Language：English   Publishing type：Research paper (scientific journal)  

Most of peroxisomal matrix proteins including a hydrogen peroxide (H₂O₂)-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 H₂O₂ in mammalian cells. The H₂O₂-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 H₂O₂. We thus suggest that the H₂O₂-induced phosphorylation of Pex14 spatiotemporally regulates peroxisomal import of catalase, functioning in counteracting action against oxidative stress by the increase of cytosolic catalase.

DOI： 10.7554/elife.55896

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1242/jcs.236943

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.celrep.2020.01.013

Language：English   Publishing type：Research paper (scientific journal)  

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 immu-nostaining 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.

DOI： 10.1101/mcs.a003483

Language：English   Publishing type：Research paper (scientific journal)  

GTP is an essential source of energy that supports a large array of cellular mechanochemical structures ranging from protein synthesis machinery to cytoskeletal apparatus for maintaining the cell cycle. However, GTP regulation during the cell cycle has been difficult to investigate because of heterogenous levels of GTP in asynchronous cell cycles and genetic redundancy of the GTP-generating enzymes. Here, in the unicellular red algae Cyanidioschyzon merolae, we demonstrated that the ATP-GTP-converting enzyme DYNAMO2 is an essential regulator of global GTP levels during the cell cycle. The cell cycle of C. merolae can be highly synchronized by light/dark stimulations to examine GTP levels at desired time points. Importantly, the genome of C. merolae encodes only two isoforms of the ATP-GTP-converting enzyme, namely DYNAMO1 and DYNAMO2. DYNAMO1 regulates organelle divisions, whereas DYNAMO2 is entirely localized in the cytoplasm. DYNAMO2 protein levels increase during the S-M phases, and changes in GTP levels are correlated with these DYNAMO2 protein levels. These results indicate that DYNAMO2 is a potential regulator of global GTP levels during the cell cycle.

DOI： 10.2183/pjab.95.007

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1093/jb/mvy073

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/s41467-018-07009-z

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1083/jcb.201708122

Language：English  

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.

DOI： 10.1007/978-981-13-2233-4_12

Language：English  

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.

DOI： 10.1007/978-981-13-2233-4_20

Language：English  

Loss of voltage-dependent anion channel 2 (VDAC2) leads to impaired peroxisome biogenesis in mammalian cells. Knockdown of BAK restores peroxisomal biogenesis in VDAC2-deficient cells, where BAK localization shifts from mitochondria to peroxisomes. Moreover, overexpression of BAK activators in wild-type cells permeabilizes peroxisomes in a BAK-dependent manner. Together, BAK most likely regulates peroxisomal membrane permeability.

Language：English  

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-XL 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.

DOI： 10.1083/jcb.201605002

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1074/jbc.M117.775916

Language：English  

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.

DOI： 10.1007/978-1-4939-6937-1_18

Language：English  

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.

DOI： 10.1007/978-1-4939-6937-1_20

Language：English  

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.

DOI： 10.1007/978-1-4939-6937-1_29

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1242/jcs.199182

Language：English  

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fphys.2014.00307

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.m113.527937

Language：Others  

DOI： 10.1007/978-3-7091-1788-0_5

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/gtc.12051

Language：English   Publishing type：Research paper (scientific journal)  

Pex5p [PTS (peroxisome-targeting signal) type 1 receptor] plays an essential role in peroxisomal matrix protein import. In the present study, we isolated a novel PEX5-deficient CHO (Chinese-hamster ovary) cell mutant, termed ZPEG101, showing typical peroxisomal import defects of both PTS1 and PTS2 proteins. ZPEG101 is distinct from other known pex5 CHO mutants in its Pex5p expression. An undetectable level of Pex5p in ZPEG101 results in unstable Pex14p, which is due to inefficient translocation to the peroxisomal membrane. All of the mutant phenotypes of ZPEG101 are restored by expression of wild-type Pex5pL, a longer form of Pex5p, suggesting a role for Pex5p in sustaining the levels of Pex14p in addition to peroxisomal matrix protein import. Complementation analysis using various Pex5p mutants revealed that in the seven pentapeptide WXXXF/Y motifs in Pex5pL, known as the multiple binding sites for Pex14p, the fifth motif is an auxiliary binding site for Pex14p and is required for Pex14p stability. Furthermore, we found that Pex5p–Pex13p interaction is essential for the import of PTS1 proteins as well as catalase, but not for that of PTS2 proteins. Therefore ZPEG101 with no Pex5p would be a useful tool for investigating Pex5p function and delineating the mechanisms underlying peroxisomal matrix protein import.

DOI： 10.1042/BJ20120911.

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1111/j.1600-0854.2011.01298.x.

Language：English  

DOI： 10.1111/j.1600-0854.2011.01298.x.

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.m111.285197

Language：English  

DOI： 10.1111/j.1600-0854.2011.01217.x

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbamcr.2006.09.012

Language：English  

DOI： 10.1016/S0006-291X(02)02936-4

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.m003303200

Language：English  

Language：English  

DOI： 10.1385/CBB:32:1-3:155

Language：English  

DOI： 10.1006/excr.1999.4413

Language：English  

DOI： 10.1074/jbc.274.18.12593

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/hmg/7.9.1399

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.273.37.24122

Language：Others   Publishing type：Research paper (scientific journal)  

ABSTRACT

Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11–20, 1997), using a transient transfection assay and an ectopic, readily visible marker, green fluorescent protein. This cDNA encodes a 359-amino-acid membrane protein of peroxisomes with two transmembrane segments and a cysteine-rich zinc finger, the RING motif. A stable transformant of ZP109 with the PEX12 was morphologically and biochemically restored for peroxisome biogenesis. Pex12p was shown by expression of bona fide as well as epitope-tagged Pex12p to expose both N- and C-terminal regions to the cytosol. Fibroblasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group III (CG-III) were also complemented for peroxisome biogenesis with PEX12 . Two unrelated patients of this group manifesting peroxisome deficiency disorders possessed homozygous, inactivating PEX12 mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for ²⁹¹ Asn and ²⁹² Ser, creating an apparently unchanged codon for Asn and a codon 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-III peroxisome deficiency. Moreover, truncation and site mutation studies, including patient PEX12 analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function.

DOI： 10.1128/mcb.18.7.4324

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/S0014-5793(98)00815-1

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1073/pnas.95.8.4350

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1006/bbrc.1997.8067

Language：English   Publishing type：Research paper (scientific journal)  

<title>ABSTRACT</title>
To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. <italic>PEX5</italic> cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R’s deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24&#37; amino acid identity with PTS1Rs from humans, <italic>Pichia pastoris</italic>, and <italic>Saccharomyces cerevisiae</italic>, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of <italic>PEX5</italic> complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations in<italic>PEX5</italic> were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

DOI： 10.1128/mcb.18.1.388

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/ng1197-265

Language：English   Publishing type：Research paper (scientific journal)  

We isolated peroxisome biogenesis mutants from Chinese hamster ovary (CHO) cells, using the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) method and wild-type CHO-K1 cells that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1). Three mutant cell clones, ZP110, ZP111, and ZP114, showed cytosolic localization of catalase, thereby indicating a defect in peroxisome biogenesis, whereas ZP112 and ZP113 contained fewer but larger catalase-positive particles. Mutant ZP115 displayed an aberrant, tubular structure immunoreactive to anti-catalase antibody. Mutants lacking morphologically recognizable peroxisomes also showed the typical peroxisome assembly-defective phenotype such as severe loss of catalase latency and resistance to 12-(1'-pyrene)dodecanoic acid (P12)/UV treatment. ZP110 and ZP111, and ZP114 were found to belong to two novel complementation groups, respectively, by complementation group analysis with cDNA transfection and cell fusion. Cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome revealed that ZP110 and ZP114 could not be classified to any of human complementation groups. Thus, ZP110/ZP111 and ZP114 are the first, two peroxisome-deficient cell mutants of newly identified complementation groups distinct from the ten mammalian groups previously characterized.

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1006/excr.1997.3552

Language：English   Publishing type：Research paper (scientific journal)  

For the study of mechanism of peroxisome biogenesis, we attempted to isolate CHO cell mutants deficient in peroxisome biogenesis. We used as the parent strain a stable CHO transformant of rat PEX2 (formerly named peroxisome assembly factor-1) cDNA, to avoid unusually frequent isolation of Pex2 mutants. Among the three peroxisome-deficient mutants obtained, ZP102 was a new CHO mutant of complementation group 2, and was restored for peroxisome assembly by the transfection of human PEX5 (formerly called PXR1 or PTS1R) cDNA. This approach would facilitate the isolation of new complementation gorups of peroxisome-deficient CHO mutants and the identification of essential genes for peroxisome biogenesis.

DOI： 10.1006/bbrc.1996.5971

Event date： 2018.8

Language：English  

Venue：Enryakuji Kaikan, Shiga   Country：Japan  

Event date： 2017.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：神戸国際会議場   Country：Japan  

Event date： 2017.6

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：仙台国際会議場（宮城）   Country：Japan  

Event date： 2016.9

Language：Japanese  

Venue：仙台国際センター   Country：Japan  

Event date： 2014.6

Language：Japanese  

Venue：東大寺総合文化センター（奈良市）   Country：Japan  

Functional analysis of P5BP1, a novel protein interacting with PTS1 recepor Pex5p

Event date： 2013.12

Language：English  

Venue：Hampshire Hotel Plaza Groningen （グローニンゲン・オランダ）   Country：Netherlands  

RING peroxin complexes comprising Pex10p and Pex12p ubiquitinate the PTS1 receptor Pex5p and regulate its shuttling between peroxisomes and the cytosol.

Event date： 2013.9

Language：Japanese  

Venue：パシフィコ横浜（横浜市）   Country：Japan  

Identification of P5BP1, a novel protein interacting with PTS1 recepor Pex5p

Event date： 2012.12

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：福岡国際会議場（福岡市）   Country：Japan  

Novel function of peroxisome-targeting signal type 1 (PTS1)-receptor Pex5p in Pex14p stability

Event date： 2012.12

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：福岡国際会議場（福岡市）   Country：Japan  

Several distinct modes of ubiquitination of peroxisome-targeting signal type 1 (PTS1)-receptor Pex5p regulate peroxisomal matrix protein import

Language：Others   Presentation type：Symposium, workshop panel (public)  

Country：Other  

Novel counteracting mechanism against oxidative stress by regulating intracellular localization of catalase via Pex14 phosphorylation

Language：Others  

Venue：名古屋国際会議場   Country：Japan  

Novel counteracting mechanism against oxidative stress by regulating intracellular localization of catalase via Pex14 phosphorylation

Language：Others   Presentation type：Symposium, workshop panel (public)  

Venue：福岡国際会議場   Country：Japan  

Oxidative stress response via peroxisome

Event date： 2019.11

Language：Japanese  

Venue：大阪市立大学、大阪   Country：Japan  

Event date： 2019.9

Language：Japanese  

Venue：パシフィコ横浜、神奈川   Country：Japan  

Event date： 2018.12

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：San Diego   Country：United States  

Event date： 2018.6 - 2018.7

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：九州大学、福岡   Country：Japan  

Event date： 2017.7

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：West Dover, VT   Country：United States  

Event date： 2017.6

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：仙台国際会議場（宮城）   Country：Japan  

Event date： 2013.12

Language：Japanese  

Venue：神戸国際会議場（神戸市）   Country：Japan  

Identification of a novel protein interacting with peroxisome targeting signal type 1 (PTS1) receptor, Pex5p.

Event date： 2012.5

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：神戸国際会議場（神戸市）   Country：Japan  

Two proteases, trypsin domain-containing 1 (Tysnd1) and peroxisomal Lon protease (PsLon), cooperatively regulate fatty acid β-oxidation in peroxisomal matrix

Event date： 2011.12

Venue：パシフィコ横浜（横浜市）   Country：Japan  

Cysteine-ubiquitination of peroxisome-targeting-signal type 1 (PTS1)-receptor Pex5p regulates Pex5p recycling

Event date： 2011.12

Venue：デンバー（アメリカ合衆国）   Country：United States  

A Novel Function of AWP1/ZFAND6: Regulation of Pex5p Export by Interacting with Cys-monoubiquitinated Pex5p and AAA ATPase, Pex6p

Event date： 2011.5

Presentation type：Oral presentation (general)  

Venue：久留米大学医学部（久留米市）   Country：Japan  

Event date： 2010.9

Venue：日航ホテル奈良（奈良市）   Country：Japan  

Event date： 2010.5

Venue：大阪国際会議場（大阪市）   Country：Japan  

Event date： 2009.12

Venue：パシフィコ横浜（横浜市）   Country：Japan  

Venue：パシフィコ横浜（横浜市）   Country：Japan  

Venue：京都国際会議場   Country：Japan  

Venue：福岡国際会議場   Country：Japan  

Venue：ワシントンコンベンションセンター（ワシントンDC）   Country：United States  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Authorship：Lead author   Language：Japanese   Publisher：(公社)日本生化学会  

ペルオキシソームは極長鎖脂肪酸のβ酸化をはじめとした多様かつ重要な代謝機能を有する細胞小器官(オルガネラ)である.ペルオキシソーム形成に必須な多数のペルオキシン遺伝子(PEX)の同定,およびその翻訳産物であるペルオキシンの機能解析が大きく進展し,ペルオキシソーム欠損症の全病因PEX遺伝子の解明に続いてペルオキシソームの形成機構が明らかとなってきた.ここでは,ペルオキシソーム構成タンパク質の輸送局在化やペルオキシソームの分裂,形態制御,分解などの統合的制御によるペルオキシソーム形成機構を概説し,ペルオキシソームタンパク質の輸送局在化機構や酸化ストレス応答におけるペルオキシソームの役割に関する最新の知見について,哺乳類ペルオキシソームを中心に我々の成果を紹介する.(著者抄録)

Language：English  

DOI： 10.5685/plmorphol.32.59

Language：Others  

Language：English