Search

Peroxisomes are organelles that are central to lipid metabolism and chemical detoxification. Despite advances in our understanding of peroxisome biogenesis, the mechanisms maintaining peroxisomal membrane proteins remain to be fully elucidated. We show here that mammalian FAF2/UBXD8, a membrane-associated cofactor of p97/VCP, maintains peroxisomal homeostasis by modulating the turnover of peroxisomal membrane proteins such as PMP70. In FAF2-deficient cells, PMP70 accumulation recruits the autophagy adaptor OPTN (Optineurin) to peroxisomes and promotes their autophagic clearance (pexophagy). Pexophagy is also induced by p97/VCP inhibition. FAF2 functions together with p97/VCP to negatively regulate pexophagy rather than as a factor for peroxisome biogenesis. Our results strongly suggest that p97/VCP FAF2 -mediated extraction of ubiquitylated peroxisomal membrane proteins (e.g., PMP70) prevents peroxisomes from inducing nonessential autophagy under steady state conditions. These findings provide insight into molecular mechanisms underlying the regulation of peroxisomal integrity by p97/VCP and its associated cofactors., (© 2024. The Author(s).)

Peroxisomes are essential organelles involved in lipid metabolisms including plasmalogen biosynthesis and β-oxidation of very long-chain fatty acids. Peroxisomes proliferate by the growth and division of pre-existing peroxisomes. The peroxisomal membrane is elongated by Pex11β and then divided by the dynamin-like GTPase, DLP1 (also known as DRP1 encoded by DNM1L gene), which also functions as a fission factor for mitochondria. Nucleoside diphosphate kinase 3 (NME3) localized in both peroxisomes and mitochondria generates GTP for DLP1 activity. Deficiencies of either of these factors induce abnormal morphology of peroxisomes and/or mitochondria, and are associated with central nervous system dysfunction. To investigate whether the impaired division of peroxisomes affects lipid metabolisms, we assessed the phospholipid composition of cells lacking each of the different division factors. In fibroblasts from the patients deficient in DLP1, NME3, or Pex11β, docosahexaenoic acid (DHA, C22:6)-containing phospholipids were found to be decreased. Conversely, the levels of several fatty acids such as arachidonic acid (AA, C20:4) and oleic acid (C18:1) were elevated. Mouse embryonic fibroblasts from Drp1- and Pex11β-knockout mice also showed a decrease in the levels of phospholipids containing DHA and AA. Collectively, these results suggest that the dynamics of organelle morphology exert marked effects on the fatty acid composition of phospholipids., (© 2022 SSIEM.)

Phos-tag, a selective phosphate-binding molecule, and Phos-tag-based methodologies have been developed to investigate the phosphoproteome. In various analytical techniques using Phos-tag derivatives, phosphate-affinity electrophoresis using Phos-tag acrylamide, called Phos-tag SDS-PAGE, enables separation of phosphorylated proteins with a slower migration from non-phosphorylated proteins in polyacrylamide gels. The procedures for Phos-tag SDS-PAGE are largely common to those for conventional SDS-PAGE, thus being readily available for all laboratories. Phos-tag SDS-PAGE is widely applied to quantitative analysis of the overall phosphorylation state depending on the number and/or sites of the phosphate group. Phos-tag SDS-PAGE has also been introduced to the field of peroxisome study, including oxidative stress-induced and mitosis-specific phosphorylation of Pex14, a central component of the translocation machinery complex for peroxisomal matrix proteins. Here, we describe a practical protocol for Phos-tag SDS-PAGE and its application to peroxisome biogenesis research., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 2 O 2 -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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mammalian PEX16 has been considered essential for generating and maintaining peroxisomal membranes. This view is based primarily on the finding that fibroblasts from several PEX16-deficient patients are devoid of peroxisomal structures but can form peroxisomes upon expression of PEX16. However, unlike these patient-derived cells, pex16 mutants in other model organisms contain partially functional peroxisomes. Here, we report that PEX16-knockout (KO) cells derived from three mammalian cultured cell lines comprise cells containing a fewer number of enlarged peroxisomes and cells lacking peroxisomes. We also suggest that PEX16 accelerates the process by which peroxisome-less cells form peroxisomal membranes and subsequently establish mature peroxisomes, independently of its ability to mediate peroxisomal targeting of PEX3. Nevertheless, PEX16 is not absolutely required for this process. Moreover, a well-known patient-derived PEX16 mutant inhibits the de novo formation of peroxisomal membranes. Our findings suggest that although PEX16 is undoubtedly important for optimal peroxisomal membrane biogenesis, mammalian cells may be able to form peroxisomes de novo and maintain the organelles without the aid of PEX16., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Recent findings provide evidence for dynamic and highly regulated dual subcellular localization of catalase, a hydrogen peroxide (H 2 O 2 )-metabolizing enzyme, in peroxisomes and the cytosol. These data suggest a number of important implications for the field of oxidative stress biology., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

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., (© 2020 Yamashita et al.)

Most of peroxisomal matrix proteins including a hydrogen peroxide (H 2 O 2 )-decomposing enzyme, catalase, are imported in a peroxisome-targeting signal type-1 (PTS1)-dependent manner. However, little is known about regulation of the membrane-bound protein import machinery. Here, we report that Pex14, a central component of the protein translocation complex in peroxisomal membrane, is phosphorylated in response to oxidative stresses such as H 2 O 2 in mammalian cells. The H 2 O 2 -induced phosphorylation of Pex14 at Ser232 suppresses peroxisomal import of catalase in vivo and selectively impairs in vitro the interaction of catalase with the Pex14-Pex5 complex. A phosphomimetic mutant Pex14-S232D elevates the level of cytosolic catalase, but not canonical PTS1-proteins, conferring higher cell resistance to H 2 O 2 . We thus suggest that the H 2 O 2 -induced phosphorylation of Pex14 spatiotemporally regulates peroxisomal import of catalase, functioning in counteracting action against oxidative stress by the increase of cytosolic catalase., Competing Interests: KO, MN, HK, RN, TM, YF No competing interests declared, ME Mahmoud El Shermely is affiliated with Basilea Pharmaceutica International Ltd. The author has no financial interests to declare., (© 2020, Okumoto et al.)

Mitochondria and peroxisomes are ubiquitous subcellular organelles that are highly dynamic and possess a high degree of plasticity. These organelles proliferate through division of pre-existing organelles. Studies on yeast, mammalian cells, and unicellular algae have led to a surprising finding that mitochondria and peroxisomes share the components of their division machineries. At the heart of the mitochondrial and peroxisomal division machineries is a GTPase dynamin-like protein, Dnm1/Drp1, which forms a contractile ring around the neck of the dividing organelles. During division, Dnm1/Drp1 functions as a motor protein and constricts the membrane. This mechanochemical work is achieved by utilizing energy from GTP hydrolysis. Over the last two decades, studies have focused on the structure and assembly of Dnm1/Drp1 molecules around the neck. However, the regulation of GTP during the division of mitochondrion and peroxisome is not well understood. Here, we review the current understanding of Dnm1/Drp1-mediated divisions of mitochondria and peroxisomes, exploring the mechanisms of GTP regulation during the Dnm1/Drp1 function, and provide new perspectives on their potential contribution to mitochondrial and peroxisomal biogenesis.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

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., (© 2020 Abe et al.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

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.

Ubiquitylation of outer mitochondrial membrane (OMM) proteins is closely related to the onset of familial Parkinson's disease. Typically, a reduction in the mitochondrial membrane potential results in Parkin-mediated ubiquitylation of OMM proteins, which are then targeted for proteasomal and mitophagic degradation. The role of ubiquitylation of OMM proteins with non-degradative fates, however, remains poorly understood. In this study, we find that the mitochondrial E3 ubiquitin ligase MITOL/March5 translocates from depolarized mitochondria to peroxisomes following mitophagy stimulation. This unusual redistribution is mediated by peroxins (peroxisomal biogenesis factors) Pex3/16 and requires the E3 ligase activity of Parkin, which ubiquitylates K268 in the MITOL C-terminus, essential for p97/VCP-dependent mitochondrial extraction of MITOL. These findings imply that ubiquitylation directs peroxisomal translocation of MITOL upon mitophagy stimulation and reveal a novel role for ubiquitin as a sorting signal that allows certain specialized proteins to escape from damaged mitochondria., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Peroxisomes cooperate with mitochondria in the performance of cellular metabolic functions, such as fatty acid oxidation and the maintenance of redox homeostasis. However, whether peroxisomes also regulate mitochondrial fission-fusion dynamics or mitochondrion-dependent apoptosis remained unclear. We now show that genetic ablation of the peroxins Pex3 or Pex5, which are essential for peroxisome biogenesis, results in mitochondrial fragmentation in mouse embryonic fibroblasts (MEFs) in a manner dependent on Drp1 (also known as DNM1L). Conversely, treatment with 4-PBA, which results in peroxisome proliferation, resulted in mitochondrial elongation in wild-type MEFs, but not in Pex3-knockout MEFs. We further found that peroxisome deficiency increased the levels of cytosolic cytochrome c and caspase activity under basal conditions without inducing apoptosis. It also greatly enhanced etoposide-induced caspase activation and apoptosis, which is indicative of an enhanced cellular sensitivity to death signals. Taken together, our data unveil a previously unrecognized role for peroxisomes in the regulation of mitochondrial dynamics and mitochondrion-dependent apoptosis. Effects of peroxin gene mutations on mitochondrion-dependent apoptosis may contribute to pathogenesis of peroxisome biogenesis disorders.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Proteins harboring peroxisome-targeting signal type-2 (PTS2) are recognized in the cytosol by mobile PTS2 receptor Pex7p and associate with a longer isoform Pex5pL of the PTS1 receptor. Trimeric PTS2 protein-Pex7p-Pex5pL complexes are translocated to peroxisomes in mammalian cells. However, it remains unclear whether Pex5pL and Pex7p are sufficient cytosolic components in transporting of PTS2 proteins to peroxisomes. Here, we construct a semi-intact cell import system to define the cytosolic components required for the peroxisomal PTS2 protein import and show that the PTS2 pre-import complexes comprising Pex7p, Pex5p, and Hsc70 isolated from the cytosol of pex14 Chinese hamster ovary cell mutant ZP161 is import-competent. PTS2 reporter proteins are transported to peroxisomes by recombinant Pex7p and Pex5pL in semi-intact cells devoid of the cytosol. Furthermore, PTS2 proteins are translocated to peroxisomes in the presence of a non-hydrolyzable ATP analogue, adenylyl imidodiphosphate, and N-ethylmaleimide, suggesting that ATP-dependent chaperones including Hsc70 are dispensable for PTS2 protein import. Taken together, we suggest that Pex7p and Pex5pL are the minimal cytosolic factors in the transport of PTS2 proteins to peroxisomes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Using clinical exome sequencing (ES), we identified an autosomal recessive missense variant, c.153C>A (p.F51L), in the peroxisome biogenesis factor 26 gene ( PEX26 ) in a 19-yr-old female of Ashkenazi Jewish descent who was referred for moderate to severe hearing loss. The proband and three affected siblings are all homozygous for the c.153C>A variant. Skin fibroblasts from this patient show normal morphology in immunostaining of matrix proteins, although the level of catalase was elevated. Import rate of matrix proteins was significantly decreased in the patient-derived fibroblasts. Binding of Pex26-F51L to the AAA ATPase peroxins, Pex1 and Pex6, is severely impaired and affects peroxisome assembly. Moreover, Pex26 in the patient's fibroblasts is reduced to ∼30% of the control, suggesting that Pex26-F51L is unstable in cells. In the patient's fibroblasts, peroxisome-targeting signal 1 (PTS1) proteins, PTS2 protein 3-ketoacyl-CoA thiolase, and catalase are present in a punctate staining pattern at 37°C and in a diffuse pattern at 42°C, suggesting that these matrix proteins are not imported to peroxisomes in a temperature-sensitive manner. Analysis of peroxisomal metabolism in the patient's fibroblasts showed that the level of docosahexaenoic acid (DHA) (C22:6n-3) in ether phospholipids is decreased, whereas other lipid metabolism, including peroxisomal fatty acid β-oxidation, is normal. Collectively, the functional data support the mild phenotype of nonsyndromic hearing loss in patients harboring the F51L variant in PEX26 ., (© 2019 Tanaka et al.; Published by Cold Spring Harbor Laboratory Press.)

A newly isolated binding protein of peroxisomal targeting signal type 2 (PTS2) receptor Pex7, termed P7BP2, is transported into peroxisomes by binding to the longer isoform of Pex5p, Pex5pL, via Pex7p. The binding to Pex7p and peroxisomal localization of P7BP2 depends on the cleavable PTS2 in the N-terminal region, suggesting that P7BP2 is a new PTS2 protein. By search on human database, three AAA+ domains are found in the N-terminal half of P7BP2. Protein sequence alignment and motif search reveal that in the C-terminal region P7BP2 contains additional structural domains featuring weak but sufficient homology to AAA+ domain. P7BP2 behaves as a monomer in gel-filtration chromatography and the single molecule observed under atomic force microscope shapes a disc-like ring. Collectively, these results suggest that P7BP2 is a novel dynein-type AAA+ family protein, of which domains are arranged into a pseudo-hexameric ring structure.

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.

Microtubule-dependent long-distance movement of peroxisomes occurs in mammalian cells. However, its molecular mechanisms remain undefined. In this study, we identified three distinct splicing variants of human mitochondrial Rho GTPase-1 (Miro1), each containing amino acid sequence insertions 1 (named Miro1-var2), 2 (Miro1-var3), and both 1 and 2 (Miro1-var4), respectively, at upstream of the transmembrane domain. Miro1-var4 and Miro1-var2 are localized to peroxisomes in a manner dependent on the insertion 1 that is recognized by the cytosolic receptor Pex19p. Exogenous expression of Miro1-var4 induces accumulation of peroxisomes at the cell periphery and augments long-range movement of peroxisomes along microtubules. Depletion of all Miro1 variants by knocking down MIRO1 suppresses the long-distance movement of peroxisomes. Such abrogated movement is restored by reexpression of peroxisomal Miro1 variants. Collectively, our findings identify for the first time peroxisome-localized Miro1 variants as adapter proteins that link peroxisomes to the microtubule-dependent transport complexes including TRAK2 in the intracellular translocation of peroxisomes in mammalian cells., (© 2018 Okumoto et al.)

Pex5 and Pex7 are cytosolic receptors for peroxisome targeting signal type-1 (PTS1) and type-2 (PTS2), respectively, and play a pivotal role in import of peroxisomal matrix proteins. Recent advance in mass spectrometry analysis has facilitated comprehensive analysis of protein-protein interaction network by a combination with immunoprecipitation or biochemical purification. In this chapter, we introduce several findings obtained by these methods applied to mammalian cells. Exploring Pex5-binding partners in mammalian cells revealed core components comprising the import machinery complex of matrix proteins and a number of PTS1-type cargo proteins. Biochemical purification of the Pex5-export stimulating factor from rat liver cytosol fraction identified Awp1, providing further insight into molecular mechanisms of the export step of mono-ubiquitinated Pex5. Identification of DDB1 (damage-specific DNA-binding protein 1), a component of CRL4 (Cullin4A-RING ubiquitin ligase) E3 complex, as a Pex7-interacting protein revealed that quality control of Pex7 by CRL4A is important for PTS2 protein import by preventing the accumulation of dysfunctional Pex7. Furthermore, analysis of binding partners of an intraperoxisomal processing enzyme, trypsin-domain containing 1 (Tysnd1), showed a protein network regulating peroxisomal fatty acid β-oxidation.

Peroxisomes contain anabolic and catabolic enzymes including oxidases that produce hydrogen peroxide as a by-product. Peroxisomes also contain catalase to metabolize hydrogen peroxide. It has been recognized that catalase is localized to cytosol in addition to peroxisomes. A recent study has revealed that loss of VDAC2 shifts localization of BAK, a pro-apoptotic member of Bcl-2 family, from mitochondria to peroxisomes and cytosol, thereby leading to release of peroxisomal matrix proteins including catalase to the cytosol. A subset of BAK is localized to peroxisomes even in wild-type cells, regulating peroxisomal membrane permeability and catalase localization. The cytosolic catalase potentially acts as an antioxidant to eliminate extra-peroxisomal hydrogen peroxide.

Nuclear Dbf2-related (NDR) kinases, comprising NDR1 and NDR2, are serine/threonine kinases that play crucial roles in the control of cell proliferation, apoptosis, and morphogenesis. We recently showed that NDR2, but not NDR1, is involved in primary cilium formation; however, the mechanism underlying their functional difference in ciliogenesis is unknown. To address this issue, we examined their subcellular localization. Despite their close sequence similarity, NDR2 exhibited punctate localization in the cytoplasm, whereas NDR1 was diffusely distributed within the cell. Notably, NDR2 puncta mostly co-localized with the peroxisome marker proteins, catalase and CFP-SKL (cyan fluorescent protein carrying the C-terminal typical peroxisome-targeting signal type-1 (PTS1) sequence, Ser-Lys-Leu). NDR2 contains the PTS1-like sequence, Gly-Lys-Leu, at the C-terminal end, whereas the C-terminal end of NDR1 is Ala-Lys. An NDR2 mutant lacking the C-terminal Leu, NDR2(ΔL), exhibited almost diffuse distribution in cells. Additionally, NDR2, but neither NDR1 nor NDR2(ΔL), bound to the PTS1 receptor Pex5p. Together, these findings indicate that NDR2 localizes to the peroxisome by using the C-terminal GKL sequence. Intriguingly, topology analysis of NDR2 suggests that NDR2 is exposed to the cytosolic surface of the peroxisome. The expression of wild-type NDR2, but not NDR2(ΔL), recovered the suppressive effect of NDR2 knockdown on ciliogenesis. Furthermore, knockdown of peroxisome biogenesis factor genes ( PEX1 or PEX3 ) partially suppressed ciliogenesis. These results suggest that the peroxisomal localization of NDR2 is implicated in its function to promote primary cilium formation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Peroxisomal biogenesis disorders (PBDs) are fatal genetic diseases consisting of 14 complementation groups (CGs). We previously isolated a peroxisome-deficient Chinese hamster ovary cell mutant, ZP114, which belongs to none of these CGs. Using a functional screening strategy, VDAC2 was identified as rescuing the peroxisomal deficiency of ZP114 where VDAC2 expression was not detected. Interestingly, knockdown of BAK or overexpression of the BAK inhibitors BCL-X L and MCL-1 restored peroxisomal biogenesis in ZP114 cells. Although VDAC2 is not localized to the peroxisome, loss of VDAC2 shifts the localization of BAK from mitochondria to peroxisomes, resulting in peroxisomal deficiency. Introduction of peroxisome-targeted BAK harboring the Pex26p transmembrane region into wild-type cells resulted in the release of peroxisomal matrix proteins to cytosol. Moreover, overexpression of BAK activators PUMA and BIM permeabilized peroxisomes in a BAK-dependent manner. Collectively, these findings suggest that BAK plays a role in peroxisomal permeability, similar to mitochondrial outer membrane permeabilization., (© 2017 Hosoi et al.)

Pex19p harbouring a prenylation CAAX box functions as a chaperone and transporter for peroxisomal membrane proteins in membrane assembly. By functional phenotype-complementation assay using a pex19 Chinese hamster ovary cell mutant ZP119, we herein cloned a rat cDNA encoding a protein similar to Pex19p, but with a C-terminal hydrophobic segment in place of the CAAX box region. The transcript of this gene was highly induced by treatment of rats with a peroxisome proliferator, clofibrate, hence termed PEX19i, while the other three less prominently inducible PEX19 variants encoded authentic Pex19p but differed in the length of 3' non-coding region. Pex19pi restored peroxisomes in ZP119 with slightly lower efficiency than Pex19p, showing apparently weaker interaction with Pex11pβ essential for peroxisome proliferation. However, the C-terminal region of Pex19p was not essential for the association of Pex19p with peroxisomal membrane and interaction with membrane assembly factors, Pex3p and Pex16p. Non-prenylated Pex19p interacted with a membrane protein cargo, Pex14p, but more weakly than Pex19pi and the farnesylated Pex19p. Thus, PEX19i most likely plays important roles involving the membrane formation at early stages, in prompt response to peroxisome proliferation. Similar types of PEX19 mRNA variants were also elevated in mouse regenerating liver., (© The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Organelle division is executed through contraction of a ring-shaped supramolecular dividing machinery. A core component of the machinery is the dynamin-based ring conserved during the division of mitochondrion, plastid and peroxisome. Here, using isolated peroxisome-dividing (POD) machinery from a unicellular red algae, Cyanidioschyzon merolae , we identified a dynamin-based ring organizing center (DOC) that acts as an initiation point for formation of the dynamin-based ring. C. merolae contains a single peroxisome, the division of which can be highly synchronized by light-dark stimulation; thus, intact POD machinery can be isolated in bulk. Dynamin-based ring homeostasis is maintained by the turnover of the GTP-bound form of the dynamin-related protein Dnm1 between the cytosol and division machinery via the DOC. A single DOC is formed on the POD machinery with a diameter of 500-700 nm, and the dynamin-based ring is unidirectionally elongated from the DOC in a manner that is dependent on GTP concentration. During the later step of membrane fission, the second DOC is formed and constructs the double dynamin-based ring to make the machinery thicker. These findings provide new insights to define fundamental mechanisms underlying the dynamin-based membrane fission in eukaryotic cells., (© 2017. Published by The Company of Biologists Ltd.)

Acyl-CoA binding domain-containing 5 (ACBD5) is a peroxisomal protein that carries an acyl-CoA binding domain (ACBD) at its N-terminal region. The recent identification of a mutation in the ACBD5 gene in patients with a syndromic form of retinal dystrophy highlights the physiological importance of ACBD5 in humans. However, the underlying pathogenic mechanisms and the precise function of ACBD5 remain unclear. We herein report that ACBD5 is a peroxisomal tail-anchored membrane protein exposing its ACBD to the cytosol. Using patient-derived fibroblasts and ACBD5 knock-out HeLa cells generated via genome editing, we demonstrate that ACBD5 deficiency causes a moderate but significant defect in peroxisomal β-oxidation of very-long-chain fatty acids (VLCFAs) and elevates the level of cellular phospholipids containing VLCFAs without affecting peroxisome biogenesis, including the import of membrane and matrix proteins. Both the N-terminal ACBD and peroxisomal localization of ACBD5 are prerequisite for efficient VLCFA β-oxidation in peroxisomes. Furthermore, ACBD5 preferentially binds very-long-chain fatty acyl-CoAs (VLC-CoAs). Together, these results suggest a direct role of ACBD5 in peroxisomal VLCFA β-oxidation. Based on our findings, we propose that ACBD5 captures VLC-CoAs on the cytosolic side of the peroxisomal membrane so that the transport of VLC-CoAs into peroxisomes and subsequent β-oxidation thereof can proceed efficiently. Our study reclassifies ACBD5-related phenotype as a novel peroxisomal disorder., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Peroxisome abundance is tightly regulated according to the physiological contexts, through regulations of both proliferation and degradation of the organelles. Here, we describe detailed methods to analyze processes for autophagic degradation of peroxisomes, termed pexophagy, in yeast organisms. The assay systems include a method for biochemical detection of pexophagy completion, and one for microscopic visualization of specialized membrane structures acting in pexophagy. As a model yeast organism utilized in studies of pexophagy, the methylotrophic yeast Komagataella phaffii (Pichia pastoris) is referred to in this chapter and related information on the studies with baker's yeast (Saccharomyces cerevisiae) is also included. The described techniques facilitate elucidation of molecular machineries for pexophagy and understanding of peroxisome-selective autophagic pathways.

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.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) is one of the useful methods to isolate protein complexes including membrane proteins under native conditions. In BN-PAGE, Coomassie Brilliant Blue G-250 binds to proteins and provides a negative charge for the electrophoretic separation without denaturing at neutral pH, allowing the analysis of molecular mass, oligomeric state, and composition of native protein complexes. BN-PAGE is widely applied to the characterization of soluble protein complexes as well as isolation of membrane protein complexes from biological membranes such as the complexes I-V of the mitochondrial respiratory chain and subcomplexes of the mitochondrial protein import machinery. BN-PAGE has also been introduced in the field of peroxisome research, for example, analysis of translocation machinery for peroxisomal matrix proteins embedded in the peroxisomal membrane. Here, we describe a basic protocol of BN-PAGE and its application to the study of peroxisome biogenesis.

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.

Cell mutants with a genetic defect affecting various cellular phenotypes are widely utilized as a powerful tool in genetic, biochemical, and cell biological research. More than a dozen complementation groups of animal somatic mutant cells defective in peroxisome biogenesis have been successfully isolated in Chinese hamster ovary (CHO) cells and used as a model system reflecting fatal human severe genetic disorders named peroxisome biogenesis disorders (PBD). Isolation and characterization of peroxisome-deficient CHO cell mutants has allowed the identification of PEX genes and the gene products peroxins, which directly leads to the accomplishment of isolation of pathogenic genes responsible for human PBDs, as well as elucidation of their functional roles in peroxisome biogenesis. Here, we describe the procedure to isolate peroxisome-deficient mammalian cell mutants from CHO cells, by making use of an effective, photo-sensitized selection method.

In mammalian cells several hundred peroxisomes are maintained by a balance between the biogenesis and turnover by peroxisome homeostasis. Pexophagy, a form of autophagy specific for peroxisomes, is the main pathway for peroxisome degradation, but molecular mechanisms of mammalian pexophagy are largely unknown. This is due to the lack of well-established pexophagy-inducing conditions in mammalian cells. Recently, several conditions that induce pexophagy were described for mammalian cells, involving ubiquitin and adaptor proteins of autophagy. In this chapter, we describe the protocol for Pex3-induced pexophagy, the more readable and highly inducible pexophagy condition in mammalian cells.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Peroxisome is a single-membrane-bounded ubiquitous organelle containing a hundred different enzymes that catalyze various metabolic pathways such as β-oxidation of very long-chain fatty acids and synthesis of plasmalogens. To investigate peroxisome biogenesis and human peroxisome biogenesis disorders (PBDs) including Zellweger syndrome, more than a dozen different complementation groups of Chinese hamster ovary (CHO) cell mutants impaired in peroxisome biogenesis are isolated as a model experimental system. By taking advantage of rapid functional complementation assay of the CHO cell mutants, successful cloning of PEX genes encoding peroxins required for peroxisome assembly invaluably contributed to the accomplishment of cloning of pathogenic genes responsible for PBDs. Peroxins are divided into three groups: 1) peroxins including Pex3p, Pex16p and Pex19p, are responsible for peroxisome membrane biogenesis via Pex19p- and Pex3p-dependent class I and Pex19p- and Pex16p-dependent class II pathways; 2) peroxins that function in matrix protein import; 3) those such as Pex11pβ are involved in peroxisome division where DLP1, Mff, and Fis1 coordinately function.

Pexophagy can be experimentally induced in mammalian cells by removing the culture serum. Pex14p, a peroxisomal membrane protein essential for matrix protein import in docking of soluble receptor Pex5p, is involved in the mammalian autophagic degradation of peroxisomes and interacts with the lipidated form of LC3, termed LC3-II, an essential factor for autophagosome formation, under the starvation condition in CHO-K1 cells. However, molecular mechanisms underlying the Pex14p-LC3-II interaction remain largely unknown. To verify whether Pex14p directly binds LC3-II, we reconstituted an in vitro conjugation system for synthesis of LC3-II. We show here that Pex14p directly interacts with LC3-II via the transmembrane domain of Pex14p. Pex5p competitively inhibited this interaction, implying that Pex14p preferentially binds to Pex5p under the nutrient-rich condition. Moreover, a Pex5p mutant defective in PTS1-protein import lost its affinity for Pex14p under the condition of nutrient deprivation, thereby more likely explaining why Pex14p prefers to interact with LC3-II under the starvation condition in vivo. Together, these results suggest that Pex14p is a unique factor that functions in the dual processes in peroxisomal biogenesis and degradation with the coordination of Pex5p in response to the environmental changes., (© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.)

Peroxisomes are degraded by a selective type of autophagy known as pexophagy. Several different types of pexophagy have been reported in mammalian cells. However, the mechanisms underlying how peroxisomes are recognized by autophagy-related machinery remain elusive. PEX3 is a peroxisomal membrane protein (PMP) that functions in the import of PMPs into the peroxisomal membrane and has been shown to interact with pexophagic receptor proteins during pexophagy in yeast. Thus, PEX3 is important not only for peroxisome biogenesis, but also for peroxisome degradation. However, whether PEX3 is involved in the degradation of peroxisomes in mammalian cells is unclear. Here, we report that high levels of PEX3 expression induce pexophagy. In PEX3-loaded cells, peroxisomes are ubiquitinated, clustered, and degraded in lysosomes. Peroxisome targeting of PEX3 is essential for the initial step of this degradation pathway. The degradation of peroxisomes is inhibited by treatment with autophagy inhibitors or siRNA against NBR1, which encodes an autophagic receptor protein. These results indicate that ubiquitin- and NBR1-mediated pexophagy is induced by increased expression of PEX3 in mammalian cells. In addition, another autophagic receptor protein, SQSTM1/p62, is required only for the clustering of peroxisomes. Expression of a PEX3 mutant with substitution of all lysine and cysteine residues by arginine and alanine, respectively, also induces peroxisome ubiquitination and degradation, hence suggesting that ubiquitination of PEX3 is dispensable for pexophagy and an endogenous, unidentified peroxisomal protein is ubiquitinated on the peroxisomal membrane.

Tail-anchored (TA) proteins are anchored into cellular membranes by a single transmembrane domain (TMD) close to the C terminus. Although the targeting of TA proteins to peroxisomes is dependent on PEX19, the mechanistic details of PEX19-dependent targeting and the signal that directs TA proteins to peroxisomes have remained elusive, particularly in mammals. The present study shows that PEX19 formed a complex with the peroxisomal TA protein PEX26 in the cytosol and translocated it directly to peroxisomes by interacting with the peroxisomal membrane protein PEX3. Unlike in yeast, the adenosine triphosphatase TRC40, which delivers TA proteins to the endoplasmic reticulum, was dispensable for the peroxisomal targeting of PEX26. Moreover, the basic amino acids within the luminal domain of PEX26 were essential for binding to PEX19 and thereby for peroxisomal targeting. Finally, our results suggest that a TMD that escapes capture by TRC40 and is followed by a highly basic luminal domain directs TA proteins to peroxisomes via the PEX19-dependent route.

Human catalase forms a 240-kDa tetrameric complex and degrades H(2) O(2) in peroxisomes. Human catalase is targeted to peroxisomes by the interaction of its peroxisomal targeting signal type 1 (PTS1)-like KANL sequence with the cytosolic PTS1 receptor Pex5p. We show herein that human catalase tetramers are formed in the cytoplasm and that the expression of a PTS signal on each of the four subunits is not necessary for peroxisomal transport. We previously demonstrated that a Pex5p mutant defective in binding to Pex13p, designated Pex5p(Mut234), imports typical PTS1-type proteins but not catalase. This impaired catalase import is not rescued by replacing its C-terminal KANL sequence with a typical PTS1 sequence, SKL, indicating that the failure of catalase import in Mut234-expressing cells is not due to its weak PTS1. In contrast, several enzymatically inactive and monomeric mutants of catalase are efficiently imported in Mut234-expressing cells. Moreover, trimeric chloramphenicol acetyltransferase (CAT) harboring SKL is not imported in Pex5p(Mut234)-expressing cells, but CAT-SKL trimers are transported to peroxisomes in the wild-type cells. These findings suggest that the Pex5p-Pex13p interaction likely plays a pivotal role in the peroxisomal import of folded and oligomeric proteins., (© 2012 John Wiley & Sons A/S.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Peroxisome division is regulated by several factors, termed fission factors, as well as the conditions of the cellular environment. Over the past decade, the idea of metabolic control of peroxisomal morphogenesis has been postulated, but remains largely undefined to date. In the current study, docosahexaenoic acid (DHA, C22:6n-3) was identified as an inducer of peroxisome division. In fibroblasts isolated from patients that carry defects in peroxisomal fatty acid β-oxidation, peroxisomes are much less abundant than normal cells. Treatment of these patient fibroblasts with DHA induced the proliferation of peroxisomes to the level seen in normal fibroblasts. DHA-induced peroxisomal proliferation was abrogated by treatment with a small inhibitory RNA (siRNA) targeting dynamin-like protein 1 and with dynasore, an inhibitor of dynamin-like protein 1, which suggested that DHA stimulates peroxisome division. DHA augmented the hyper-oligomerization of Pex11pβ and the formation of Pex11pβ-enriched regions on elongated peroxisomes. Time-lapse imaging analysis of peroxisomal morphogenesis revealed a sequence of steps involved in peroxisome division, including elongation in one direction followed by peroxisomal fission. DHA enhanced peroxisomal division in a microtubule-independent manner. These results suggest that DHA is a crucial signal for peroxisomal elongation, a prerequisite for subsequent fission and peroxisome division.

During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator. In the in vitro Pex5 export assay, recombinant AWP1 stimulated Pex5 export and an anti-AWP1 antibody interfered with Pex5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1-Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of Pex5 rather than to unmodified Pex5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, Pex5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for Pex5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of Pex5 export during peroxisome biogenesis., (© 2011 John Wiley & Sons A/S.)

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders such as Zellweger syndrome. Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for PBDs of complementation groups 1 and 4, respectively. PEX26 responsible for peroxisome biogenesis disorders of complementation group 8 codes for C-tail-anchored type-II membrane peroxin Pex26p, the recruiter of Pex1p-Pex6p complexes to peroxisomes. Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by ATPase cycle. Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome targeting signal type-1 and shuttles between the cytosol and peroxisomes. AAA peroxins are involved in the export from peroxisomes of Pex5p. Pex5p is ubiquitinated at the conserved cysteine11 in a form associated with peroxisomes. Pex5p with a mutation of the cysteine11 to alanine, termed Pex5p-C11A, abrogates peroxisomal import of proteins harboring peroxisome targeting signals 1 and 2 in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, hence suggesting an essential role of the cysteine residue in the export of Pex5p., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The molecular mechanisms underlying protein turnover and enzyme regulation in the peroxisomal matrix remain largely unknown. Trypsin domain-containing 1 (Tysnd1) and peroxisomal Lon protease (PsLon) are newly identified peroxisomal matrix proteins that harbor both a serine protease-like domain and a peroxisome-targeting signal 1 (PTS1) sequence. Tysnd1 processes several PTS1-containing proteins and cleaves N-terminal presequences from PTS2-containing protein precursors. Here we report that knockdown of Tysnd1, but not PsLon, resulted in accumulation of endogenous β-oxidation enzymes in their premature form. The protease activity of Tysnd1 was inactivated by intermolecular self-conversion of the 60-kDa form to 15- and 45-kDa chains, which were preferentially degraded by PsLon. Peroxisomal β-oxidation of a very long fatty acid was significantly decreased by knockdown of Tysnd1 and partially lowered by PsLon knockdown. Taken together, these data suggest that Tysnd1 is a key regulator of the peroxisomal β-oxidation pathway via proteolytic processing of β-oxidation enzymes. The proteolytic activity of oligomeric Tysnd1 is in turn controlled by self-cleavage of Tysnd1 and degradation of Tysnd1 cleavage products by PsLon.

Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome-targeting signal (PTS) type 1 and shuttles between the cytosol and peroxisomes. Here, we show that Pex5p is ubiquitinated at the conserved cysteine(11) in a manner sensitive to dithiothreitol, in a form associated with peroxisomes. Pex5p with a mutation of the cysteine(11) to alanine, termed Pex5p-C11A, abrogates peroxisomal import of PTS1 and PTS2 proteins in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, resulting in its accumulation in peroxisomes. These results suggest an essential role of the cysteine residue in the export of Pex5p. Furthermore, domain mapping indicates that N-terminal 158-amino-acid region of Pex5p-C11A, termed 158-CA, is sufficient for such dominant-negative activity by binding to membrane peroxin Pex14p via its two pentapeptide WXXXF/Y motifs. Stable expression of either Pex5p-C11A or 158-CA likewise inhibits the wild-type Pex5p import into peroxisomes, strongly suggesting that Pex5p-C11A exerts the dominant-negative effect at the translocation step via Pex14p. Taken together, these findings show that the cysteine(11) of Pex5p is indispensable for two distinct steps, its import and export. The Pex5p-C11A would be a useful tool for gaining a mechanistic insight into the matrix protein import into peroxisomes., (© 2011 John Wiley & Sons A/S.)

A peroxisomal C-tail-anchored type-II membrane protein, Pex26p, recruits AAA ATPase Pex1p-Pex6p complexes to peroxisomes. We herein attempted to gain mechanistic insight into Pex26p function. Pex26pΔ33-40 truncated in amino-acid residues at 33-40 abolishes the recruiting of Pex1p-Pex6p complex to peroxisomes and fails to complement the impaired phenotype of pex26 CHO cell mutant ZP167, thereby suggesting that peroxisomal localization of Pex1p and Pex6p is indispensable for the transport of matrix proteins. In in vitro transport assay using semipermeabilized CHO cells, Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. This finding is confirmed by the assay using Walker-motif mutants. Transport of Pex1p and Pex6p is temperature-dependent. In vitro binding assays with glutathione-S-transferase-fused Pex26p, Pex1p and Pex6p bind to Pex26p in a manner dependent on ATP binding but not ATP hydrolysis. These results suggest that ATP hydrolysis is required for stable localization of Pex1p to peroxisomes, but not for binding to Pex26p. Moreover, Pex1p and Pex6p are altered to a more compact conformation upon binding to ATP, as verified by limited proteolysis. Taken together, Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by the ATPase cycle., (© 2011 John Wiley & Sons A/S.)

We earlier isolated peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, ZPEG241, by the 9-(1'-pyrene)nonanol/ultraviolet selection method, from TKaEG2, the wild-type CHO-K1 cells transformed with two cDNAs encoding rat Pex2p and peroxisome targeting signal type 2 (PTS2)-tagged enhanced green fluorescent protein (EGFP). Peroxisomal localization of PTS2-EGFP was specifically impaired in ZPEG241 due to the failure of Pex5pL expression. Analysis of partial genomic sequence of PEX5 revealed one-point nucleotide-mutation from G to A in the 3'-acceptor splice site located at 1 nt upstream of exon 7 encoding Pex5pL specific 37-amino acid insertion, thereby generating 21-nt deleted mRNA of PEX5L in ZPEG241. When ZPEG241-derived Pex5pL was ectopically expressed in ZPEG241, PTS2 import was not restored because of no interaction with Pex7p. Together, we confirm the pivotal role of Pex5pL in PTS2 import, showing that the N-terminal 7-amino acid residues in the 37-amino acid insertion of Pex5pL are essential for the binding to Pex7p.

Reactive oxygen species are generated within peroxisomes during peroxisomal metabolism. However, due to technological difficulties, the intraperoxisomal redox state remain elusive, and the effect of peroxisome deficiency on the intracellular redox state is controversial. A newly developed, genetically encoded fluorescence resonance energy transfer (FRET) probe, Redoxfluor, senses the physiological redox state via its internal disulfide bonds, resulting in a change in the conformation of the protein leading to a FRET response. We made use of Redoxfluor to measure the redox states at the subcellular level in yeast and Chinese hamster ovary (CHO) cells. In wild-type peroxisomes harboring an intact fatty acid beta-oxidation system, the redox state within the peroxisomes was more reductive than that in the cytosol, despite the fact that reactive oxygen species were generated within the peroxisomes. Interestingly, we observed that the redox state of the cytosol of cell mutants for peroxisome assembly, regarded as models for a neurological metabolic disorder, was more reductive than that of the wild-type cells in yeast and CHO cells. Furthermore, Redoxfluor was utilized to develop an efficient system for the screening of drugs that moderate the abnormal cytosolic redox state in the mutant CHO cell lines for peroxisome assembly without affecting the redox state of normal cells.