Kyushu University Academic Staff Educational and Research Activities Database
Researcher information (To researchers) Need Help? How to update
Okumoto Kanji Last modified date:2024.06.03

Assistant Professor / Informational biology
Department of Biology
Faculty of Sciences

Graduate School
Undergraduate School

E-Mail *Since the e-mail address is not displayed in Internet Explorer, please use another web browser:Google Chrome, safari.
 Reseacher Profiling Tool Kyushu University Pure
Home Page of Laboratory of Functional Cell Biology, Department of Biology, Graduate School of Sciences, Kyushu University. .
Academic Degree
Studies on RING finger peroxins, Pex10p and Pex12p: cDNA cloning and functional analysis
Country of degree conferring institution (Overseas)
Field of Specialization
Molecular cell biology
Total Priod of education and research career in the foreign country
Outline Activities
Research: The system regulating homeostasis of peroxisomes, including the biogenesis, morphology, and metabolic functions. Peroxisome biogenesis and its dysfunction in human disorders.
Educatuion: Two experimental classes (fundamental biological experiments and applied cell biology); A class of molecular cell biology.
Social activity: "ESSP ver.2", the high-level scientific program for high school students.
Research Interests
  • Studies on peroxisome biogenesis and the homeostasis
    keyword : Peroixsome, homeostasis, ubiquitin, phosphorylation
    2003.02Studies on intracellular protein traffic using peroxisome as a model organelle. Most focused issue is how three RING finger peroxins (peroxisome assembly factors) function in import of peroxisome matrix proteins..
Academic Activities
1. Yukio Fujiki, 奥本 寛治, 向井 悟, 田村 茂彦, Molecular basis for peroxisome biogenesis disorders, Molecular machines involved in peroxisome biogenesis and maintenance, Springer-Verlag, Wien, Austria. pp. 91-110, 2014.10.
1. Kanji Okumoto, Yukio Fujiki, Analysis of Peroxisome Biogenesis by Phos-Tag SDS-PAGE., Methods in molecular biology (Clifton, N.J.), 10.1007/978-1-0716-3048-8_15, 2643, 207-215, 2023.03, 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..
2. Yukito Aiba, Jihoon Kim, Arata Imamura, Kanji Okumoto, Nobushige Nakajo, Regulation of Myt1 kinase activity via its N-terminal region in Xenopus meiosis and mitosis., Cells and Development, 10.1016/j.cdev.2021.203754, 169, 203754, 2022.03, Immature animal oocytes are naturally arrested at the first meiotic prophase (Pro-I), which corresponds to the G2 phase of the cell cycle. In Xenopus oocytes, Myt1 kinase phosphorylates and inactivates cyclin-dependent kinase 1 (Cdk1) at Pro-I, thereby preventing oocytes from entering meiosis I (MI) prematurely. Previous studies have shown that, upon resuming MI, Cdk1 and p90rsk, which is a downstream kinase of the Mos-MAPK pathway, in turn phosphorylate the C-terminal region of Myt1, to suppress its activity, thereby ensuring high Cdk1 activity during M phase. However, the roles of the N-terminal region of Myt1 during meiosis and mitosis remain to be elucidated. In the present study, we show that the N-terminal region of Myt1 participates in the regulation of Myt1 activity in the Xenopus cell cycle. In particular, we found that a short, conserved sequence in the N-terminal region, termed here as the PAYF motif, is required for the normal activity of Myt1 in oocytes. Furthermore, multiple phosphorylations by Cdk1 at the Myt1 N-terminal region were found to be involved in the negative regulation of Myt1. In particular, phosphorylations at Thr11 and Thr16 of Myt1, which are adjacent to the PAYF motif, were found to be important for the inactivation of Myt1 in the M phase of the cell cycle. These results suggest that in addition to the regulation of Myt1 activity via the C-terminal region, the N-terminal region of Myt1 also plays an important role in the regulation of Myt1 activity..
3. Kanji Okumoto, Mahmoud El Shermely, Masanao Natsui, Hidetaka Kosako, Ryuichi Natsuyama, Toshihiro Marutani, Yukio Fujiki, The peroxisome counteracts oxidative stresses by suppressing catalase import via Pex14 phosphorylation, eLife, 10.7554/elife.55896, 9, 2020.08, 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..
4. Yukio Fujiki, Yuichi Abe, Yuuta Imoto, Akemi J. Tanaka, Kanji Okumoto, Masanori Honsho, Shigehiko Tamura, Non Miyata, Toshihide Yamashita, Wendy K. Chung, Tsuneyoshi Kuroiwa, Recent insights into peroxisome biogenesis and associated diseases, Journal of Cell Science, 10.1242/jcs.236943, 133, 9, jcs236943, 2020.05, 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..
5. Michael M. Dubreuil, David W. Morgens, Kanji Okumoto, Masanori Honsho, Kévin Contrepois, Brittany Lee-McMullen, Gavin Mc Allister Traber, Ria S. Sood, Scott J. Dixon, Michael P. Snyder, Yukio Fujiki, Michael C. Bassik, Systematic Identification of Regulators of Oxidative Stress Reveals Non-canonical Roles for Peroxisomal Import and the Pentose Phosphate Pathway, Cell Reports, 10.1016/j.celrep.2020.01.013, 30, 5, 1417-1433.e7, 2020.02, 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..
6. Akemi J. Tanaka, Kanji Okumoto, Shigehiko Tamura, Yuichi Abe, Yoel Hirsch, Liyong Deng, Joseph Ekstein, Wendy K. Chung, Yukio Fujiki, A newly identified mutation in the PEX26 gene is associated with a milder form of Zellweger spectrum disorder, Cold Spring Harbor Molecular Case Studies, 10.1101/mcs.a003483, 5, 1, a003483-a003483, 2019.01, 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..
7. Yuuta Imoto, Yuichi Abe, Kanji Okumoto, Mio Ohnuma, Haruko Kuroiwa, Tsuneyoshi Kuroiwa, Yukio Fujiki, Dynamics of the nucleoside diphosphate kinase protein DYNAMO2 correlates with the changes in the global GTP level during the cell cycle of Cyanidioschyzon merolae, Proceedings of the Japan Academy Series B: Physical and Biological Sciences, 10.2183/pjab.95.007, 95, 2, 75-85, 2019.01, 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..
8. Hajime Niwa, Yasuhiro Miyauchi-Nanri, Kanji Okumoto, Satoru Mukai, Kentaro Noi, Teru Ogura, Yukio Fujiki, A newly isolated Pex7-binding, atypical PTS2 protein P7BP2 is a novel dynein-type AAA+ protein, Journal of Biochemistry, 10.1093/jb/mvy073, 164, 6, 437-447, 2018.12, 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..
9. Yuuta Imoto, Yuichi Abe, Masanori Honsho, Kanji Okumoto, Mio Ohnuma, Haruko Kuroiwa, Tsuneyoshi Kuroiwa, Yukio Fujiki, Onsite GTP fuelling via DYNAMO1 drives division of mitochondria and peroxisomes, Nature Communications, 10.1038/s41467-018-07009-z, 9, 1, 2018.12, 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..
10. Kanji Okumoto, Tatsuaki Ono, Ryusuke Toyama, Ayako Shimomura, Aiko Nagata, Yukio Fujiki, New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes, Journal of Cell Biology, 10.1083/jcb.201708122, 217, 2, 619-633, 2018.02, 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..
11. Non Miyata, Kanji Okumoto, Yukio Fujiki, Cell death or survival against oxidative stress, Subcellular Biochemistry, 10.1007/978-981-13-2233-4_20, 463-471, 2018.01, 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..
12. Kanji Okumoto, Non Miyata, Yukio Fujiki, Identification of peroxisomal protein complexes with PTS receptors, pex5 and pex7, in mammalian cells, Subcellular Biochemistry, 10.1007/978-981-13-2233-4_12, 287-298, 2018.01, 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..
13. Yukio Fujiki, Non Miyata, Satoru Mukai, Kanji Okumoto, Emily H. Cheng, BAK regulates catalase release from peroxisomes, Molecular & Cellular Oncology, 4, 3, 2017.03, 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..
14. Shoko Abe, Tomoaki Nagai, Moe Masukawa, Kanji Okumoto, Yuta Homma, Yukio Fujiki, Kensaku Mizuno, Localization of protein kinase NDR2 to peroxisomes and its role in ciliogenesis, Journal of Biological Chemistry, 10.1074/jbc.M117.775916, 292, 10, 4089-4098, 2017.03, 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..
15. Ken-ichiro Hosoi, Non Miyata, Satoru Mukai, Satomi Furuki, Kanji Okumoto, Emily H. Cheng, Yukio Fujiki, The VDAC2-BAK axis regulates peroxisomal membrane permeability, Journal of Cell Biology, 10.1083/jcb.201605002, 216, 3, 709-722, 2017.03, 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..
16. Kanji Okumoto, Shigehiko Tamura, Yukio Fujiki, Blue native PAGE Applications to study peroxisome biogenesis, Methods in Molecular Biology, 10.1007/978-1-4939-6937-1_18, 1595, 197-205, 2017.01, 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..
17. Yuuta Imoto, Yuichi Abe, Kanji Okumoto, Masanori Honsho, Haruko Kuroiwa, Tsuneyoshi Kuroiwa, Yukio Fujiki, Defining the dynamin-based ring organizing center on the peroxisome-dividing machinery isolated from Cyanidioschyzon merolae, Journal of Cell Science, 10.1242/jcs.199182, 130, 5, 853-867, 2017.01, 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..
18. Kanji Okumoto, Yukio Fujiki, Generation of peroxisome-deficient somatic animal cell mutants, Methods in Molecular Biology, 10.1007/978-1-4939-6937-1_29, 1595, 319-327, 2017.01, 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..
19. Kanji Okumoto, Masanori Honsho, Yuqiong Liu, Yukio Fujiki, Peroxisomal membrane and matrix protein import using a semi-intact mammalian cell system, Methods in Molecular Biology, 10.1007/978-1-4939-6937-1_20, 1595, 213-219, 2017.01, 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..
20. Yukio Fujiki, Kanji Okumoto, Masanori Honsho, Protein Import into Peroxisomes: the principles and methods of studying (version 2.0), Encyclopedia of Life Sciences, 2015.04.
21. Masafumi Noguchi, Kanji Okumoto, Yukio Fujiki, System to quantify the import of peroxisomal matrix proteins by fluorescence intensity, GENES TO CELLS, 10.1111/gtc.12051, 18, 6, 476-492, 2013.06.
22. Ryuichi Natsuyama, Kanji Okumoto, Yukio Fujiki, Pex5p stabilizes Pex14p: a study using a newly isolated pex5 CHO cell mutant, ZPEG101, Biochem J., 10.1042/BJ20120911., 449, 1, 195-207, 2013.01, 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..
23. 宮田暖, 奥本寛治, 向井悟, 野口雅史, 藤木幸夫, AWP1/ZFAND6 functions in Pex5 export by interacting with cys-monoubiquitinated Pex5 and Pex6 AAA ATPase, Traffic, 10.1111/j.1600-0854.2011.01298.x., 13, 1, 168-183, 2012.01, 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..
24. 藤木幸夫, 名城千加, 宮田暖, 田村茂彦, 奥本寛治, New insights into dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p in shuttling of PTS1-receptor Pex5p during peroxisome biogenesis, Biochim. Biophys. Acta, 10.1111/j.1600-0854.2011.01298.x., 1823, 1, 145-149, 2012.01.
25. Kanji Okumoto, Sachi Misono, Non Miyata, Yui Matsumoto, Satoru Mukai, Yukio Fujiki, Cysteine-ubiquitination of peroxisome-targeting-signal type 1 (PTS1)-receptor Pex5p regulates Pex5p import and export, Traffic, 10.1111/j.1600-0854.2011.01217.x, 2011.05.
26. 田中敦, 奥本寛治, 藤木幸夫, cDNA cloning and characterization of the third isoform in human peroxin Pex11p., Biochem. Biophys. Res. Commun., 10.1016/S0006-291X(02)02936-4, 300, 4, 819-823, 300(4), 819-823, 2003.01.
27. Ghaedi Kamran, 田村茂彦, 奥本寛治, 松園裕嗣, 藤木幸夫, The peroxin Pex3p initiates membrane assembly in peroxisome biogenesis., Mol. Biol. Cell, 11, 6, 2085-2102, 11(6),2085-102, 2000.06.
28. 藤木幸夫, 奥本寛治, 大寺秀典, 田村茂彦, Peroxisome biogenesis and molecular defects in peroxisome assembly disorders, Cell Biochem. Biophys., 10.1385/CBB:32:1-3:155, 32, 1, 155-164, 2000.04.
29. Ghaedi Kamran, 川井淳, 奥本寛治, 他4名, 藤木幸夫, Isolation and characterization of novel peroxisome biogenesis-defective Chinese hamster ovary cell mutants using green fluorescent protein., Exp, Cell Res., 10.1006/excr.1999.4413, 248, 2, 489-497, 248(2), 489-497, 1999.05.
30. 志水信弘, 伊藤竜太, 他5名, 奥本寛治, 原野友之, 藤木幸夫, The peroxin Pex14p, cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis., J. Biol. Chem, 10.1074/jbc.274.18.12593, 274, 18, 12593-12604, 274(18), 12593-15604, 1999.04.
31. 木下尚彦, Ghaedi Kamran, 下沢伸行, Wanders R.J.A., 松園裕嗣, 今中常雄, 奥本寛治, 他2名, 藤木幸夫, Newly identified Chinese hamster ovary cell mutants are defective in biogenesis of peroxisomal membrane vesicles (peroxisomal ghosts), representing a novel complementation group in mammals., J. Biol. Chem., 10.1074/jbc.273.37.24122, 273, 37, 24122-24130, 273(37), 24122-24130, 1998.09.
32. 阿部巧, 奥本寛治, 田村茂彦, 藤木幸夫, Clofibrate-inducible, 28-kDa peroxisomal integral membrane protein is encoded by PEX11., FEBS Lett., 10.1016/S0014-5793(98)00815-1, 431, 3, 468-472, 431(3), 468-472, 1998.07.
33. 田村茂彦, 奥本寛治, 他6名, 藤木幸夫, Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I., Proc. Natl. Acad. Sci. USA., 10.1073/pnas.95.8.4350, 95, 8, 4350-4355, 95(8), 4350-4355, 1998.04.
34. 下沢伸行, 鈴木康之, 他5名, 奥本寛治, 藤木幸夫, 折井忠夫, Barth P.G., Wanders R.J.A., 近藤直美, Peroxisome biogenesis disorders: identification of a new complementation group distinct from peroxisome-deficient CHO mutants and not complemented by human PEX13., Biochem. Biophys. Res. Commun., 10.1006/bbrc.1997.8067, 243, 2, 368-371, 243(2), 368-371, 1998.02.
35. 大手秀典, 奥本寛治, 他8名, 藤木幸夫, Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants., Mol. Cell. Biol., 10.1128/mcb.18.1.388, 18, 1, 388-399, 18(1), 388-399, 1998.01, ABSTRACT
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. PEX5 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% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, 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 PEX5 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 inPEX5 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..
1. Oxidative stress response via peroxisome.
2. Novel counteracting mechanism against oxidative stress by regulating intracellular localization of catalase via Pex14 phosphorylation.
3. Novel counteracting mechanism against oxidative stress by regulating intracellular localization of catalase via Pex14 phosphorylation.
4. Kanji Okumoto, Yuichi Yagita, Masanori Honsho, and Yukio Fujiki, Biogenesis of peroxisomal tail-anchored membrane proteins, new splicing variants of Miro1, acyl-CoA binding domain containing protein 5 (ACBD5), and peroxin Pex26p., International Symposium on “Proteins: From the Cradle to the Grave”, 2018.08.
5. Functional analysis of P5BP1, a novel protein interacting with PTS1 recepor Pex5p.
6. Kanji Okumoto, Hiromi Noda, Yukio Fujiki, RING peroxin complexes comprising Pex10p and Pex12p ubiquitinate the PTS1 receptor Pex5p and regulate its shuttling between peroxisomes and the cytosol., PerFuMe Kick-off Conference, 2013.12.
7. Identification of P5BP1, a novel protein interacting with PTS1 recepor Pex5p.
8. Novel function of peroxisome-targeting signal type 1 (PTS1)-receptor Pex5p in Pex14p stability .
9. Several distinct modes of ubiquitination of peroxisome-targeting signal type 1 (PTS1)-receptor Pex5p regulate peroxisomal matrix protein import.
Membership in Academic Society
  • Japan Society for Cell Biology
  • The Japanese Biochemistry Society
  • The Molecular Biology Society of Japan
Educational Activities
A experimental class of Applied Cell Physiology.
Classes of General Science Experiments, Molecular Cell Biology, and Research Administration I .
Professional and Outreach Activities
ESSP (Excellent Students in Science) project for high school students.