteen complementation groups of PBDs are found, and their complementing genes termed PEXs are isolated. Several new mutations in peroxins from patients with mild PBD phenotype or patients with phenotypes unrelated to the commonly observed impairments of PBD patients are found by next-generation sequencing. Exploring a dysfunctional step(s) caused by the mutation is important for unveiling the pathogenesis of novel mutation by means of cellular and biochemical analyses.

Peroxisome is an organelle conserved in almost all eukaryotic cells with a variety of functions in cellular metabolism, including fatty acid β-oxidation, synthesis of ether glycerolipid plasmalogens, and redox homeostasis. Such metabolic functions and the exclusive importance of peroxisomes have been highlighted in fatal human genetic disease called peroxisomal biogenesis disorders (PBDs). Recent advances in this field have identified over 30 PEX genes encoding peroxins as essential factors for peroxisome biogenesis in various species from yeast to humans. Functional delineation of the peroxins has revealed that peroxisome biogenesis comprises the processes, involving peroxisomal membrane assembly, matrix protein import, division, and proliferation. Catalase, the most abundant peroxisomal enzyme, catalyzes decomposition of hydrogen peroxide. Peroxisome plays pivotal roles in the cellular redox homeostasis and the response to oxidative stresses, depending on intracellular localization of catalase.

Peroxisomes proliferate by sequential processes comprising elongation, constriction, and scission of peroxisomal membrane. It is known that the constriction step is mediated by a GTPase named dynamin-like protein 1 (DLP1) upon efficient loading of GTP. However, mechanism of fuelling GTP to DLP1 remains unknown in mammals. We earlier show that nucleoside diphosphate (NDP) kinase-like protein, termed dynamin-based ring motive-force organizer 1 (DYNAMO1), generates GTP for DLP1 in a red alga, Cyanidioschyzon merolae. In the present study, we identified that nucleoside diphosphate kinase 3 (NME3), a mammalian homologue of DYNAMO1, localizes to peroxisomes. Elongated peroxisomes were observed in cells with suppressed expression of NME3 and fibroblasts from a patient lacking NME3 due to the homozygous mutation at the initiation codon of NME3. Peroxisomes proliferated by elevation of NME3 upon silencing the expression of ATPase family AAA domain containing 1, ATAD1. In the wild-type cells expressing catalytically-inactive NME3, peroxisomes were elongated. These results suggest that NME3 plays an important role in peroxisome division in a manner dependent on its NDP kinase activity. Moreover, the impairment of peroxisome division reduces the level of ether-linked glycerophospholipids, ethanolamine plasmalogens, implying the physiological importance of regulation of peroxisome morphology.

Plasmalogens are a subclass of ether glycerophospholipids characterized by a vinyl-ether bond at the sn-1 position of the glycerol backbone. Plasmalogen biosynthesis is initiated in peroxisomes. At the third step of plasmalogen synthesis, alkyl-dihydroxyacetonephosphate (DHAP) is enzymatically reduced to 1-alkyl-sn-glycero-3-phospate by acyl/alkyl DHAP reductase (ADHAPR), whose activity is found in both peroxisomes and microsomes. We herein show that knockdown of ADHAPR in HeLa cells reduced the synthesis of ethanolamine plasmalogen (PlsEtn), similar to the Chinese hamster ovary cell mutant FAA.K1B deficient in ADHAPR activity. Endogenous ADHAPR and ectopically expressed FLAG-tagged ADHAPR were localized to peroxisomes and endoplasmic reticulum (ER) as a type I integral membrane protein in HeLa cells. ADHAPR targets to peroxisomes via a Pex19p-dependent class I pathway. In addition, it is also inserted into the ER via the SRP-dependent mechanism. The ADHAPR mutant lacking the N-terminal domain preferentially targets to the ER, restoring the reduced level of PlsEtn synthesis in FAA.K1B cell. In contrast, the expression of full-length ADHAPR in the mutant cells elevates the synthesis of phosphatidylethanolamine, but not PlsEtn. Taken together, these results suggest that the third step of plasmalogen synthesis is mediated by ER-localized ADHAPR.

Peroxisomes are single-membrane organelles present in eukaryotes. The functional importance of peroxisomes in humans is represented by peroxisome-deficient peroxisome biogenesis disorders (PBDs), including Zellweger syndrome. Defects in the genes that encode the 14 peroxins that are required for peroxisomal membrane assembly, matrix protein import and division have been identified in PBDs. A number of recent findings have advanced our understanding of the biology, physiology and consequences of functional defects in peroxisomes. In this Review, we discuss a cooperative cell defense mechanisms against oxidative stress that involves the localization of BAK (also known as BAK1) to peroxisomes, which alters peroxisomal membrane permeability, resulting in the export of catalase, a peroxisomal enzyme. Another important recent finding is the discovery of a nucleoside diphosphate kinase-like protein that has been shown to be essential for how the energy GTP is generated and provided for the fission of peroxisomes. With regard to PBDs, we newly identified a mild mutation, Pex26-F51L that causes only hearing loss. We will also discuss findings from a new PBD model mouse defective in Pex14, which manifested dysregulation of the BDNF–TrkB pathway, an essential signaling pathway in cerebellar morphogenesis. Here, we thus aim to provide a current view of peroxisome biogenesis and the molecular pathogenesis of PBDs.

The peroxisome is a subcellular organelle that functions in essential metabolic pathways, including biosynthesis of plasmalogens, fatty acid β-oxidation of very-long-chain fatty acids, and degradation of hydrogen peroxide. Peroxisome biogenesis disorders (PBDs) manifest as severe dysfunction in multiple organs, including the central nervous system (CNS), but the pathogenic mechanisms in PBDs are largely unknown. Because CNS integrity is coordinately established and maintained by neural cell interactions, we here investigated whether cell-cell communication is impaired and responsible for the neurological defects associated with PBDs. Results from a noncontact co-culture system consisting of primary hippocampal neurons with glial cells revealed that a peroxisome-deficient astrocytic cell line secretes increased levels of brain-derived neurotrophic factor (BDNF), resulting in axonal branching of the neurons. Of note, the BDNF expression in astrocytes was not affected by defects in plasmalogen biosynthesis and peroxisomal fatty acid β-oxidation in the astrocytes. Instead, we found that cytosolic reductive states caused by a mislocalized catalase in the peroxisome-deficient cells induce the elevation in BDNF secretion. Our results suggest that peroxisome deficiency dysregulates neuronal axogenesis by causing a cytosolic reductive state in astrocytes. We conclude that astrocytic peroxisomes regulate BDNF expression and thereby support neuronal integrity and function.

Fourteen PEX genes are currently identified as genes responsible for peroxisome biogenesis disorders (PBDs). Patients with PBDs manifest as neurodegenerative symptoms such as neuronal migration defect and malformation of the cerebellum. To address molecular mechanisms underlying the pathogenesis of PBDs, mouse models for the PBDs have been generated by targeted disruption of Pex genes. Pathological phenotypes and metabolic abnormalities in Pex-knockout mice well resemble those of the patients with PBDs. The mice with tissue- or cell type-specific inactivation of Pex genes have also been established by using a Cre-loxP system. The genetically modified mice reveal that pathological phenotypes of PBDs are mediated by interorgan and intercellular communications. Despite the illustrations of detailed pathological phenotypes in the mutant mice, mechanistic insights into pathogenesis of PBDs are still underway. In this chapter, we overview the phenotypes of Pex-inactivated mice and the current understanding of the pathogenesis underlying PBDs.

Peroxisomes are presented in all eukaryotic cells and play essential roles in many of lipid metabolic pathways, including β-oxidation of fatty acids and synthesis of ether-linked glycerophospholipids, such as plasmalogens. Impaired peroxisome biogenesis, including defects of membrane assembly, import of peroxisomal matrix proteins, and division of peroxisome, causes peroxisome biogenesis disorders (PBDs). Fourteen complementation groups of PBDs are found, and their complementing genes termed PEXs are isolated. Several new mutations in peroxins from patients with mild PBD phenotype or patients with phenotypes unrelated to the commonly observed impairments of PBD patients are found by next-generation sequencing. Exploring a dysfunctional step(s) caused by the mutation is important for unveiling the pathogenesis of novel mutation by means of cellular and biochemical analyses.

Peroxisome is an organelle conserved in almost all eukaryotic cells with a variety of functions in cellular metabolism, including fatty acid β-oxidation, synthesis of ether glycerolipid plasmalogens, and redox homeostasis. Such metabolic functions and the exclusive importance of peroxisomes have been highlighted in fatal human genetic disease called peroxisomal biogenesis disorders (PBDs). Recent advances in this field have identified over 30 PEX genes encoding peroxins as essential factors for peroxisome biogenesis in various species from yeast to humans. Functional delineation of the peroxins has revealed that peroxisome biogenesis comprises the processes, involving peroxisomal membrane assembly, matrix protein import, division, and proliferation. Catalase, the most abundant peroxisomal enzyme, catalyzes decomposition of hydrogen peroxide. Peroxisome plays pivotal roles in the cellular redox homeostasis and the response to oxidative stresses, depending on intracellular localization of catalase.

Plasmalogens, mostly ethanolamine-containing alkenyl ether phospholipids, are a major subclass of glycerophospholipids. Plasmalogen synthesis is initiated in peroxisomes and completed in the endoplasmic reticulum. The absence of plasmalogens in several organs of peroxisome biogenesis-defective patients suggests that the de novo synthesis of plasmalogens plays a pivotal role in its homeostasis in tissues. Plasmalogen synthesis is regulated by modulating the stability of fatty acyl-CoA reductase 1 on peroxisomal membranes, a rate-limiting enzyme in plasmalogen synthesis, by sensing plasmalogens in the inner leaflet of plasma membranes. Dysregulation of plasmalogen homeostasis impairs cholesterol biosynthesis by altering the stability of squalene monooxygenase, a key enzyme in cholesterol biosynthesis, implying physiological consequences of plasmalogen homeostasis with respect to cholesterol metabolism in cells, as well as in organs such as the liver.

Neuroinflammation characterized by activation of glial cells is observed in various neurodegenerative diseases including Alzheimer's disease (AD). Although the reduction of ether-type glycerophospholipids, plasmalogens (Pls), in the brain is reported in AD patients, the mechanism of the reduction and its impact on neuroinflammation remained elusive. In the present study, we found for the first time that various inflammatory stimuli reduced Pls levels in murine glial cells via NF-κB activation, which then downregulated a Pls-synthesizing enzyme, glycerone phosphate O-acyltransferase (Gnpat) through increased c-Myc recruitment onto theGnpatpromoter. We also found that systemic injection of lipopolysaccharide, aging, and chronic restraint stress reduced brain Pls contents that were associated with glial NF-κB activation, an increase in c-Myc expression, and downregulation ofGnpatin the mouse cortex and hippocampus. More interestingly, the reduction of Pls contents in the murine cortex itself could increase the activated phenotype of microglial cells and the expression of proinflammatory cytokines, suggesting further acceleration of neuroinflammation by reduction of brain Pls. A similar mechanism ofGnpatreduction was also found in human cell lines, triple-transgenic AD mouse brain, and postmortem human AD brain tissues. These findings suggest a novel mechanism of neuroinflammation that may explain prolonged progression of AD and help us to explore preventive and therapeutic strategies to treat neurodegenerative diseases.SIGNIFICANCE STATEMENTEther-type glycerophospholipids, plasmalogens (Pls), are reduced in the brain of Alzheimer disease (AD) patients. We found that inflammatory stimuli reduced Pls contents by downregulation of the Pls-synthesizing enzyme glycerone phosphate O-acyltransferase (Gnpat) through NF-κB-mediated recruitment of c-Myc onto theGnpatpromoter in both murine and human cell lines. Murine brains after systemic lipopolysaccharide, chronic stress, and aging, as well as triple-transgenic AD mice and postmortem human AD brain tissues all showed increased c-Myc and reducedGnpatexpression. Interestingly, knockdown ofGnpatitself activated NF-κB in glial cell lines and microglia in mouse cortex. Our findings provide a new insight into the mechanism of neuroinflammation and may help to develop a novel therapeutic approach for neurodegenerative diseases such as AD.

Peroxisome number and quality are maintained by its biogenesis and turnover and are important for the homeostasis of peroxisomes. Peroxisomes are increased in number by division with dynamic morphological changes including elongation, constriction, and fission. In the course of peroxisomal division, peroxisomal morphogenesis is orchestrated by Pex11β, dynamin-like protein 1 (DLP1), and mitochondrial fission factor (Mff). Conversely, peroxisome number is reduced by its degradation. Peroxisomes are mainly degraded by pexophagy, a type of autophagy specific for peroxisomes. Upon pexophagy, an adaptor protein translocates on peroxisomal membrane and connects peroxisomes to autophagic machineries. Molecular mechanisms of pexophagy are well studied in yeast systems where several specific adaptor proteins are identified. Pexophagy in mammals also proceeds in a manner dependent on adaptor proteins. In this review, we address the recent progress in studies on peroxisome morphogenesis and pexophagy.

The peroxisomal membrane protein Pex14p is phosphorylated in vivo, whereas no function has been assigned to Pex14p phosphorylation in yeast and mammalian cells. Mitotic phosphorylation of Pex14p and consequent suppression of catalase import are a mechanism of protecting DNA upon nuclear envelope breakdown at mitosis., Peroxisomal matrix proteins are imported into peroxisomes via membrane-bound docking/translocation machinery. One central component of this machinery is Pex14p, a peroxisomal membrane protein involved in the docking of Pex5p, the receptor for peroxisome targeting signal type 1 (PTS1). Studies in several yeast species have shown that Pex14p is phosphorylated in vivo, whereas no function has been assigned to Pex14p phosphorylation in yeast and mammalian cells. Here, we investigated peroxisomal protein import and its dynamics in mitotic mammalian cells. In mitotically arrested cells, Pex14p is phosphorylated at Ser-232, resulting in a lower import efficiency of catalase, but not the majority of proteins including canonical PTS1 proteins. Conformational change induced by the mitotic phosphorylation of Pex14p more likely increases homomeric interacting affinity and suppresses topological change of its N-terminal part, thereby giving rise to the retardation of Pex5p export in mitotic cells. Taken together, these data show that mitotic phosphorylation of Pex14p and consequent suppression of catalase import are a mechanism of protecting DNA upon nuclear envelope breakdown at mitosis.

Synthesis of ethanolamine plasmalogen (PlsEtn) is regulated by modulating the stability of fatty acyl-CoA reductase 1 (Far1) on peroxisomal membrane, a rate-limiting enzyme in plasmalogen synthesis. Dysregulation of plasmalogen homeostasis impairs cholesterol biosynthesis in cultured cells by altering the stability of squalene epoxidase (SQLE). However, regulation of PlsEtn synthesis and physiological consequences of plasmalogen homeostasis in tissues remain unknown. In the present study, we found that the protein but not the transcription level of Far1 in the cerebellum of the Pex14 mutant mouse expressing Pex14p lacking its C-terminal region (Pex14ΔC/ΔC) is higher than that from wild-type mouse, suggesting that Far1 is stabilized by the lowered level of PlsEtn. The protein level of SQLE was increased, whereas the transcriptional activity of the liver X receptors (LXRs), ligand-activated transcription factors of the nuclear receptor superfamily, is lowered in the cerebellum of Pex14ΔC/ΔC and the mice deficient in dihydroxyacetonephosphate acyltransferase, the initial enzyme for the synthesis of PlsEtn. These results suggest that the reduction of plasmalogens in the cerebellum more likely compromises the cholesterol homeostasis, thereby reducing the transcriptional activities of LXRs, master regulators of cholesterol homeostasis.

Ether glycerolipids, plasmalogens are found in various mammalian cells and tissues. However, physiological role of plasmalogens in epithelial cells remains unknown. We herein show that synthesis of ethanolamine-containing plasmalogens, plasmenylethanolamine (PlsEtn), is deficient in MCF7 cells, an epithelial cell line, with severely reduced expression of alkyl-dihydroxyacetonephosphate synthase (ADAPS), the second enzyme in the PlsEtn biosynthesis. Moreover, expression of ADAPS or supplementation of PlsEtn containing C18-alkenyl residue delays the migration of MCF7 cells as compared to that mock-treated MCF7 and C16-alkenyl-PlsEtn-supplemented MCF7 cells. Localization of E-cadherin to cell–cell junctions is highly augmented in cells containing C18-alkenyl-PlsEtn. Together, these results suggest that PlsEtn containing C18-alkenyl residue plays a distinct role in the integrity of E-cadherin-mediated adherens junction.

Mitochondria and peroxisomes proliferate by division. During division, a part of their membrane is pinched off by constriction of the ring-shaped mitochondrial division (MD) and peroxisome-dividing (POD) machinery. This constriction is mediated by a dynamin-like GTPase Dnm1 that requires a large amount of GTP as an energy source. Here, via proteomics of the isolated division machinery, we show that the 17-kDa nucleoside diphosphate kinase-like protein, dynamin-based ring motive-force organizer 1 (DYNAMO1), locally generates GTP in MD and POD machineries. DYNAMO1 is widely conserved among eukaryotes and colocalizes with Dnm1 on the division machineries. DYNAMO1 converts ATP to GTP, and disruption of its activity impairs mitochondrial and peroxisomal fissions. DYNAMO1 forms a ring-shaped complex with Dnm1 and increases the magnitude of the constricting force. Our results identify DYNAMO1 as an essential component of MD and POD machineries, suggesting that local GTP generation in Dnm1-based machinery regulates motive force for membrane severance., Mitochondria and peroxisomes require a dynamin-like GTPase to remodel membranes during division. Here the authors identify DYNAMO1, a nucleoside diphosphate kinase-like protein that generates a local source of GTP to promote constriction of the division machinery and produce daughter organelles.

Fatty acyl-CoA reductase 1 (Far1) is an ubiquitously expressed peroxisomal membrane protein generating fatty alcohols required for the biosynthesis of ether lipids. Lipid droplet localization of exogenously expressed and endogenous human Far1 was observed by fluorescence microscopy under conditions of increased triglyceride synthesis in tissue culture cells. This unexpected finding was supported further by correlative light electron microscopy and subcellular fractionation. Selective permeabilization, protease sensitivity and N-glycosylation tagging suggested that Far1 is able to assume two different membrane topologies, differing in the orientation of the short hydrophilic C-terminus towards the lumen or the cytosol, respectively. Two closely spaced hydrophobic domains are contained within the C-terminal region. When analyzed separately, the second domain was sufficient for the localization of a fluorescent reporter to lipid droplets. Targeting of Far1 to lipid droplets was not impaired in either pex19 or TRC40/ASNA1 CRISPR/Cas9 knockout cells. In conclusion, our data suggest that Far1 is a novel member of the rather exclusive group of dual topology membrane proteins. At the same time, Far1 shows lipid metabolism-dependent differential subcellular localizations to peroxisomes and lipid droplets.

Plasmalogen biosynthesis is regulated by modulating fatty acyl-CoA reductase 1 stability in a manner dependent on cellular plasmalogen level. However, physiological significance of the regulation of plasmalogen biosynthesis remains unknown. Here we show that elevation of the cellular plasmalogen level reduces cholesterol biosynthesis without affecting the isoprenylation of proteins such as Rab and Pex19p. Analysis of intermediate metabolites in cholesterol biosynthesis suggests that the first oxidative step in cholesterol biosynthesis catalyzed by squalene monooxygenase (SQLE), an important regulator downstream HMG-CoA reductase in cholesterol synthesis, is reduced by degradation of SQLE upon elevation of cellular plasmalogen level. By contrast, the defect of plasmalogen synthesis causes elevation of SQLE expression, resulting in the reduction of 2,3-epoxysqualene required for cholesterol synthesis, hence implying a novel physiological consequence of the regulation of plasmalogen biosynthesis.

Peroxisomes are essential intracellular organelles that involve many metabolic processes, such as β-oxidation of very long-chain fatty acids and synthesis of plasmalogen and bile acids as well as generation and degradation of hydrogen peroxide. These peroxisomal functions are fulfilled by strictly and spatiotemporally regulated compartmentation of the proteins catalysing these reactions. Defects in peroxisomal protein import results in inherited peroxisome biogenesis disorders in humans. Peroxisomal matrix and membrane proteins are synthesised on free ribosomes but transported into peroxisomes by distinct pathways determined by specific targeting signals and their receptors. The mechanism by which this is achieved has been clarified by identification of many PEX genes and the products named peroxins, the essential factors for peroxisome biogenesis. This article introduces several basic methods to investigate protein import into peroxisomes. Key Concepts Peroxisome plays an essential role in various metabolic pathways. Deficiency of peroxisomes in human causes severe foetal genetic diseases, peroxisome-deficient disorders. Functions and the integrity of peroxisomes are archived by proper import of both matrix and membrane proteins into peroxisomes. Peroxisomal proteins are encoded by nuclear DNA, synthesised in cytosolic free ribosomes and post-translationally imported into pre-existing peroxisomes. Peroxisomal matrix and membrane proteins are translocated into peroxisomes via different pathways in a manner dependent on their distinct targeting signals. Many of peroxins encoded by PEX genes are responsible factors for peroxisome biogenesis and are involved in the import of peroxisomal proteins. Various experimental approaches have elucidated the molecular mechanisms underlying peroxisome biogenesis. Post-translational import of peroxisomal proteins into isolated peroxisomes can be reproduced in vitro. Semi-permeabilised cells, in which only the plasma membrane is selectively permeabilised, are used to investigate peroxisomal protein import, as with living cells. Keywords: peroxisomes; protein translocation; peroxisome targeting signals; import receptors; PEX genes; peroxins; peroxisome biogenesis disorders

Pex11p family proteins are key players in peroxisomal fission, but their molecular mechanisms remains mostly unknown. In the present study, overexpression of Pex11pβ caused substantial vesiculation of peroxisomes in mammalian cells. This vesicle formation was dependent on dynamin-like protein 1 (DLP1) and mitochondrial fission factor (Mff), as knockdown of these proteins diminished peroxisomal fission after Pex11pβ overexpression. The fission-deficient peroxisomes exhibited an elongated morphology, and peroxisomal marker proteins, such as Pex14p or matrix proteins harboring peroxisomal targeting signal 1, were discernible in a segmented staining pattern, like beads on a string. Endogenous Pex11pβ was also distributed a striped pattern, but which was not coincide with Pex14p and PTS1 matrix proteins. Altered morphology of the lipid membrane was observed when recombinant Pex11p proteins were introduced into proteo-liposomes. Constriction of proteo-liposomes was observed under confocal microscopy and electron microscopy, and the reconstituted Pex11pβ protein localized to the membrane constriction site. Introducing point mutations into the N-terminal amphiphathic helix of Pex11pβ strongly reduced peroxisomal fission, and decreased the oligomer formation. These results suggest that Pex11p contributes to the morphogenesis of the peroxisomal membrane, which is required for subsequent fission by DLP1.

Plasmalogen synthesis can be analyzed by metabolic labeling, followed by the separation of ethanolamine plasmalogens from glycerophospholipids on one-dimensional thin-layer chromatography. The vinyl-ether bond of plasmalogens is acid-labile, which allows separating plasmalogens as 2-acyl-glycerophospholipids from diacyl-glycerophospholipids.

The Pex19p- and Pex3p-dependent direct import of peroxisomal membrane proteins (PMPs), termed the class I pathway, can be reconstituted in vitro by incubating cell-free synthesized PMPs with highly purified peroxisomes at 26 °C for 1 h. This method ensures that the proteins targeted to peroxisomes are imported directly without involvement of other organelles.

Peroxisomes are essential intracellular organelles that catalyze a number of essential metabolic pathways including β-oxidation of very long chain fatty acids, synthesis of plasmalogen, bile acids, and generation and degradation of hydrogen peroxide. These peroxisomal functions are accomplished by strictly and spatiotemporally regulated compartmentalization of the enzymes catalyzing these reactions. Defects in peroxisomal protein import result in inherited peroxisome biogenesis disorders in humans. Peroxisomal matrix and membrane proteins are synthesized on free ribosomes and transported to peroxisomes in a manner dependent on their specific targeting signals and their receptors. Peroxisomal protein import can be analyzed using a semi-intact assay system, in which targeting efficiency is readily monitored by immunofluorescence microscopy. Furthermore, cytosolic factors required for peroxisomal protein import can be manipulated, suggesting that the semi-intact system is a useful and convenient system to uncover the molecular mechanisms of peroxisomal protein import.

Alkenyl ether phospholipids are a major sub-class of ethanolamine- and choline-phospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. Biosynthesis of ethanolamine-containing alkenyl ether phospholipids, plasmalogens, is regulated by modulating the stability of fatty acyl-CoA reductase 1 (Far1) in a manner dependent on the level of cellular plasmalogens. However, precise molecular mechanisms underlying the regulation of plasmalogen synthesis remain poorly understood. Here we show that degradation of Far1 is accelerated by inhibiting dynamin-, Src kinase-, or flotillin-1-mediated endocytosis without increasing the cellular level of plasmalogens. By contrast, Far1 is stabilized by sequestering cholesterol with nystatin. Moreover, abrogation of the asymmetric distribution of plasmalogens in the plasma membrane by reducing the expression of CDC50A encoding a β-subunit of flippase elevates the expression level of Far1 and plasmalogen synthesis without reducing the total cellular level of plasmalogens. Together, these results support a model that plasmalogens localised in the inner leaflet of the plasma membranes are sensed for plasmalogen homeostasis in cells, thereby suggesting that plasmalogen synthesis is spatiotemporally regulated by monitoring cellular level of plasmalogens.

Plasmalogen synthesis can be analyzed by metabolic labeling, followed by the separation of ethanolamine plasmalogens from glycerophospholipids on one-dimensional thin-layer chromatography. The vinyl-ether bond of plasmalogens is acid-labile, which allows separating plasmalogens as 2-acyl-glycerophospholipids from diacyl-glycerophospholipids.

Peroxisomes are essential intracellular organelles that catalyze a number of essential metabolic pathways including β-oxidation of very long chain fatty acids, synthesis of plasmalogen, bile acids, and generation and degradation of hydrogen peroxide. These peroxisomal functions are accomplished by strictly and spatiotemporally regulated compartmentalization of the enzymes catalyzing these reactions. Defects in peroxisomal protein import result in inherited peroxisome biogenesis disorders in humans. Peroxisomal matrix and membrane proteins are synthesized on free ribosomes and transported to peroxisomes in a manner dependent on their specific targeting signals and their receptors. Peroxisomal protein import can be analyzed using a semi-intact assay system, in which targeting efficiency is readily monitored by immunofluorescence microscopy. Furthermore, cytosolic factors required for peroxisomal protein import can be manipulated, suggesting that the semi-intact system is a useful and convenient system to uncover the molecular mechanisms of peroxisomal protein import.

The Pex19p- and Pex3p-dependent direct import of peroxisomal membrane proteins (PMPs), termed the class I pathway, can be reconstituted in vitro by incubating cell-free synthesized PMPs with highly purified peroxisomes at 26 °C for 1 h. This method ensures that the proteins targeted to peroxisomes are imported directly without involvement of other organelles.

Rhizomelic chondrodysplasia punctata (RCDP) is an autosomal recessive disorder due to the deficiency in ether lipid synthesis. RCDP type 1, the most prominent type, is caused by the dysfunction of the receptor of peroxisome targeting signal type 2, Pex7 (peroxisomal biogenesis factor 7), and the rest of the patients, RCDP types 2 and 3, have defects in peroxisomal enzymes catalyzing the initial two steps of alkyl-phospholipid synthesis, glyceronephosphate O-acyltransferase and alkylglycerone phosphate synthase (Agps). We herein investigated defects of two patients with RCDP type 3. Patient 1 had a novel missense mutation, T1533G, resulting in the I511M substitution in Agps. The plasmalogen level was mildly reduced, whereas the protein level and peroxisomal localization of Agps-I511M in fibroblasts were normal as in the control fibroblasts. Structure prediction analysis suggested that the mutated residue was located in the helix α15 on the surface of V-shaped active site tunnel in Agps, likely accounting for the mild defects of plasmalogen synthesis. These results strongly suggest that an individual with mildly affected level of plasmalogen synthesis develops RCDP. In fibroblasts from patient 2, the expression of AGPS mRNA and Agps protein was severely affected, thereby giving rise to the strong reduction of plasmalogen synthesis.

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

Peroxisomal fatty acyl-CoA reductase 1 (Far1) is essential for supplying fatty alcohols required for ether bond formation in ether glycerophospholipid synthesis. The stability of Far1 is regulated by a mechanism that is dependent on cellular plasmalogen levels. However, the membrane topology of Far1 and how Far1 is targeted to membranes remain largely unknown. Here, Far1 is shown to be a peroxisomal tail-anchored protein. The hydrophobic C terminus of Far1 binds to Pex19p, a cytosolic receptor harboring a C-terminal CAAX motif, which is responsible for the targeting of Far1 to peroxisomes. Far1, but not Far2, was preferentially degraded in response to the cellular level of plasmalogens. Experiments in which regions of Far1 or Far2 were replaced with the corresponding region of the other protein showed that the region flanking the transmembrane domain of Far1 is required for plasmalogen-dependent modulation of Far1 stability. Expression of Far1 increased plasmalogen synthesis in wild-type Chinese hamster ovary cells, strongly suggesting that Far1 is a rate-limiting enzyme for plasmalogen synthesis.