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

Mitophagy plays an important role in the maintenance of mitochondrial homeostasis and can be categorized into two types: ubiquitin-mediated and receptor-mediated pathways. During receptor-mediated mitophagy, mitophagy receptors facilitate mitophagy by tethering the isolation membrane to mitochondria. Although at least five outer mitochondrial membrane proteins have been identified as mitophagy receptors, their individual contribution and interrelationship remain unclear. Here, we show that HeLa cells lacking BNIP3 and NIX, two of the five receptors, exhibit a complete loss of mitophagy in various conditions. Conversely, cells deficient in the other three receptors show normal mitophagy. Using BNIP3/NIX double knockout (DKO) cells as a model, we reveal that mitophagy deficiency elevates mitochondrial reactive oxygen species (mtROS), which leads to activation of the Nrf2 antioxidant pathway. Notably, BNIP3/NIX DKO cells are highly sensitive to ferroptosis when Nrf2-driven antioxidant enzymes are compromised. Moreover, the sensitivity of BNIP3/NIX DKO cells is fully rescued upon the introduction of wild-type BNIP3 and NIX, but not the mutant forms incapable of facilitating mitophagy. Consequently, our results demonstrate that BNIP3 and NIX-mediated mitophagy plays a role in regulating mtROS levels and protects cells from ferroptosis.

DOI： 10.1038/s41418-024-01280-y

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Language：English  

DOI： 10.1007/978-981-97-4584-5_16

PubMed

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

Target of rapamycin complex 1 (TORC1) is activated in response to nutrient availability and growth factors, promoting cellular anabolism and proliferation. To explore the mechanism of TORC1-mediated proliferation control, we performed a genetic screen in fission yeast and identified Sfp1, a zinc-finger transcription factor, as a multicopy suppressor of temperature-sensitive TORC1 mutants. Our observations suggest that TORC1 phosphorylates Sfp1 and protects Sfp1 from proteasomal degradation. Transcription analysis revealed that Sfp1 positively regulates genes involved in ribosome production together with two additional transcription factors, Ifh1/Crf1 and Fhl1. Ifh1 physically interacts with Fhl1, and the nuclear localization of Ifh1 is regulated in response to nutrient levels in a manner dependent on TORC1 and Sfp1. Taken together, our data suggest that the transcriptional regulation of the genes involved in ribosome biosynthesis by Sfp1, Ifh1, and Fhl1 is one of the key pathways through which nutrient-activated TORC1 promotes cell proliferation.

DOI： 10.1080/10985549.2023.2282349

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

Atg: autophagy related; IMM: inner mitochondrial membrane; IMS: intermembrane space; PAS: phagophore assembly site; SAR: selective autophagy receptor.

DOI： 10.1080/15548627.2023.2237343

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

Accumulating evidence has demonstrated the presence of intertissue-communication regulating systemic aging, but the underlying molecular network has not been fully explored. We and others previously showed that two basic helix-loop-helix transcription factors, MML-1 and HLH-30, are required for lifespan extension in several longevity paradigms, including germlineless Caenorhabditis elegans. However, it is unknown what tissues these factors target to promote longevity. Here, using tissue-specific knockdown experiments, we found that MML-1 and its heterodimer partners MXL-2 and HLH-30 act primarily in neurons to extend longevity in germlineless animals. Interestingly, however, the downstream cascades of MML-1 in neurons were distinct from those of HLH-30. Neuronal RNA interference (RNAi)-based transcriptome analysis revealed that the glutamate transporter GLT-5 is a downstream target of MML-1 but not HLH-30. Furthermore, the MML-1-GTL-5 axis in neurons is critical to prevent an age-dependent collapse of proteostasis and increased oxidative stress through autophagy and peroxidase MLT-7, respectively, in long-lived animals. Collectively, our study revealed that systemic aging is regulated by a molecular network involving neuronal MML-1 function in both neural and peripheral tissues.

DOI： 10.1073/pnas.2221553120

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.molcel.2023.05.016

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Language：Japanese   Publisher：日本基礎老化学会  

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.molcel.2023.04.022

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.celrep.2023.112454

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

DOI： 10.1080/15548627.2023.2214029

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Human parechovirus (PeV-A) is an RNA virus that belongs to the family Picornaviridae and it is currently classified into 19 genotypes. PeV-As usually cause mild illness in children and adults. Among the genotypes, PeV-A3 can cause severe diseases in neonates and young infants, resulting in neurological sequelae and death. In this study, we identify the human myeloid-associated differentiation marker (MYADM) as an essential host factor for the entry of six PeV-As (PeV-A1 to PeV-A6), including PeV-A3. The infection of six PeV-As (PeV-A1 to PeV-A6) to human cells is abolished by knocking out the expression of MYADM. Hamster BHK-21 cells are resistant to PeV-A infection, but the expression of human MYADM in BHK-21 confers PeV-A infection and viral production. Furthermore, VP0 capsid protein of PeV-A3 interacts with one extracellular domain of human MYADM on the cell membrane of BHK-21. The identification of MYADM as an essential entry factor for PeV-As infection is expected to advance our understanding of the pathogenesis of PeV-As.

DOI： 10.1038/s41467-023-37399-8

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Other Link： https://www.nature.com/articles/s41467-023-37399-8

Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00125-022-05800-8

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Other Link： https://link.springer.com/article/10.1007/s00125-022-05800-8/fulltext.html

Language：Japanese   Publisher：新潟医学会  

【緒言】病原体等を取り扱う機関においては,バイオセーフティおよびバイオセキュリティの観点からそれらの保管や取扱いの管理体制を整備する責任がある.バイオリスク管理の概念は研究者の間に浸透しているものの,実際の管理運用体制は必ずしも十分に整備されていない.本研究の目的は,新潟大学のライフサイエンス研究に関連するバイオリスクを把握し,それらを包括的に管理するトータルリスクマネジメント体制の構築を促進することである.【対象と方法】始めに新潟大学における病原体等使用の現状把握を行った.全学の教職員に対し,使用している病原体等の種類や管理方法,関連法令の理解度について,アンケート調査を行った.実情に即した病原体等の管理を運用するため,それぞれの病原体等の管理基準となる学内バイオセーフティーレベル(Bio Safety Level;BSL)を定めたほか,病原体等ごとの法的規制の情報をBSL一覧表に集約化した.また,実験室における設備要件や取扱い方法の基準を定め,実験室の評価とモニタリングのための包括的な実験室要件のチェックシートを作成した.さらに法令遵守および適正管理のため,オンライン管理システムの構築を行った.【結果】新潟大学において使用されている病原体等の種類は,細菌,ウイルス,真菌,毒素,プリオンであり,寄生虫の保有はなかった.研究者により病原体等を取り扱う管理基準が統一されていないほか,病原体等を使用する実験室要件の基準の不統一が明らかになった.また,病原体等の取扱いに関する法令への理解度が低いことが明らかになった.これらの研究リスクに対応するため,病原体等の取扱基準として学内BSLを整備したほか,病原体等を用いる実験室の設備要件・取扱い要件を示した実験室チェックシートを作成した.さらに,法令規制を明確にするために,病原体等ごとの法的規制を一覧化した.関連法令や学内規程の遵守を包括的に管理するために,オンラインの研究リスク管理システムを構築した.【結論】本取り組みにより,新潟大学における病原体等使用実験を行う際に対処すべきバイオリスクおよび法令リスクが明らかとなった.それらの研究リスクへ対応するための具体的方法が明確化され,行動規範や関連法令の遵守を適正管理するための円滑なトータルリスクマネジメント体制が整備された.本成果は,今後,高度化・複雑化していくと考えられる研究コンプライアンスへ対応するための組織づくりの基盤となる.(著者抄録)

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

Mitophagy is a type of autophagy that selectively degrades mitochondria. Mitochondria, known as the “powerhouse of the cell”, supply the majority of the energy required by cells. During energy production, mitochondria produce reactive oxygen species (ROS) as byproducts. The ROS damage mitochondria, and the damaged mitochondria further produce mitochondrial ROS. The increased mitochondrial ROS damage cellular components, including mitochondria themselves, and leads to diverse pathologies. Accordingly, it is crucial to eliminate excessive or damaged mitochondria to maintain mitochondrial homeostasis, in which mitophagy is believed to play a major role. Recently, the molecular mechanism and physiological role of mitophagy have been vigorously studied in yeast and mammalian cells. In yeast, Atg32 and Atg43, mitochondrial outer membrane proteins, were identified as mitophagy receptors in budding yeast and fission yeast, respectively. Here we summarize the molecular mechanisms of mitophagy in yeast, as revealed by the analysis of Atg32 and Atg43, and review recent progress in our understanding of mitophagy induction and regulation in yeast.

DOI： 10.3390/cells10123569

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

Male germline development involves choreographed changes to mitochondrial number, morphology and organization. Mitochondrial reorganization during spermatogenesis was recently shown to require mitochondrial fusion and fission. Mitophagy, the autophagic degradation of mitochondria, is another mechanism for controlling mitochondrial number and physiology, but its role during spermatogenesis is largely unknown. During post-meiotic spermatid development, restructuring of the mitochondrial network results in packing of mitochondria into a tight array in the sperm midpiece to fuel motility. Here, we show that disruption of mouse Fis1 in the male germline results in early spermatid arrest that is associated with increased mitochondrial content. Mutant spermatids coalesce into multinucleated giant cells that accumulate mitochondria of aberrant ultrastructure and numerous mitophagic and autophagic intermediates, suggesting a defect in mitophagy. We conclude that Fis1 regulates mitochondrial morphology and turnover to promote spermatid maturation.

DOI： 10.1242/dev.199686

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

Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.

DOI： 10.1038/s41594-021-00614-5

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

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease characterized by the formation of cytoplasmic ubiquitinated TDP-43 protein aggregates in motor neurons. Stress granules (SGs) are stress-induced cytoplasmic protein aggregates containing various neuropathogenic proteins, including TDP-43. Several studies have suggested that SGs are the initial site of the formation of pathogenic ubiquitinated TDP-43 aggregates in ALS neurons. Mutations in the optineurin (OPTN) and TIA1 genes are causative factors of familial ALS with TDP-43 aggregation pathology. We found that both OPTN depletion and ALS-associated OPTN mutations upregulated the TIA1 level in cells recovered from heat shock, and this upregulated TIA1 increased the amount of ubiquitinated TDP-43. Ubiquitinated TDP-43 induced by OPTN depletion was localized in SGs. Our study suggests that ALS-associated loss-of-function mutants of OPTN increase the amount of ubiquitinated TDP-43 in neurons by increasing the expression of TIA1, thereby promoting the aggregation of ubiquitinated TDP-43.

DOI： 10.1016/j.isci.2021.102733

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

Muscle disuse induces atrophy through increased reactive oxygen species (ROS) released from damaged mitochondria. Mitophagy, the autophagic degradation of mitochondria, is associated with increased ROS production. However, the mitophagy activity status during disuse-induced muscle atrophy has been a subject of debate. Here, we developed a new mitophagy reporter mouse line to examine how disuse affected mitophagy activity in skeletal muscles. Mice expressing tandem mCherry-EGFP proteins on mitochondria were then used to monitor the dynamics of mitophagy activity. The reporter mice demonstrated enhanced mitophagy activity and increased ROS production in atrophic soleus muscles following a 14-day hindlimb immobilization. Results also showed an increased expression of multiple mitophagy genes, including Bnip3, Bnip3l, and Park2. Our findings thus conclude that disuse enhances mitophagy activity and ROS production in atrophic skeletal muscles and suggests that mitophagy is a potential therapeutic target for disuse-induced muscle atrophy.

DOI： 10.1002/jcp.30404

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

Mitochondrial autophagy (mitophagy) selectively degrades mitochondria and plays an important role in mitochondrial homeostasis. In the yeast Saccharomyces cerevisiae, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for mitophagy, whereas this phosphorylation is counteracted by the protein phosphatase Ppg1. Although Ppg1 functions cooperatively with the Far complex (Far3, Far7, Far8, Vps64/Far9, Far10 and Far11), their relationship and the underlying phosphoregulatory mechanism of Atg32 remain unclear. Our recent study revealed: (i) the Far complex plays its localization-dependent roles, regulation of mitophagy and target of rapamycin complex 2 (TORC2) signaling, via the mitochondria- and endoplasmic reticulum (ER)-localized Far complexes, respectively; (ii) Ppg1 and Far11 form a subcomplex, and Ppg1 activity is required to assemble the sub- and core-Far complexes; (iii) association and dissociation between the Far complex and Atg32 are crucial determinants for mitophagy regulation. Here, we summarize our findings and discuss unsolved issues.

DOI： 10.1080/15548627.2021.1885184

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

Mitophagy is a selective type of autophagy in which damaged or unnecessary mitochondria are sequestered by double-membranous structures called phagophores and delivered to vacuoles/lysosomes for degradation. The molecular mechanisms underlying mitophagy have been studied extensively in budding yeast and mammalian cells. To gain more diverse insights, our recent study identified Atg43 as a mitophagy receptor in the fission yeast Schizosaccharomyces pombe. Atg43 is localized on the mitochondrial outer membrane through the Mim1-Mim2 complex and binds to Atg8, a ubiquitin-like protein conjugated to phagophore membranes. Artificial tethering of Atg8 to mitochondria can bypass the requirement of Atg43 for mitophagy, suggesting that the main role of Atg43 in mitophagy is to stabilize phagophore expansion on mitochondria by interacting with Atg8. Atg43 shares no sequence similarity with mitophagy receptors in other organisms and has a mitophagy-independent function, raising the possibility that Atg43 has acquired the mitophagic function by convergent evolution.

DOI： 10.1080/15548627.2021.1874662

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

Parkin promotes cell survival by removing damaged mitochondria via mitophagy. However, although some studies have suggested that Parkin induces cell death, the regulatory mechanism underlying the dual role of Parkin remains unknown. Herein, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) regulates Parkin-mediated cell death through the FKBP38-dependent dynamic translocation from the mitochondria to the ER during mitophagy. Mechanistically, MITOL mediates ubiquitination of Parkin at lysine 220 residue, which promotes its proteasomal degradation, and thereby fine-tunes mitophagy by controlling the quantity of Parkin. Deletion of MITOL leads to accumulation of the phosphorylated active form of Parkin in the ER, resulting in FKBP38 degradation and enhanced cell death. Thus, we have shown that MITOL blocks Parkin-induced cell death, at least partially, by protecting FKBP38 from Parkin. Our findings unveil the regulation of the dual function of Parkin and provide a novel perspective on the pathogenesis of PD.

DOI： 10.15252/embr.201949097

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

Mammalian target of rapamycin complex 1 (TORC1) is controlled by the GATOR complex composed of the GATOR1 subcomplex and its inhibitor, the GATOR2 subcomplex, sensitive to amino acid starvation. Previously, we identified fission yeast GATOR1 that prevents deregulated activation of TORC1 (Chia et al., 2017). Here, we report identification and characterization of GATOR2 in fission yeast. Unexpectedly, the GATOR2 subunit Sea3, an ortholog of mammalian WDR59, is physically and functionally proximal to GATOR1, rather than GATOR2, attenuating TORC1 activity. The fission yeast GATOR complex is dispensable for TORC1 regulation in response to amino acid starvation, which instead activates the Gcn2 pathway to inhibit TORC1 and induce autophagy. On the other hand, nitrogen starvation suppresses TORC1 through the combined actions of the GATOR1-Sea3 complex, the Gcn2 pathway, and the TSC complex, another conserved TORC1 inhibitor. Thus, multiple, parallel signaling pathways implement negative regulation of TORC1 to ensure proper cellular starvation responses.

DOI： 10.7554/eLife.60969

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

Mitophagy plays an important role in mitochondrial homeostasis. In yeast, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for mitophagy. This phosphorylation is counteracted by the yeast equivalent of the STRIPAK complex consisting of the PP2A-like protein phosphatase Ppg1 and Far3-7-8-9-10-11 (Far complex), but the underlying mechanism remains elusive. Here we show that two subpopulations of the Far complex reside in the mitochondria and endoplasmic reticulum, respectively, and play distinct roles; the former inhibits mitophagy via Atg32 dephosphorylation, and the latter regulates TORC2 signaling. Ppg1 and Far11 form a subcomplex, and Ppg1 activity is required for the assembling integrity of Ppg1-Far11-Far8. The Far complex preferentially interacts with phosphorylated Atg32, and this interaction is weakened by mitophagy induction. Furthermore, the artificial tethering of Far8 to Atg32 prevents mitophagy. Taken together, the Ppg1-mediated Far complex formation and its dissociation from Atg32 are crucial for mitophagy regulation.

DOI： 10.7554/eLife.63694

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

Degradation of mitochondria through mitophagy contributes to the maintenance of mitochondrial function. In this study, we identified that Atg43, a mitochondrial outer membrane protein, serves as a mitophagy receptor in the model organism Schizosaccharomyces pombe to promote the selective degradation of mitochondria. Atg43 contains an Atg8-family-interacting motif essential for mitophagy. Forced recruitment of Atg8 to mitochondria restores mitophagy in Atg43-deficient cells, suggesting that Atg43 tethers expanding isolation membranes to mitochondria. We found that the mitochondrial import factors, including the Mim1-Mim2 complex and Tom70, are crucial for mitophagy. Artificial mitochondrial loading of Atg43 bypasses the requirement of the import factors, suggesting that they contribute to mitophagy through Atg43. Atg43 not only maintains growth ability during starvation but also facilitates vegetative growth through its mitophagy-independent function. Thus, Atg43 is a useful model to study the mechanism and physiological roles, as well as the origin and evolution, of mitophagy in eukaryotes.

DOI： 10.7554/eLife.61245

Language：English  

Mitochondrial quality control maintains mitochondrial function by regulating mitochondrial dynamics and mitophagy. Despite the identification of mitochondrial quality control factors, little is known about the crucial regulators coordinating both mitochondrial fission and mitophagy. Through a cell-based functional screening assay, FK506 binding protein 8 (FKBP8) was identified to target microtubule-associated protein 1 light chain 3 (LC3) to the mitochondria and to change mitochondrial morphology. Microscopy analysis revealed that the formation of tubular and enlarged mitochondria was observed in FKBP8 knockdown HeLa cells and the cortex of Fkbp8 heterozygote-knockout mouse embryos. Under iron depletion-induced stress, FKBP8 was recruited to the site of mitochondrial division through budding and colocalized with LC3. FKBP8 was also found to be required for mitochondrial fragmentation and mitophagy under hypoxic stress. Conversely, FKBP8 overexpression induced mitochondrial fragmentation in HeLa cells, human fibroblasts and mouse embryo fibroblasts (MEFs), and this fragmentation occurred in Drp1 knockout MEF cells, FIP200 knockout HeLa cells and BNIP3/NIX double knockout HeLa cells, but not in Opa1 knockout MEFs. Interestingly, we found an LIR motif-like sequence (LIRL), as well as an LIR motif, at the N-terminus of FKBP8 and LIRL was essential for both inducing mitochondrial fragmentation and binding of FKBP8 to OPA1. Together, we suggest that FKBP8 plays an essential role in mitochondrial fragmentation through LIRL during mitophagy and this activity of FKBP8 together with LIR is required for mitophagy under stress conditions.

DOI： 10.1096/fj.201901735R

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

Mitophagy plays an important role in the maintenance of mitochondrial homeostasis. PTEN-induced kinase (PINK1), a key regulator of mitophagy, is degraded constitutively under steady-state conditions. During mitophagy, it becomes stabilized in the outer mitochondrial membrane, particularly under mitochondrial stress conditions, such as in treatment with uncouplers, generation of excessive mitochondrial reactive oxygen species, and formation of protein aggregates in mitochondria. Stabilized PINK1 recruits and activates E3 ligases, such as Parkin and mitochondrial ubiquitin ligase (MUL1), to ubiquitinate mitochondrial proteins and induce ubiquitin-mediated mitophagy. Here, we found that the anticancer drug gemcitabine induces the stabilization of PINK1 and subsequent mitophagy, even in the absence of Parkin. We also found that gemcitabine-induced stabilization of PINK1 was not accompanied by mitochondrial depolarization. Interestingly, the stabilization of PINK1 was mediated by MUL1. These results suggest that gemcitabine induces mitophagy through MUL1-mediated stabilization of PINK1 on the mitochondrial membrane independently of mitochondrial depolarization.

DOI： 10.1038/s41598-020-58315-w

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

Glaucoma-Associated Mutations in the Optineurin Gene Have Limited Impact on Parkin-Dependent Mitophagy.

DOI： 10.1167/iovs.19-27184

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

Mitophagy plays an important role in mitochondrial quality control. In yeast, phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 (CK2) upon induction of mitophagy is a prerequisite for interaction of Atg32 with Atg11 (an adaptor protein for selective autophagy) and following delivery of mitochondria to the vacuole for degradation. Because CK2 is constitutively active, Atg32 phosphorylation must be precisely regulated to prevent unrequired mitophagy. We found that the PP2A (protein phosphatase 2A)-like protein phosphatase Ppg1 was essential for dephosphorylation of Atg32 and inhibited mitophagy. We identified the Far complex proteins, Far3, Far7, Far8, Far9, Far10, and Far11, as Ppg1-binding proteins. Deletion of Ppg1 or Far proteins accelerated mitophagy. Deletion of a cytoplasmic region (amino acid residues 151–200) of Atg32 caused the same phenotypes as in ppg1Δ cells, which suggested that dephosphorylation of Atg32 by Ppg1 required this region. Therefore, Ppg1 and the Far complex cooperatively dephosphorylate Atg32 to prevent excessive mitophagy. Mitophagy in yeast is initiated by CK2-mediated phosphorylation of the mitophagy receptor Atg32. However, how this phosphorylation is prevented under non-mitophagy-inducing conditions is unclear. Furukawa et al. show that the PP2A-like protein phosphatase Ppg1 and the Far complex negatively regulate mitophagy by counteracting CK2-mediated phosphorylation of Atg32.

DOI： 10.1016/j.celrep.2018.05.064

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

Cdc14 protein phosphatase is critical for late mitosis progression in budding yeast, although its orthologs in other organisms, including mammalian cells, function as stress-responsive phosphatases. We found herein unexpected roles of Cdc14 in autophagy induction after nutrient starvation and target of rapamycin complex 1 (TORC1) kinase inactivation. TORC1 kinase phosphorylates Atg13 to repress autophagy under nutrient-rich conditions, but if TORC1 becomes inactive upon nutrient starvation or rapamycin treatment, Atg13 is rapidly dephosphorylated and autophagy is induced. Cdc14 phosphatase was required for optimal Atg13 dephosphorylation, pre-autophagosomal structure formation, and autophagy induction after TORC1 inactivation. In addition, Cdc14 was required for sufficient induction of ATG8 and ATG13 expression. Moreover, Cdc14 activation provoked autophagy even under normal conditions. This study identified a novel role of Cdc14 as the stress-responsive phosphatase for autophagy induction in budding yeast.

DOI： 10.1016/j.jmb.2018.04.007

Language：Others  

Mechanisms and Physiological Roles of Mitophagy in Yeast.

DOI： 10.14348/molcells.2018.2214

Language：Japanese  

Language：Japanese  

Language：Others  

Mitophagy in Yeast: A Screen of Mitophagy-Deficient Mutants.

DOI： 10.1007/7651_2017_13

Language：Others  

Detection of Hypoxia-Induced and Iron Depletion-Induced Mitophagy in Mammalian Cells.

DOI： 10.1007/7651_2017_19

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

Mitophagy is thought to play an important role in mitochondrial quality control. Mitochondrial division is believed to occur first, and autophagosome formation subsequently occurs to enwrap mitochondria as a process of mitophagy. However, there has not been any temporal analysis of mitochondrial division and autophagosome formation in mitophagy. Therefore, the relationships among these processes remain unclear. We show that the mitochondrial division factor Dnm1 in yeast or Drp1 in mammalian cells is dispensable for mitophagy. Autophagosome formation factors, such as FIP200, ATG14, and WIPIs, were essential for the mitochondrial division for mitophagy. Live-cell imaging showed that isolation membranes formed on the mitochondria. A small portion of the mitochondria then divided from parental mitochondria simultaneously with the extension of isolation membranes and autophagosome formation. These findings suggest the presence of a mitophagy process in which mitochondrial division for mitophagy is accomplished together with autophagosome formation.

DOI： 10.1083/jcb.201605093

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

Phosphatase and tensin homolog-induced putative kinase 1 (PINK1), a Ser/Thr kinase, and PARKIN, a ubiquitin ligase, are causal genes for autosomal recessive early-onset parkinsonism. Multiple lines of evidence indicate that PINK1 and PARKIN cooperatively control the quality of the mitochondrial population via selective degradation of damaged mitochondria by autophagy. Here, we report that PINK1 and PARKIN induce cell death with a 12-h delay after mitochondrial depolarization, which differs from the time profile of selective autophagy of mitochondria. This type of cell death exhibited definite morphologic features such as plasma membrane rupture, was insensitive to a pan-caspase inhibitor, and did not involve mitochondrial permeability transition. Expression of a constitutively active form of PINK1 caused cell death in the presence of a pan-caspase inhibitor, irrespective of the mitochondrial membrane potential. PINK1-mediated cell death depended on the activities of PARKIN and proteasomes, but it was not affected by disruption of the genes required for autophagy. Furthermore, fluorescence and electron microscopic analyses revealed that mitochondria were still retained in the dead cells, indicating that PINK1-mediated cell death is not caused by mitochondrial loss. Our findings suggest that PINK1 and PARKIN play critical roles in selective cell death in which damaged mitochondria are retained, independent of mitochondrial autophagy.

DOI： 10.1074/jbc.M116.714923

Language：Others  

Mitophagy in yeast: Molecular mechanisms and physiological role.

DOI： 10.1016/j.bbamcr.2015.01.005

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

In cultured cells, not many mitochondria are degraded by mitophagy induced by physiological cellular stress. We observed mitophagy in HeLa cells using a method that relies on the pH-sensitive fluorescent protein Keima. With this approach, we found that mitophagy was barely induced by carbonyl cyanide m-chlorophenyl hydrazone treatment, which is widely used as an inducer of PARK2/Parkin-related mitophagy, whereas a small but modest amount of mitochondria were degraded by mitophagy under conditions of starvation or hypoxia. Mitophagy induced by starvation or hypoxia was marginally suppressed by knockdown of ATG7 and ATG12, or MAP1LC3B, which are essential for conventional macroautophagy. In addition, mitophagy was efficiently induced in Atg5 knockout mouse embryonic fibroblasts. However, knockdown of RAB9A and RAB9B, which are essential for alternative autophagy, but not conventional macroautophagy, severely suppressed mitophagy. Finally, we found that the MAPKs MAPK1/ERK2 and MAPK14/p38 were required for mitophagy. Based on these findings, we conclude that mitophagy in mammalian cells predominantly occurs through an alternative autophagy pathway, requiring the MAPK1 and MAPK14 signaling pathways.

DOI： 10.1080/15548627.2015.1023047

Language：English  

Mitochondria are organelles that supply a large amount of energy required for cellular activities. Accumulation of dysfunctional mitochondria within cells inhibits cellular functions and causes several diseases. Thus, the cell has devised specific mechanisms to ensure proper quality and quantity control of this organelle. Mitochondrial autophagy (mitophagy) is thought to be one of the primary mechanisms for mitochondrial quality control that selectively eliminate dysfunctional or excess mitochondria via an autophagic process. ATG32 is a mitophagy-specific gene identified by a yeast genome-wide screen for mitophagy. Atg32, a protein encoded by ATG32, is a transmembrane protein localized in the mitochondrial outer membrane. During mitophagy induction, Atg32 functions as a mitochondrial receptor protein that interacts with cytosolic adaptor protein Atg11, which recruits mitochondria to the autophagic machinery for degradation. In this chapter, we describe the molecular mechanism and physiology of mitophagy in yeast, with an emphasis on the role of Atg32.

DOI： 10.1016/B978-0-12-405528-5.00010-9

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

When mitophagy is induced in Saccharomyces cerevisiae, the mitochondrial outer membrane protein ScAtg32 interacts with the cytosolic adaptor protein ScAtg11. ScAtg11 then delivers the mitochondria to the pre-autophagosomal structure for autophagic degradation. Despite the importance of ScAtg32 for mitophagy, the expression and functional regulation of ScAtg32 are poorly understood. In this study, we identified and characterized the ScAtg32 homolog in Pichia pastoris (PpAtg32). Interestingly, we found that PpAtg32 was barely expressed before induction of mitophagy and was rapidly expressed after induction of mitophagy by starvation. Additionally, PpAtg32 was phosphorylated when mitophagy was induced. We found that PpAtg32 expression was suppressed by Tor and the downstream PpSin3-PpRpd3 complex. Inhibition of Tor by rapamycin induced PpAtg32 expression, but could neither phosphorylate PpAtg32 nor induce mitophagy. Based on these findings, we conclude that the Tor and PpSin3-PpRpd3 pathway regulates PpAtg32 expression, but not PpAtg32 phosphorylation.

DOI： 10.1242/jcs.153254

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

Mitophagy is a process that selectively degrades mitochondria. When mitophagy is induced in yeast, the mitochondrial outer membrane protein Atg32 is phosphorylated, interacts with the adaptor protein Atg11 and is recruited into the vacuole with mitochondria. We screened kinase-deleted yeast strains and found that CK2 is essential for Atg32 phosphorylation, Atg32-Atg11 interaction and mitophagy. Inhibition of CK2 specifically blocks mitophagy, but not macroautophagy, pexophagy or the Cvt pathway. In vitro, CK2 phosphorylates Atg32 at serine 114 and serine 119. We conclude that CK2 regulates mitophagy by directly phosphorylating Atg32.

DOI： 10.1038/embor.2013.114

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

DubinJohnson syndrome (DJS) is a recessive inherited disorder characterized by conjugated hyperbilirubinemia. It is caused by dysfunction of adenosine triphosphate-binding cassette, sub-family C, member 2 (ABCC2/MRP2) on the canalicular membrane of hepatocytes. We performed mutational analysis of the ABCC2/MRP2 gene in a Japanese female with DJS. Furthermore, we investigated the effects of the two identified DJS-associated mutations on MRP2 function. We found a compound heterozygous mutation in the patient: W709R (c.2124T>C), a missense mutation in exon 17, and R1310X (c.3928C>T), a nonsense mutation in exon 28. DJS-associated mutations have been shown to impair the protein maturation and transport activity of ABCC2/MRP2. We established HEK293 cell lines stably expressing one of the two identified DJS-associated mutations. Expressed W709R MRP2 was mainly core-glycosylated, predominantly retained in the endoplasmic reticulum, and exhibited no transport activity, suggesting that this mutation causes deficient maturation and impaired protein sorting. No MRP2 protein was expressed from HEK293 cells transfected with an R1310X-containing construct. This compound heterozygous mutation of the MRP2 gene causes dysfunction of the MRP2 protein and the hyperbilirubinemia seen in DJS.

DOI： 10.1111/j.1872-034X.2012.01103.x

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

Mitochondrial transcription factor A (TFAM) plays a role in the maintenance of mitochondrial DNA (mtDNA) by packaging mtDNA, forming the mitochondrial nucleoid. There have been many reports about a function of TFAM at the cellular level, but only a few studies have been done in individual organisms. Here we examined the effects of TFAM on the Drosophila lifespan and oxidative stress response, by overexpressing TFAM using the GAL4/UAS system. Under standard conditions, the lifespan of TFAM-overexpressing flies was shorter than that of the control flies. However, the lifespan of TFAM-overexpressing flies was longer when they were treated with 1% H2O2. These results suggest that even though excess TFAM has a negative influence on lifespan, it has a defensive function under strong oxidative stress. In the TFAM-overexpressing flies, no significant changes in mtDNA copy number or mtDNA transcription were observed. However, the results of a total antioxidant activity assay suggest the possibility that TFAM is involved in the elimination of oxidative stress. The present results clearly show the effects of TFAM overexpression on the lifespan of Drosophila under both standard conditions and oxidative stress conditions, and our findings contribute to the understanding of the physiological mechanisms involving TFAM in mitochondria. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2012.11.084

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

Miller syndrome is a recessive inherited disorder characterized by postaxial acrofacial dysostosis. It is caused by dysfunction of the DHODH (dihydroorotate dehydrogenase) gene, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway and is localized at mitochondria intermembrane space. We investigated the consequence of three missense mutations, G202A, R346W and R135C of DHODH, which were previously identified in patients with Miller syndrome. First, we established He La cell lines stably expressing DHODH with Miller syndrome-causative mutations: G202A, R346W and R135C. These three mutant proteins retained the proper mitochondrial localization based on immunohistochemistry and mitochondrial subfractionation studies. The G202A, R346W DHODH proteins showed reduced protein stability. On the other hand, the third one R135C, in which the mutation lies at the ubiquinone-binding site, was stable but possessed no enzymatic activity. In conclusion, the G202A and R346W mutation causes deficient protein stability, and the R135C mutation does not affect stability but impairs the substrate-induced enzymatic activity, suggesting that impairment of DHODH activity is linked to the Miller syndrome phenotype.

DOI： 10.1042/BSR20120046

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

p32 is an evolutionarily conserved and ubiquitously expressed multifunctional protein. Although p32 exists at diverse intra and extracellular sites, it is predominantly localized to the mitochondrial matrix near the nucleoid associated with mitochondrial transcription factor A. Nonetheless, its function in the matrix is poorly understood. Here, we determined p32 function via generation of p32-knockout mice. p32-deficient mice exhibited mid-gestation lethality associated with a severe developmental defect of the embryo. Primary embryonic fibroblasts isolated from p32-knockout embryos showed severe dysfunction of the mitochondrial respiratory chain, because of severely impaired mitochondrial protein synthesis. Recombinant p32 binds RNA, not DNA, and endogenous p32 interacts with all mitochondrial messenger RNA species in vivo. The RNA-binding ability of p32 is well correlated with the mitochondrial translation. Co-immunoprecipitation revealed the close association of p32 with the mitoribosome. We propose that p32 is required for functional mitoribosome formation to synthesize proteins within mitochondria.

DOI： 10.1093/nar/gks774

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

The mitochondrial oxidative phosphorylation (OXPHOS) proteins are encoded by both nuclear and mitochondrial DNA. The nuclear-encoded OXPHOS mRNAs have specific subcellular localizations, but little is known about which localize near mitochondria. Here, we compared mRNAs in mitochondria-bound polysome fractions with those in cytosolic, free polysome fractions. mRNAs encoding hydrophobic OXPHOS proteins, which insert into the inner membrane, were localized near mitochondria. Conversely, OXPHOS gene which mRNAs were predominantly localized in cytosol had less than one transmembrane domain. The RNA-binding protein Y-box binding protein-1 is localized at the mitochondrial outer membrane and bound to the OXPHOS mRNAs. Our findings offer new insight into mitochondrial co-translational import in human cells. (C) 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

DOI： 10.1016/j.mito.2012.02.004

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

Mitochondria play key roles in essential cellular functions, such as energy production, metabolic pathways and aging. Growth factor-mediated expression of the mitochondria] OXPHOS (oxidative phosphorylation) complex proteins has been proposed to play a fundamental role in metabolic homeostasis. Although protein translation is affected by general RNA-binding proteins, very little is known about the mechanism involved in mitochondrial OXPHOS protein translation. In the present study, serum stimulation induced nuclear-encoded OXPHOS protein expression, such as NDUFA9 [NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39 kDa], NDUFB8 [NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8, 19 kDa], SDHB [succinate dehydrogenase complex, subunit B, iron sulfur (Ip)] and UQCRFS1 (ubiquinol-cytochrome c reductase, Rieske iron sulfur polypeptide 1), and mitochondrial ATP production, in a translation-dependent manner. We also observed that the major ribonucleoprotein YB-1 (Y-box-binding protein-1) preferentially bound to these OXPHOS mRNAs and regulated the recruitment of mRNAs from inactive mRNPs (messenger ribonucleoprotein particles) to active polysomes. YB-1 depletion led to up-regulation of mitochondrial function through induction of OXPHOS protein translation from inactive mRNP release. In contrast, YB-1 overexpression suppressed the translation of these OXPHOS mRNAs through reduced polysome formation, suggesting that YB-1 regulated the translation of mitochondrial OXPHOS mRNAs through mRNA binding. Taken together, our findings suggest that YB-1 is a critical factor for translation that may control OXPHOS activity.

DOI： 10.1042/BJ20111728

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

In mammalian cells, the autophagy-dependent degradation of mitochondria (mitophagy) is thought to maintain mitochondrial quality by eliminating damaged mitochondria. However, the physiological importance of mitophagy has not been clarified in yeast. Here, we investigated the physiological role of mitophagy in yeast using mitophagy-deficient atg32- or atg11-knock-out cells. When wild-type yeast cells in respiratory growth encounter nitrogen starvation, mitophagy is initiated, excess mitochondria are degraded, and reactive oxygen species (ROS) production from mitochondria is suppressed; as a result, the mitochondria escape oxidative damage. On the other hand, in nitrogen-starved mitophagy-deficient yeast, excess mitochondria are not degraded and the undegraded mitochondria spontaneously age and produce surplus ROS. The surplus ROS damage the mitochondria themselves and the damaged mitochondria produce more ROS in a vicious circle, ultimately leading to mitochondrial DNA deletion and the so-called "petitemutant" phenotype. Cells strictly regulate mitochondrial quantity and quality because mitochondria produce both necessary energy and harmful ROS. Mitophagy contributes to this process by eliminating the mitochondria to a basal level to fulfill cellular energy requirements and preventing excess ROS production.

DOI： 10.1074/jbc.M111.280156

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

Mitophagy, which selectively degrades mitochondria via autophagy, has a significant role in mitochondrial quality control. When mitophagy is induced in yeast, mitochondrial residential protein Atg32 binds Atg11, an adaptor protein for selective types of autophagy, and it is recruited into the vacuole along with mitochondria. The Atg11-Atg32 interaction is believed to be the initial molecular step in which the autophagic machinery recognizes mitochondria as a cargo, although how this interaction is mediated is poorly understood. Therefore, we studied the Atg11-Atg32 interaction in detail. We found that the C-terminus region of Atg11, which included the fourth coiled-coil domain, interacted with the N-terminus region of Atg32 (residues 100-120). When mitophagy was induced, Ser-114 and Ser-119 on Atg32 were phosphorylated, and then the phosphorylation of Atg32, especially phosphorylation of Ser-114 on Atg32, mediated the Atg11-Atg32 interaction and mitophagy. These findings suggest that cells can regulate the amount of mitochondria, or select specific mitochondria (damaged or aged) that are degraded by mitophagy, by controlling the activity and/or localization of the kinase that phosphorylates Atg32. We also found that Hog1 and Pbs2, which are involved in the osmoregulatory signal transduction cascade, are related to Atg32 phosphorylation and mitophagy.

DOI： 10.1091/mbc.E11-02-0145

Language：English  

The mitochondrion is an organelle that carries out a number of important metabolic processes such as fatty acid oxidation, the citric acid cycle, and oxidative phosphorylation. However, this multitasking organelle also generates reactive oxygen species (ROS), which can cause oxidative stress resulting in self-damage. This type of mitochondrial damage can lead to the further production of ROS and a resulting downward spiral with regard to mitochondrial capability. This is extremely problematic because the accumulation of dysfunctional mitochondria is related to aging, cancer, and neurodegenerative diseases. Accordingly, appropriate quality control of this organelle is important to maintain proper cellular homeostasis. It has been thought that selective mitochondria autophagy (mitophagy) contributes to the maintenance of mitochondrial quality by eliminating damaged or excess mitochondria, although little is known about the mechanism. Recent studies in yeast identified several mitophagy-related proteins, which have been characterized with regard to their function and regulation. In this article, we review recent advances in the physiology and molecular mechanism of mitophagy and discuss the similarities and differences of this degradation process between yeast and mammalian cells. Antioxid. Redox Signal. 14, 1989-2001.

DOI： 10.1089/ars.2010.3762

Language：English  

Mitochondria are important organelles that supply energy to the cell. However, these organelles are also the major source of cellular reactive oxygen species (ROS). Thus, the elimination of damaged or excess mitochondria is essential for maintaining cellular homeostasis. Selective autophagy of mitochondria (mitophagy) plays an important role in the quality control of mitochondria. However, little is known about the molecular mechanism of mitophagy in mammalian cells. Nix is a mitochondrial outer membrane protein that is required for mitochondrial clearance during erythrocyte maturation. Recently, it was reported that Nix is a mitochondrial receptor that can directly connect to one of the autophagic machinery components, the Atg8 homologs LC3 and GABARAP.

DOI： 10.4161/auto.6.3.11420

Language：English  

Mitochondria autophagy(mitophagy) is the process of selective degradation of mitochondria that has an important role in mitochondrial quality control. To gain insight into the molecular mechanism of mitophagy, we screened a yeast knockout library for strains that are defective in mitophagy. We found 32 strains that showed a complete or partial block of mitophagy. One of the genes identified, YLR356W, is required for mitophagy, but not for macroautophagy or other types of selective autophagy. The deletion of YLR356W partially inhibits mitophagy during starvation, whereas there is almost complete inhibition at post-log phase. Accordingly, we hypothesize that Ylr356w is required to detect or present aged or dysfunctional mitochondria when cells reach the post-log phase.

DOI： 10.1091/mbc.E09-03-0225

Language：English  

P>Mitochondria are critical for supplying energy to the cell, but during catabolism this organelle also produces reactive oxygen species that can cause oxidative damage. Accordingly, quality control of mitochondria is important to maintain cellular homeostasis. It has been assumed that autophagy is the pathway for mitochondrial recycling, and that the selective degradation of mitochondria via autophagy (mitophagy) is the primary mechanism for mitochondrial quality control, although there is little experimental evidence to support this idea. Recent studies in yeast identified several mitophagy-related genes and have uncovered components involved in the molecular mechanism and regulation of mitophagy. Similarly, studies of Parkinson disease and reticulocyte maturation reveal that Parkin and Nix, respectively, are required for mitophagy in mammalian cells, and these analyses have revealed important physiological roles for mitophagy. Here, we review the current knowledge on mitophagy, in particular on the molecular mechanism and regulation of mitophagy in yeast. We also discuss some of the differences between yeast and mammalian mitophagy.

DOI： 10.1111/j.1365-2958.2009.07035.x

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

Mitophagy is the process of selective mitochondrial degradation via autophagy, which has an important role in mitochondrial quality control. Very little is known, however, about the molecular mechanism of mitophagy. A genome-wide yeast mutant screen for mitophagy-defective strains identified 32 mutants with a block in mitophagy, in addition to the known autophagy-related (ATG) gene mutants. We further characterized one of these mutants, ylr356w Delta that corresponds to a gene whose function has not been identified. YLR356W is a mitophagy-specific gene that was not required for other types of selective autophagy or macroautophagy. The deletion of YLR356W partially inhibited mitophagy during starvation, whereas there was an almost complete inhibition at post-log phase. Accordingly, we have named this gene ATG33. The new mutants identified in this analysis will provide a useful foundation for researchers interested in the study of mitochondrial homeostasis and quality control.

DOI： 10.1091/mbc.E09-03-0225

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

Macroautophagy (hereafter autophagy) is a ubiquitous degradative process in eukaryotic cells.' Mitochondria autophagy (mitophagy) is a type of specific autophagy that degrades mitochondria selectively.(2) Mitophagy is thought to play an important role for maintaining the quality of these organelles by eliminating damaged mitochondria, and it is involved in cellular differentiation, whereas dysfunctional mitophagy is related with neurodegenerative diseases;(3-5) however, the mechanism of mitophagy is poorly understood. To facilitate the analysis of mitophagy, we recently established a simple method to monitor mitophagy in yeast, the Om45-GFP processing assay.(6) Om45-GFP is a mitochondrial outer membrane protein. Following the uptake of mitochondria into the vacuole, Om45-GFP is degraded, releasing the intact form of GFP, which is detected by immunoblotting. Therefore, the amount of free GFP reflects the level of mitophagy.

DOI： 10.4161/auto.5.8.9854

Language：English  

The elimination of aged, damaged, or excess mitochondria is an important subcellular event to maintain proper cellular homeostasis. Recent studies reveal that autophagy-dependent degradation of mitochondria (mitophagy) plays an important role in removing these organelles. Very little is known, however, about the molecular mechanism of mitophagy. We found a novel protein, Atg32, that is required for mitophagy but not for other types of selective autophagy or nonselective autophagy. Atg32 is a mitochondrial outer membrane protein. When mitophagy is induced, Atg32 binds to Atg11, an adaptor protein for selective autophagy. Eventually, this interaction results in the recruitment of mitochondria to the vacuole for degradation.

DOI： 10.4161/auto.5.8.9747

Language：Others  

[Molecular mechanism of mitochondria autophagy].

Language：English  

DOI： 10.1038/cr.2009.94

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

Mitochondrial quality control is important in maintaining proper cellular homeostasis. Although selective mitochondrial degradation by autophagy (mitophagy) is suggested to have an important role in quality control, and though there is evidence for a direct relation between mitophagy and neurodegenerative diseases, the molecular mechanism of mitophagy is poorly understood. Using a screen for mitophagy-deficient mutants, we found that YIL146C/ECM37 is essential for mitophagy. This gene is not required for other types of selective autophagy or for nonspecific macroautophagy. We designated this autophagy-related (ATG) gene as ATG32. The Atg32 protein localizes on mitochondria. Following the induction of mitophagy, Atg32 binds Atg11, an adaptor protein for selective types of autophagy, and is then recruited to and imported into the vacuole along with mitochondria. Therefore, Atg32 confers selectivity for mitochondrial sequestration as a cargo and is necessary for recruitment of this organelle by the autophagy machinery for mitophagy.

DOI： 10.1016/j.devcel.2009.06.014

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

The regulation of mitochondrial degradation through autophagy is expected to be a tightly controlled process, considering the significant role of this organelle in many processes ranging from energy production to cell death. However, very little is known about the specific nature of the degradation process. We developed a new method to detect mitochondrial autophagy (mitophagy) by fusing the green fluorescent protein at the C terminus of two endogenous mitochondrial proteins and monitored vacuolar release of green fluorescent protein. Using this method, we screened several atg mutants and found that ATG11, a gene that is essential only for selective autophagy, is also essential for mitophagy. In addition, we found that mitophagy is blocked even under severe starvation conditions, if the carbon source makes mitochondria essential for metabolism. These findings suggest that the degradation of mitochondria is a tightly regulated process and that these organelles are largely protected from nonspecific autophagic degradation.

DOI： 10.1074/jbc.M802403200

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

Mitochondrial DNA (mtDNA) is highly susceptible to injury induced by reactive oxygen species (ROS). During aging, mutations of mtDNA accumulate to induce dysfunction of the respiratory chain, resulting in the enhanced ROS production. Therefore, age-dependent memory impairment may result from oxidative stress derived from the respiratory chain. Mitochondrial transcription factor A (TFAM) is now known to have roles not only in the replication of mtDNA but also its maintenance. We herein report that an overexpression of TFAM in HeLa cells significantly inhibited rotenone-induced mitochondrial ROS generation and the subsequent NF-kappa B (nuclear factor-kappa B) nuclear translocation. Furthermore, TFAM transgenic (TG) mice exhibited a prominent amelioration of an age-dependent accumulation of lipid peroxidation products and a decline in the activities of complexes I and IV in the brain. In the aged TG mice, deficits of the motor learning memory, the working memory, and the hippocampal long-term potentiation (LTP) were also significantly improved. The expression level of interleukin-1 beta(IL-1 beta) and mtDNA damages, which were predominantly found in microglia, significantly decreased in the aged TG mice. The IL-1 beta amount markedly increased in the brain of the TG mice after treatment with lipopolysaccharide (LPS), whereas its mean amount was significantly lower than that of the LPS-treated aged wild-type mice. At the same time, an increased mtDNA damage in microglia and an impaired hippocampal LTP were also observed in the LPS-treated aged TG mice. Together, an overexpression of TFAM is therefore considered to ameliorate age-dependent impairment of the brain functions through the prevention of oxidative stress and mitochondrial dysfunctions in microglia.

DOI： 10.1523/JNEUROSCI.1957-08.2008

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

Mitochondrial transcription factor A (TFAM) contains a basic C-terminal tail which is essential for the promoter-specific transcription. TFAM is also a major component of a protein-mitochondrial DNA (mtDNA) complex, called nucleoid, as a non-specific DNA-binding protein. However, little is known about a role of the C-tail in the nucleoid. Overexpression of full-length TFAM decreased the amount of a D-loop form of mtDNA in cells, while overexpression of TFAM lacking its C-tail (TFAM-AC) did not, suggesting that the C-tail is involved in destabilization or formation of the D-loop. An mRNA for mtDNA-derived ND1 was hardly decreased in the former but rather decreased in the latter. Given that the D-loop formation is coupled with the transcription, the decrease in the D-loop is likely due to its destabilization. The recombinant full-length TFAM much strongly unwound DNA than TFAM-AC, which is consistent with the above idea because D-loop is resolved by unwinding of the supercoiling state. Notably, truncation of the C-tail decreased DNA-binding activity of TFAM by three orders of magnitude. Thus, the C-terminal tail of TFAM is important for the strong general binding to mtDNA. This strong DNA-binding conferred by the C-tail may play an important role in the nucleoid structure.

DOI： 10.1093/jb/mvm020

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

Coenzyme Q(10) (CoQ(10)) is a vital lipophilic molecule that transfers electrons from mitochondrial respiratory chain complexes I and II to complex III. Deficiency of CoQ(10) has been associated with diverse clinical phenotypes, but, in most patients, the molecular cause is unknown. The first defect in a CoQ(10) biosynthetic gene, COQ2, was identified in a child with encephalomyopathy and nephrotic syndrome and in a younger sibling with only nephropathy. Here, we describe an infant with severe Leigh syndrome, nephrotic syndrome, and CoQ(10) deficiency in muscle and fibroblasts and compound heterozygous mutations in the PDSS2 gene, which encodes a subunit of decaprenyl diphosphate synthase, the first enzyme of the CoQ(10) biosynthetic pathway. Biochemical assays with radiolabeled substrates indicated a severe defect in decaprenyl diphosphate synthase in the patient's fibroblasts. This is the first description of pathogenic mutations in PDSS2 and confirms the molecular and clinical heterogeneity of primary CoQ(10) deficiency.

DOI： 10.1086/510023

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

Human mitochondrial DNA takes on a large protein-DNA complex called a nucleoid or mitochromosome. Mitochondrial transcription factor A (TFAM) is a major component of the complex. During an attempt to search for proteins associated with the TFAM-containing complex by a proteomic method, we found one protein that has not been considered to be mitochondrial: PDIP38. PDIP38 was initially identified as a binding protein to nuclear DNA polymerase delta. PDIP38 is almost exclusively recovered from the mitochondrial fraction of human HeLa cells. PDIP38 is completely cleaved when TritonX-100-solubilized mitochondria are treated with proteinase K, but not when mitoplasts devoid of outer membranes are treated, indicating that PDIP38 is located in the mitochondrial matrix. TFAM and mitochondrial single-stranded DNA binding protein (mtSSB) are co-immunoprecipitated with PDIP38 by anti-PDIP38 antibodies. On the other hand, only the latter is crosslinked to PDIP38 when mitochondria are treated with a crosslinker, formaldehyde. In addition to mtSSB, 60 kDa heat shock protein and a Lon protease homolog, both of which have single-stranded DNA binding activity, are also crosslinked. PDIP38 associates with the nucleoid components and could be involved in the metabolism of mitochondrial DNA.

DOI： 10.1093/jb/mvi169

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

Mitochondrial transcription factor A (TFAM), a transcription factor for mitochondrial DNA (mtDNA) that also possesses the property of nonspecific DNA binding, is essential for maintenance of mtDNA. To clarify the role of TFAM, we repressed the expression of endogenous TFAM in HeLa cells by RNA interference. The amount of TFAM decreased maximally to about 15% of the normal level at day 3 after RNA interference and then recovered gradually. The amount of mtDNA changed closely in parallel with the daily change in TFAM while in organello transcription of mtDNA at day 3 was maintained at about 50% of the normal level. TFAM lacking its C-terminal 25 amino acids (TFAM-DeltaC) marginally activated transcription in vitro. When TFAM-DeltaC was expressed at levels comparable to those of endogenous TFAM in HeLa cells, mtDNA increased twofold, suggesting that TFAM-DeltaC is as competent in maintaining mtDNA as endogenous TFAM under these conditions. The in organello transcription of TFAM-DeltaC-expressing cells was no more than that in the control. Thus, the mtDNA amount is finely correlated with the amount of TFAM but not with the transcription level. We discuss an architectural role for TFAM in the maintenance of mtDNA in addition to its role in transcription activation.

DOI： 10.1128/MCB.24.22.9823-9834.2004

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

Mitochondrial transcription factor A (TFAM), a member of the high mobility group proteins, is essential for maintenance of mitochondrial DNA (mtDNA). Most TFAM and mtDNA (both of which are normally soluble) was recovered from the particulate fraction of human placental mitochondria when extracted with the non-ionic detergent Nonidet P-40. mtDNA and TFAM were co-immunoprecipitated by anti-TFAM antibodies. TFAM was released into the supernatant by DNase I digestion of mtDNA in the particulate fraction. Thus, TFAM and mtDNA are tightly associated with each other, and it is likely that few TFAM or mtDNA molecules exist in an unbound form in mitochondria. Based on the fact that TFAM is abundant enough to wrap mtDNA entirely, these results suggest that human mtDNA is packaged with TFAM.

DOI： 10.1093/nar/gkg251

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

We studied the role of the N-terminal region of the transmembrane domain of the human erythrocyte anion exchanger (band 3; residues 361-408) in the insertion, folding, and assembly of the first transmembrane span (TM1) to give rise to a transport-active molecule. We focused on the sequence around the 9-amino acid region deleted in Southeast Asian ovalocytosis (Ala-400 to Ala-408), which gives rise to nonfunctional band 3, and also on the portion of the protein N-terminal to the transmembrane domain (amino acids 361-396). We examined the effects of mutations in these regions on endoplasmic reticulum. insertion (using cell-free translation), chloride transport, and cell-surface movement in Xenopus oocytes. We found that the hydrophobic length of TM1 was critical for membrane insertion and that formation of a transport-active structure also depended on the presence of specific amino acid sequences in TM1. Deletions of 2 or 3 amino acids including Pro-403 retained transport activity provided that a polar residue was located 2 or 3 amino acids on the C-terminal side of Asp-399. Finally, deletion of the cytoplasmic surface sequence G(381) LVRD abolished chloride transport, but not surface expression, indicating that this sequence makes an essential structural contribution to the anion transport site of band 3.

DOI： 10.1074/jbc.M211662200

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

Band 3 is a typical polytopic membrane protein that mediates anion exchange activity [anion exchanger 1 (AE1)]. Although the topology and topogenesis of similar to40 residues just after transmembrane (TM) 9 have been extensively studied, the topogenesis of this region [tenth region (10thR)] has been unclear. Glycosylation sites created in the 10thR were efficiently glycosylated in a cell-free transcription/ translation system, whereas the glycosylation efficiencies were quite low in a cultured cell system. When TM12-14 was deleted or when cycloheximide was added to the culture medium, however, the glycosylation efficiency in the cultured cells increased to the same level as in the cell-free system, indicating that TM12 is essential for the sequestration from oligosaccharyl transferase into membrane and that cycloheximide treatment of the cells can mimic the cell-free system by reducing the rate of chain elongation. The glycosylation efficiency in cultured cells also increased with deletion of TM1-3. These results suggest that the 10thR is transiently extruded into the lumen and then inserted into the membrane. Both TM12 and the distant TM1-3 affect the membrane insertion of the 10thR. This indicates that during the folding of the protein, the 10thR is inserted into the membrane after the TM1-12 segments are properly assembled.

DOI： 10.1021/bi026619q

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

Background: In eclampsia, it is mandatory to recognize specific cerebrovascular complications before initiation of treatment. Diffusion- weighted magnetic resonance imaging (MRI) is a new technique that differentiates between cerebral infarction and hypertensive encephalopathy with vasogenic edema. Case: A 23-year-old primigravida developed eclampsia at 29 weeks' gestation. Focal neurologic signs and neuroimaging findings by computed tomography and MRI were consistent with acute infarction or vasogenic edema. Diffusion-weighted MRI did not show an abnormal signal, indicating vasogenic edema. Control of the severe hypertension without anticoagulation therapy was begun. After delivery, the woman's neurologic abnormalities disappeared. Conclusion: Diffusion-weighted MRI differentiated between cerebral infarction and vasogenic edema, helping in the management of eclampsia.

DOI： 10.1016/S0029-7844(98)00575-4

Language：Japanese  

【オートファジーと疾患】 ミトコンドリア恒常性の維持とマイトファジー
マイトファジーは、異常なミトコンドリアを分解することでミトコンドリア恒常性の維持に寄与している。マイトファジーには、ParkinとPINK1が関与する経路と、マイトファジーレセプターが関与する経路に大別される。Parkin-PINK1依存的マイトファジーの発見は、パーキンソン病の原因が異常ミトコンドリアの蓄積であることを示唆している。一方、マイトファジーレセプターNIXを介したマイトファジーは赤血球の正常な分化に必要であり、NIX欠損マウスでは貧血性疾患が見られる。このように、マイトファジーの破綻が様々な疾患の原因となることが知られている。(著者抄録)

Language：Japanese  

【オートファジーと疾患】 Parkinに依存しないマイトファジー
マイトファジーは,オートファジーによる選択的ミトコンドリア分解機構である.パーキンソン病の原因遺伝子産物であるParkinとPINK1は重要なマイトファジー因子であり,精力的に研究されてきた.一方で,ParkinやPINK1がすべてのマイトファジーに必要ではなく,これらに依存しないマイトファジーの理解も重要である.ここでは,Parkinに依存しないマイトファジーに関して概説する.(著者抄録)

Language：Japanese  

Digestシリーズ オートファジー その発見から未解決問題まで(Vol.4) 選択的オートファジー

Language：Japanese  

酵母におけるマイトファジーの分子機構と生理的役割

Language：English  

Mitochondria autophagy (mitophagy) is a process that selectively degrades mitochondria via autophagy. Recently, there has been significant progress in the understanding of mitophagy in yeast. Atg32, a mitochondrial outer membrane receptor, is indispensable for mitophagy. Phosphorylation of Atg32 is an initial cue for selective mitochondrial degradation. Atg32 expression and phosphorylation regulate the induction and efficiency of mitophagy. In addition to Atg32-related processes, recent studies have revealed that mitochondrial fission and the mitochondria-endoplasmic reticulum (ER) contact site may play important roles in mitophagy. Mitochondrial fission is required to regulate mitochondrial size. Mitochondria-ER contact is mediated by the ER-mitochondria encounter structure and is important to supply lipids from the ER for autophagosome biogenesis for mitophagy. Mitophagy is physiologically important for regulating the number of mitochondria, diminishing mitochondrial production of reactive oxygen species, and extending chronological lifespan under caloric restriction. These findings suggest that mitophagy contributes to maintain mitochondrial homeostasis. However, whether mitophagy selectively degrades damaged or dysfunctional mitochondria in yeast is unknown. This article is part of a Special Issue entitled: Mitophagy. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamcr.2015.01.005

Language：Japanese  

ミトコンドリアオートファジー ミトコンドリア恒常性維持機構
ミトコンドリアは、細胞活動に必須なATPの大部分を作り出す重要なオルガネラである。その機能が維持されること、即ち、ミトコンドリア恒常性維持は、細胞が正常に機能するため必須である。私たちは、新しいミトコンドリア恒常性維持機構としてミトコンドリアオートファジー(マイトファジー)に着目し研究を行ってきた。ここでは、これまでに明らかにされてきた、マイトファジーの生理的意義、疾患との関わり、分子機構を概説し、私たちが目指している、マイトファジーを用いた疾患治療への応用の可能性について簡単に紹介したい。(著者抄録)

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マイトファジー ミトコンドリアを選択的に分解する機構

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培養細胞を用いたオートファジーの活性測定法の開発とその臨床応用

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生化学・分子生物学 マイトファジー 基礎から臨床応用まで

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オートファジーによるミトコンドリア分解機構
出芽酵母の研究から、ミトコンドリアのオートファジーによる選択的な分解機構が明らかになりつつある。一方、哺乳類細胞の研究から、マイトファジー(オートファジーによるミトコンドリア分解)が機能低下したミトコンドリアを除去するというミトコンドリア品質管理に貢献していることが明らかになってきている。著者等は出芽酵母から、マイトファジー関連遺伝子としてATG32、ATG33を同定した。

Language：Japanese  

ミトコンドリアオートファジーの分子機構
オートファジーは飢餓を含む様々な細胞ストレスにより誘導される。ミトコンドリアのオートファジー(マイトファジー)は選択的オートファジーの一種で、傷害をうけたミトコンドリアを除去し、ミトコンドリア傷害の蓄積に起因する老化や神経変性疾患から個体を防御する機能があると考えられている。オートファジーの概論と、酵母を用いて行ったマイトファジーの解析、現在までに明らかにされてきたマイトファジーのメカニズムについて概説した。

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Language：Japanese  

偽膜性腸炎を契機に中毒性巨大結腸症,腸穿孔をきたした1例
子宮頸癌放射線治療後の直腸腟瘻に対する人工肛門造設術後に偽膜性腸炎を発症し中毒性巨大結腸症,腸穿孔を続発した60歳症例を経験した.本症例では偽膜性腸炎の発症後早期に診断し,薬物療法を行い,CRP値が低下し,便細菌培養およびCDトキシンが陰性化したにも拘わらず中毒性巨大結腸症,さらには腸穿孔をきたし外科的治療を必要とした.これは,炎症が高度で,全層に及び漿膜まで波及していたために,炎症が鎮静化した時点で既に筋は弛緩し腸管は拡張していたものと考えられる.また偽膜性腸炎の発症と炎症の重症化に,汎血球減少という宿主側の生体防御能の低下も大きく関与していた可能性が考えられた