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• W4376604805 abstract "•Mitofissin is a mitochondrial fission factor that resides in the IMS•The yeast mitofissin Atg44 promotes mitochondrial fission essential for mitophagy•Atg44 directly introduces membrane fragility to facilitate membrane fission•Mechanisms and roles of membrane fission by Atg44 are different from those by Dnm1 Mitophagy plays an important role in mitochondrial homeostasis by selective degradation of mitochondria. During mitophagy, mitochondria should be fragmented to allow engulfment within autophagosomes, whose capacity is exceeded by the typical mitochondria mass. However, the known mitochondrial fission factors, dynamin-related proteins Dnm1 in yeasts and DNM1L/Drp1 in mammals, are dispensable for mitophagy. Here, we identify Atg44 as a mitochondrial fission factor that is essential for mitophagy in yeasts, and we therefore term Atg44 and its orthologous proteins mitofissin. In mitofissin-deficient cells, a part of the mitochondria is recognized by the mitophagy machinery as cargo but cannot be enwrapped by the autophagosome precursor, the phagophore, due to a lack of mitochondrial fission. Furthermore, we show that mitofissin directly binds to lipid membranes and brings about lipid membrane fragility to facilitate membrane fission. Taken together, we propose that mitofissin acts directly on lipid membranes to drive mitochondrial fission required for mitophagy. Mitophagy plays an important role in mitochondrial homeostasis by selective degradation of mitochondria. During mitophagy, mitochondria should be fragmented to allow engulfment within autophagosomes, whose capacity is exceeded by the typical mitochondria mass. However, the known mitochondrial fission factors, dynamin-related proteins Dnm1 in yeasts and DNM1L/Drp1 in mammals, are dispensable for mitophagy. Here, we identify Atg44 as a mitochondrial fission factor that is essential for mitophagy in yeasts, and we therefore term Atg44 and its orthologous proteins mitofissin. In mitofissin-deficient cells, a part of the mitochondria is recognized by the mitophagy machinery as cargo but cannot be enwrapped by the autophagosome precursor, the phagophore, due to a lack of mitochondrial fission. Furthermore, we show that mitofissin directly binds to lipid membranes and brings about lipid membrane fragility to facilitate membrane fission. Taken together, we propose that mitofissin acts directly on lipid membranes to drive mitochondrial fission required for mitophagy. Autophagy is a catabolic process that is induced by various cellular stresses and that recycles cytoplasmic components through vacuolar/lysosomal degradation. Upon the induction of autophagy, cup-shaped membranous structures—called phagophores—emerge in the cytosol, expand, and engulf cytoplasmic proteins and organelles to form autophagosomes. The autophagosome fuses with the vacuole in yeast and plants or the lysosome in metazoans, leading to the degradation of its sequestered materials by resident hydrolases.1Nakatogawa H. Mechanisms governing autophagosome biogenesis.Nat. Rev. Mol. Cell Biol. 2020; 21: 439-458https://doi.org/10.1038/s41580-020-0241-0Crossref PubMed Scopus (314) Google Scholar Mitophagy is the selective autophagic degradation of mitochondria, which plays an important role in maintaining mitochondrial quality and quantity.2Pickles S. Vigié P. Youle R.J. Mitophagy and quality control mechanisms in mitochondrial maintenance.Curr. Biol. 2018; 28: R170-R185https://doi.org/10.1016/j.cub.2018.01.004Abstract Full Text Full Text PDF PubMed Scopus (967) Google Scholar Mitophagy shares most of the molecular processes with non-selective autophagy. However, mitophagy requires at least two additional steps: (1) the selection of particular mitochondrial regions as cargo and (2) the adjustment of the cargo morphology to fit the shape and size of the phagophore and autophagosome. The cargo selection process of mitophagy relies on receptor proteins localized on the outer mitochondrial membrane (OMM). In Saccharomyces cerevisiae (Sc), Atg32 is the sole mitophagy receptor,3Kanki T. Wang K. Cao Y. Baba M. Klionsky D.J. Atg32 is a mitochondrial protein that confers selectivity during mitophagy.Dev. Cell. 2009; 17: 98-109https://doi.org/10.1016/j.devcel.2009.06.014Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar,4Okamoto K. Kondo-Okamoto N. Ohsumi Y. Mitochondria-anchored receptor Atg32 mediates degradation of mitochondria via selective autophagy.Dev. Cell. 2009; 17: 87-97https://doi.org/10.1016/j.devcel.2009.06.013Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar which is phosphorylated upon mitophagy induction, enabling it to interact with the cytosolic scaffold/adaptor protein Atg11,5Kanki T. Kurihara Y. Jin X. Goda T. Ono Y. Aihara M. Hirota Y. Saigusa T. Aoki Y. Uchiumi T. Kang D. Casein kinase 2 is essential for mitophagy.EMBO Rep. 2013; 14: 788-794https://doi.org/10.1038/embor.2013.114Crossref PubMed Scopus (112) Google Scholar,6Aoki Y. Kanki T. Hirota Y. Kurihara Y. Saigusa T. Uchiumi T. Kang D. Phosphorylation of serine 114 on Atg32 mediates mitophagy.Mol. Biol. Cell. 2011; 22: 3206-3217https://doi.org/10.1091/mbc.E11-02-0145Crossref PubMed Scopus (167) Google Scholar and which focally accumulates on the mitochondrion that is targeted for degradation.7Furukawa K. Fukuda T. Yamashita S.I. Saigusa T. Kurihara Y. Yoshida Y. Kirisako H. Nakatogawa H. Kanki T. The PP2A-like protein phosphatase Ppg1 and the far complex cooperatively counteract CK2-mediated phosphorylation of Atg32 to inhibit mitophagy.Cell Rep. 2018; 23: 3579-3590https://doi.org/10.1016/j.celrep.2018.05.064Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Subsequently, Atg11 interacts with the Atg1 complex consisting of core autophagy proteins that assemble at the phagophore assembly site (PAS) to initiate phagophore formation.3Kanki T. Wang K. Cao Y. Baba M. Klionsky D.J. Atg32 is a mitochondrial protein that confers selectivity during mitophagy.Dev. Cell. 2009; 17: 98-109https://doi.org/10.1016/j.devcel.2009.06.014Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar,8Yorimitsu T. Klionsky D.J. Atg11 links cargo to the vesicle-forming machinery in the cytoplasm to vacuole targeting pathway.Mol. Biol. Cell. 2005; 16: 1593-1605https://doi.org/10.1091/mbc.e04-11-1035Crossref PubMed Scopus (0) Google Scholar In mammals, several mitophagy receptors, such as BNIP3L/NIX, BNIP3, FUNDC1, BCL2L13, and FKBP8 have been identified. These receptors interact with mammalian Atg8-family proteins located on the phagophore membrane to ensure selective entrapment of mitochondria as autophagy cargo.2Pickles S. Vigié P. Youle R.J. Mitophagy and quality control mechanisms in mitochondrial maintenance.Curr. Biol. 2018; 28: R170-R185https://doi.org/10.1016/j.cub.2018.01.004Abstract Full Text Full Text PDF PubMed Scopus (967) Google Scholar Besides receptor-mediated mitophagy, ubiquitin-mediated mitophagy, in which mitochondrial cargo tagged by PINK1- and PRKN/parkin-mediated ubiquitination is recognized by the autophagy machinery, serves as another subset of mitophagy pathways in mammals.2Pickles S. Vigié P. Youle R.J. Mitophagy and quality control mechanisms in mitochondrial maintenance.Curr. Biol. 2018; 28: R170-R185https://doi.org/10.1016/j.cub.2018.01.004Abstract Full Text Full Text PDF PubMed Scopus (967) Google Scholar Because mitochondria are typically larger than the autophagosome, it was suggested that mitochondrial fission generates mitochondrial fragments of appropriate size in advance of their engulfment by a phagophore.9Ashrafi G. Schwarz T.L. The pathways of mitophagy for quality control and clearance of mitochondria.Cell Death Differ. 2013; 20: 31-42https://doi.org/10.1038/cdd.2012.81Crossref PubMed Scopus (1040) Google Scholar,10Liesa M. Shirihai O.S. Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure.Cell Metab. 2013; 17: 491-506https://doi.org/10.1016/j.cmet.2013.03.002Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar,11Sebastián D. Palacín M. Zorzano A. Mitochondrial dynamics: coupling mitochondrial fitness with healthy aging.Trends Mol. Med. 2017; 23: 201-215https://doi.org/10.1016/j.molmed.2017.01.003Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar,12Shirihai O.S. Song M. Dorn 2nd, G.W. How mitochondrial dynamism orchestrates mitophagy.Circ. Res. 2015; 116: 1835-1849https://doi.org/10.1161/CIRCRESAHA.116.306374Crossref PubMed Scopus (207) Google Scholar However, well-studied mitochondrial fission factors, such as dynamin-related proteins Dnm1 in yeasts and DNM1L/Drp1 in mammals,13Wai T. Langer T. Mitochondrial dynamics and metabolic regulation.Trends Endocrinol. Metab. 2016; 27: 105-117https://doi.org/10.1016/j.tem.2015.12.001Abstract Full Text Full Text PDF PubMed Scopus (744) Google Scholar are dispensable for receptor-mediated mitophagy,14Burman J.L. Pickles S. Wang C. Sekine S. Vargas J.N.S. Zhang Z. Youle A.M. Nezich C.L. Wu X. Hammer J.A. Youle R.J. Mitochondrial fission facilitates the selective mitophagy of protein aggregates.J. Cell Biol. 2017; 216: 3231-3247https://doi.org/10.1083/jcb.201612106Crossref PubMed Scopus (272) Google Scholar,15Mendl N. Occhipinti A. Müller M. Wild P. Dikic I. Reichert A.S. Mitophagy in yeast is independent of mitochondrial fission and requires the stress response gene WHI2.J. Cell Sci. 2011; 124: 1339-1350https://doi.org/10.1242/jcs.076406Crossref PubMed Scopus (127) Google Scholar,16Yamashita S.I. Jin X. Furukawa K. Hamasaki M. Nezu A. Otera H. Saigusa T. Yoshimori T. Sakai Y. Mihara K. Kanki T. Mitochondrial division occurs concurrently with autophagosome formation but independently of Drp1 during mitophagy.J. Cell Biol. 2016; 215: 649-665https://doi.org/10.1083/jcb.201605093Crossref PubMed Scopus (128) Google Scholar raising the possibility that an unidentified machinery/mechanism also acts to promote mitochondrial fission. In this study, we identified Atg44 in fission yeast and budding yeast as a mitochondrial intermembrane space (IMS) protein that drives Dnm1/DNM1L-independent mitochondrial fission essential for receptor-mediated mitophagy. As Atg44-like proteins can be found in fungi, algae, and some plants, we termed this family of proteins “mitofissin.” In vitro reconstitution and structural analysis suggested that the yeast mitofissin Atg44 has the ability to directly bind to lipid membranes and brings about lipid membrane fragility to facilitate membrane fission. Our study sheds light on the process in which mitochondrial fission contributes to efficient engulfment of mitochondrial cargo by a phagophore and provides insights into the molecular basis for the function of mitofissin in mitochondrial membrane fission. To identify genes required for mitophagy, we screened the fission yeast Schizosaccharomyces pombe (Sp) genome-wide deletion library for mitophagy-deficient mutants and found SPAC26A3.14C, an uncharacterized gene, to be essential for mitophagy. Based on the unified nomenclature for autophagy-related genes in yeast,17Klionsky D.J. Cregg J.M. Dunn Jr., W.A. Emr S.D. Sakai Y. Sandoval I.V. Sibirny A. Subramani S. Thumm M. Veenhuis M. Ohsumi Y. A unified nomenclature for yeast autophagy-related genes.Dev. Cell. 2003; 5: 539-545https://doi.org/10.1016/s1534-5807(03)00296-xAbstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar we named this gene atg44+. This gene encodes a protein (Atg44) that is composed of 73 amino acids without any known functional domains (Figure S1A). In S. pombe, nitrogen starvation induces mitophagy, which can be monitored by the vacuolar processing of a chimeric mitochondrial protein Tuf1-RFP or Sdh2-GFP to produce free RFP or GFP18Fukuda T. Ebi Y. Saigusa T. Furukawa K. Yamashita S.I. Inoue K. Kobayashi D. Yoshida Y. Kanki T. Atg43 tethers isolation membranes to mitochondria to promote starvation-induced mitophagy in fission yeast.eLife. 2020; 9e61245https://doi.org/10.7554/eLife.61245Crossref Scopus (24) Google Scholar (Figures 1A and S1B). In atg44Δ cells, mitophagy was completely blocked similarly to cells lacking Atg1, a core autophagy protein (Figures 1A and S1B). Atg44 is conserved among fungi, algae, and some plants (Figure S1A), and Sc-Atg44 has recently been reported as a mitochondrial protein of unknown function (designated as Mco8).19Morgenstern M. Stiller S.B. Lübbert P. Peikert C.D. Dannenmaier S. Drepper F. Weill U. Höß P. Feuerstein R. Gebert M. et al.Definition of a high-confidence mitochondrial proteome at quantitative scale.Cell Rep. 2017; 19: 2836-2852https://doi.org/10.1016/j.celrep.2017.06.014Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar Exogenous expression of Sc-ATG44 can rescue mitophagy in Sp-atg44Δ cells, suggesting functional conservation (Figure 1B). Consistently, in S. cerevisiae, deletion of Sc-ATG44 resulted in loss of mitophagy, which was rescued by exogenous expression of Sp-Atg44 or Atg44 from Komagataella phaffii/Pichia pastoris (Kp-ATG44) (Figures 1C and 1D). Loss of Atg44 in either S. pombe or S. cerevisiae did not or only marginally affected non-selective macroautophagy, as measured by GFP/RFP processing (Figures S1C, S1D, and S1F) or the Pho8Δ60 assay (Figure 1E), or other types of selective autophagy including the Cvt pathway that delivers the precursor form of the hydrolase aminopeptidase I to the vacuole (Figure S1G), endoplasmic reticulum-phagy/reticulophagy (Figures S1E and S1H), and pexophagy (Figure S1I), suggesting that Atg44 is specifically required for mitophagy. To investigate the localization of Atg44, we expressed Sc-Atg44-FLAG, whose functionality was verified (Figure S1J), in S. cerevisiae and found that Sc-Atg44-FLAG colocalized with the mitochondrial protein Idh1-GFP (Figure 1F). By cell fractionation, Sc-Atg44 was collected in the 6,500 × g pellet fraction where mitochondrial Atg33 was enriched (Figure 1G). These findings suggest that Atg44 is a mitochondrial protein. We then treated isolated mitochondria with 0.1 M sodium carbonate (pH 11) and separated the membrane and supernatant fractions, and we found that Atg44 was released into the supernatant along with Atp2, the beta subunit of the F1Fo ATPase (Figure S1K). Finally, we isolated mitochondria from S. cerevisiae or S. pombe and studied the intramitochondrial localization of Atg44 using a Proteinase K (ProK) protection assay. Treatment with ProK alone caused degradation of the OMM protein Atg33 or Tom70-GFP but not of Sc-Atg44-FLAG or Sp-Atg44. However, ProK treatment under the hypotonic condition that disrupts the OMM but maintains the inner mitochondrial membrane (IMM) caused the degradation of Sc-Atg44-FLAG or Sp-Atg44, as well as the IMS protein Mia40-GFP or Mic60-FLAG, but not of the matrix protein Atp2 or Tuf1-RFP (Figures S1L and S1M); the latter were degraded when all mitochondrial membranes were disrupted with Triton X-100. Based on these results, we conclude that Atg44 localizes in the IMS and is not a transmembrane protein. Next, we examined a role for Atg44 in mitophagy using S. cerevisiae. We initially tested whether Sc-Atg44 is required for the cargo selection process that involves the phosphorylation, Atg11 interaction, and focal accumulation of Atg32.5Kanki T. Kurihara Y. Jin X. Goda T. Ono Y. Aihara M. Hirota Y. Saigusa T. Aoki Y. Uchiumi T. Kang D. Casein kinase 2 is essential for mitophagy.EMBO Rep. 2013; 14: 788-794https://doi.org/10.1038/embor.2013.114Crossref PubMed Scopus (112) Google Scholar,6Aoki Y. Kanki T. Hirota Y. Kurihara Y. Saigusa T. Uchiumi T. Kang D. Phosphorylation of serine 114 on Atg32 mediates mitophagy.Mol. Biol. Cell. 2011; 22: 3206-3217https://doi.org/10.1091/mbc.E11-02-0145Crossref PubMed Scopus (167) Google Scholar,7Furukawa K. Fukuda T. Yamashita S.I. Saigusa T. Kurihara Y. Yoshida Y. Kirisako H. Nakatogawa H. Kanki T. The PP2A-like protein phosphatase Ppg1 and the far complex cooperatively counteract CK2-mediated phosphorylation of Atg32 to inhibit mitophagy.Cell Rep. 2018; 23: 3579-3590https://doi.org/10.1016/j.celrep.2018.05.064Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar The phosphorylation status of Atg32 in Sc-atg44Δ cells, upon nitrogen starvation, was the same as that in wild-type cells (Figure S2A). The Atg32-Atg11 interaction was also unaffected in the absence of Sc-Atg44 (Figure S2B). After interacting with Atg11, Atg32 accumulates at one or multiple sites on mitochondria where mitophagy is expected to proceed.7Furukawa K. Fukuda T. Yamashita S.I. Saigusa T. Kurihara Y. Yoshida Y. Kirisako H. Nakatogawa H. Kanki T. The PP2A-like protein phosphatase Ppg1 and the far complex cooperatively counteract CK2-mediated phosphorylation of Atg32 to inhibit mitophagy.Cell Rep. 2018; 23: 3579-3590https://doi.org/10.1016/j.celrep.2018.05.064Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar This accumulation of Atg32 was comparable between Sc-atg44Δ and wild-type cells (Figure S2C). These results suggest that the absence of Atg44 does not affect the cargo selection processes. We then tested whether Atg44 is required for formation of autophagosomes that contain mitochondria as cargo (hereafter referred to as mitophagosomes). Autophagosomes/mitophagosomes are accumulated in cells lacking Ypt7, a Rab GTPase-family protein required for fusion of autophagosomes/mitophagosomes with vacuoles.16Yamashita S.I. Jin X. Furukawa K. Hamasaki M. Nezu A. Otera H. Saigusa T. Yoshimori T. Sakai Y. Mihara K. Kanki T. Mitochondrial division occurs concurrently with autophagosome formation but independently of Drp1 during mitophagy.J. Cell Biol. 2016; 215: 649-665https://doi.org/10.1083/jcb.201605093Crossref PubMed Scopus (128) Google Scholar,20Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. Formation process of autophagosome is traced with Apg8/Aut7p in yeast.J. Cell Biol. 1999; 147: 435-446https://doi.org/10.1083/jcb.147.2.435Crossref PubMed Scopus (720) Google Scholar Upon mitophagy induction, Sc-ypt7Δ cells accumulated mitophagosomes, which were observed as punctate structures labeled with Idh1-GFP that overlapped with RFP-Atg8, a marker for the phagophore and autophagosome membranes (Figure S2D). By contrast, mitophagosomes were completely absent in Sc-atg44Δ Sc-ypt7Δ cells (Figure S2D), suggesting that Atg44 is required for the process between cargo selection and mitophagosome formation. Notably, the Sc-atg44Δ Sc-ypt7Δ mutant exhibited mitochondrial protrusions that extended toward the center of the cell (Figure 2A). Such protrusions were also observed in the Sc-atg44Δ single mutant labeled with Idh1-GFP (matrix protein), Om14-RFP (OMM protein), or MitoTracker (membrane potential-dependent dye) (Figures 2B, S2E, and S2F). Formation of the protrusions required the core autophagy proteins Atg1 and Atg8 as well as mitophagy factors such as Atg32 and Atg11 (Figure 2B), indicating that the mitochondrial protrusion formation depends on the mitophagy process. We found that GFP-Atg32 accumulated around the protrusions and that GFP-Atg8 and GFP-Atg11 puncta colocalized with the protrusions with high frequency (Figure 2C), suggesting that the protrusions correspond to the mitochondrial regions that are destined to be delivered by mitophagy. These results also suggested the possible interaction between Atg44 and Atg32 at the sites of Atg32 accumulation, but such interaction was not detected under our experimental conditions (Figure S2G). We also found that the protrusions made contacts with the vacuolar surface presumably at the PAS, the site of phagophore membrane formation (Figure 2D). Detailed analysis with focused ion beam scanning electron microscopy (FIB-SEM) revealed that the mitochondrial protrusion, which was detectable specifically in atg44Δ cells, was formed from a part of the mitochondria, and its tip was in close proximity to the vacuole (Figure 2E; Videos S1 and S2). The cristae structure appeared to be substantially maintained inside the mitochondrial protrusion (Figure 2E; Video S2). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyMjc5Yzc3MGY0NWRkNjc3OTA5YTY3OGNlYTliNzhmMCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjg4MzQ0NjQ2fQ.o7mJADn18K0hj2tyil6FyLcCqY4Vb_IKsPSbZWDSlEdiyYlOGPbTEUeSj6l0kzgv0Do2nzw-i_Da7mQP3c8d4b2pdxEEWFZiouxhYdg-QWeykHx4iB-SVT1FlpXznOjc1KAnGJyLXX3wUYvCwYu27sovWK1Fv5e6L3tHu-uFeh4lZH1RAxS3AEnIDv9quKkxN95zhPu5nf0tw9-0_aEXTknTGI0v_429NGXqrFVtmZjPSNfHGf5htaxT0a6nfsVGs07neHstRWc_2Hrrmvcx6u8cMliTaN3h22zJksW0coV1pKMIA2ZhVR1spuIobMKFu-5f-1V24eFwt5VjGmEejg Download .mp4 (48.42 MB) Help with .mp4 files Video S1. Three-dimensional representation of S. cerevisiae WT cells upon mitophagy induction, related to Figure 2EThree-dimensional representation of S. cerevisiae WT cells upon mitophagy induction, reconstructed from serial-sectioned images obtained using FIB-SEM. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI0MzQwZDg2YjkyMmJkNTIyOTUyMWQ1YjllMjg3NmZlYiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjg4MzQ0NjQ2fQ.o8fEkKdFC-4oA7fBg5ICr4G8RTXYcYJIVw1lfYkJtZEno4BZPjsGsmGMVfUnqh4eS9bgDoavCOGHrRLpaH3pMl5Xea2quKJ1yNWt4dikNB7sQGLBMvSjS3iyKex9Z0AfvbjV_sS0PC1v25YqIKavDD1wKieilIuj2P_y37BqY1-TI7s2KGbTKRqHFsi-4gqSPAkgeh0m62MUnsXyyKeBj7-6-eOXVGHB06r1of0Cbyp7krJOgIxRfY_f98VgqmrQc1njOrEmjDlQgNLVNZs1U3oWVQsofjcnbUpTQSTHVItpvxnaMHdO9dMCR7aF9ZTgBGZR3v8bvxpFC0lrenWuDg Download .mp4 (10.65 MB) Help with .mp4 files Video S2. Three-dimensional representation of S. cerevisiae atg44Δ cells upon mitophagy induction, related to Figure 2EThree-dimensional representation of S. cerevisiae atg44Δ cells upon mitophagy induction, reconstructed from serial-sectioned images obtained using FIB-SEM. Yellow: mitochondria; blue: vacuoles. Taken together, these findings confirm that the autophagy machinery assembles at particular sites on mitochondria, and these portions are targeted to the vacuolar surface as mitophagy proceeds. In the absence of Atg44, however, these parts remain connected to the main body of the mitochondrion, resulting in formation of the mitochondrial protrusions that are not enwrapped by the phagophore. During our analysis of Sc-atg44Δ cells, we realized that their mitochondria were spherically enlarged and similar to those in dnm1Δ cells, which are defective in mitochondrial fission (Figures 3A, 3B, and S3A). As atg44Δ cells show a defect in the engulfment of mitochondria by the phagophore, we hypothesized that Atg44 is involved in mitochondrial fission that produces fragments suitable for phagophore engulfment. To further examine the involvement of Atg44 in mitochondrial morphology, we observed mitochondria in Sc-atg44Δ cells by electron microscopy. Consistent with the results of fluorescence microscopy, mitochondria in Sc-atg44Δ cells were spherically enlarged, although the cristae structure was unaffected (Figure 3C; Videos S3 and S4). Similarly, loss of Atg44 in S. pombe affected mitochondrial morphology; some of the Sp-atg44Δ cells showed spherically enlarged mitochondria like Sp-dnm1Δ cells (Figure 3D). Because enlarged mitochondria are a critical feature of a deficiency in mitochondrial fission, these findings support the hypothesis that Atg44 has a positive role in mitochondrial fission. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyZmIyMWI1ZmI5Y2NjMDNiY2YzNDlhNTJhYTc3ZWMzNiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjg4MzQ0NjQ2fQ.WxcmNsDPrF7r1rlF5Njbr5I_1_8rEsuVL6KFYRPLDiZcWUZPzYpuVzqFLI3S4aB6x3EFOHHgptSRsge-zIFlYk0ARrC2D50WvSaGXmg6REOXbiIgrUyJtlFyvMeAxNTWbvxd78_GtmAL_i2gGdgLmnRKM9ffknJRZFJ1P1a8QIiKuSE6c2wZIe48IJltIahUd4ezES_CQUMXDM4uxmYmg_jYdwx_b9TmhF5JuChLiWHcVa7DwsLKD2yYyo9OM5-YLWqSmCUxinjhOVWWJVOZ0SoCDoOf5PfvVGfAeFVVl6WQOIvppWM1yPb4cFejPDvm5mWYXujP2NtWVOl1vIGErQ Download .mp4 (10.67 MB) Help with .mp4 files Video S3. Three-dimensional representation of whole S. cerevisiae WT cells grown in YPL, related to Figure 3CThree-dimensional representation of whole S. cerevisiae WT cells grown in YPL, reconstructed from serial-sectioned images obtained using SBF-SEM. Brown: mitochondria; red: nucleus; blue: vacuoles. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIwZjYwMzJjM2Y4MmVmYzZhMWUxMDllNzQzZTdkZjM4YSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjg4MzQ0NjQ2fQ.T7EZY-X-27mRofyiUHfuaFzjl9l12t_cGqIbLj0Z7tzd4hbzM4dVhkSigqKX08tOU8UNHIvtDL6NB7N0DHIWBKzPHIPsBcDG9kyrHRe9NcwXEy2hjxzwyhmD2jaBP-L6QaQGeAdE4s5kLVqITfx8g1STdpuywOKaNTx4_Q4xnXSXbuvLMiGifMSnOhhBNOvUibynTKZ4siTFbbYSu9mIpcK8BG0bU3gYSCHYSQeYxTmRk-BX6Bt9LuuwFLLr6mpSYpRKMHQ_oZbvF8uyiGjKdOuItyJ1kb3wQ15rYO5mYXJUu5ZvyLbMpqXsL0Uc-zoGM8kJ7x6rIX2QGoTvJrIs4A Download .mp4 (7.23 MB) Help with .mp4 files Video S4. Three-dimensional representation of whole S. cerevisiae atg44Δ cells grown in YPL, related to Figure 3CThree-dimensional representation of whole S. cerevisiae atg44Δ cells grown in YPL reconstructed from serial-sectioned images obtained using SBF-SEM. Brown: mitochondria; red: nucleus; blue: vacuoles. To further confirm that Atg44 acts in mitochondrial fission, Atg44 was overexpressed in S. cerevisiae and S. pombe cells. Overexpression of Atg44 in both species caused mitochondrial fragmentation not only in wild-type cells but also in Dnm1-deficient cells (Figures 3E and 3F). We next expressed Sc-Atg44 in HeLa cells to test whether Atg44 causes mitochondrial fission in mammalian cells. Another mitochondrial protein with a similar size as Atg44, LACTB_N68-FLAG (N-terminal 68 amino acids of LACTB with a FLAG tag),21Polianskyte Z. Peitsaro N. Dapkunas A. Liobikas J. Soliymani R. Lalowski M. Speer O. Seitsonen J. Butcher S. Cereghetti G.M. et al.LACTB is a filament-forming protein localized in mitochondria.Proc. Natl. Acad. Sci. USA. 2009; 106: 18960-18965https://doi.org/10.1073/pnas.0906734106Crossref PubMed Scopus (58) Google Scholar was used as a control. We confirmed that LACTB_N68 localizes in the IMS (Figure S3B). Unexpectedly, HeLa cells expressing any of these proteins exhibited fragmented mitochondria (Figure S3C). We assumed that the observed fragmentation was attributed to the mitochondrial division that occurred in response to mitochondrial stress caused by overloading exogenous proteins in the IMS. To prevent such a type of mitochondrial division, we performed the same analysis using mitochondrial fission-deficient DNM1L/Drp1 knockout (DNM1L KO) HeLa cells. DNM1L KO cells exhibited tubular or spherically enlarged mitochondria due to the loss of mitochondrial fission (Figure 3G).22Otera H. Miyata N. Kuge O. Mihara K. Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling.J. Cell Biol. 2016; 212: 531-544https://doi.org/10.1083/jcb.201508099Crossref PubMed Scopus (156) Google Scholar Expression of Sc-Atg44 and Sc-Atg44-FLAG, but not LACTB_N68-FLAG, brought about fragmentation of mitochondria (Figure 3G). These findings suggest that Atg44 promotes mitochondrial fission without a need for DNM1L/Dnm1. Involvement of Atg44 in mitochondrial fission supports our hypothesis that Atg44 produces fragments that are dimensionally suitable for efficient engulfment during mitophagy. As Dnm1 is not essential for mitophagy (Figures S3D and S3E),15Mendl N. Occhipinti A. Müller M. Wild P. Dikic I. Reichert A.S. Mitophagy in yeast is independent of mitochondrial fission and requires the stress response gene WHI2.J. Cell Sci. 2011; 124: 1339-1350https://doi.org/10.1242/jcs.076406Crossref PubMed Scopus (127) Google Scholar,16Yamashita S.I. Jin X. Furukawa K. Hamasaki M. Nezu A. Otera H. Saigusa T. Yoshimori T. Sakai Y. Mihara K. Kanki T. Mitochondrial division occurs concurrently with autophagosome formation but independently of Drp1 during mitophagy.J. Cell Biol. 2016; 215: 649-665https://doi.org/10.1083/jcb.201605093Crossref PubMed Scopus (128) Google Scholar we infer that Atg44 and Dnm1 mediate mitochondrial fission events with distinct physiological roles. To test whether mitophagy could take place without Atg44 if mitochondria were artificially divided into fragments small enough for phagophore engulfment, we utilized cells lacking the mitochondrial fusion factor Mgm1 or Fzo1. In the absence of Mgm1-mediated mitochondrial fusion, mitochondrial fission predominates over fusion, leading to increased fragmentation (Figures S3F and S3G).23Sesaki H. Southard S.M. Yaffe M.P. Jensen R.E. Mgm1p, a dynamin-related GTPase, is essential for fusion of the mitochondrial outer membrane.Mol. Biol. Cell. 2003; 14: 2342-2356https://doi.org/10.1091/mbc.e02-12-0788Crossref PubMed Scopus (0) Google Scholar As expected, in S. pombe atg44Δ cells lacking Mgm1, some of the mitochondria became fragmented and mitophagy was partially rescued (Figures 4A and S3F). Similarly, in S. cerevisiae, the absence of Mgm1 or Fzo1 in atg44Δ cells rescued mitophagy to levels comparable to cells deficient for only Mgm1 or Fzo1 (Figures 4B and S3G). These findings further support the concept that Atg44 is a mitochondrial fission factor that generates mitochondrial fragments suitable for mitophagy. The maintenance of mitochondrial morphology by balancing mitochondrial fusion and fission is important for proper mitochondrial function.13Wai T. Langer T. Mitochondrial dynamics and metabolic regulation.Trends Endocrinol. Metab. 2016; 27: 10" @default.
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• W4376604805 title "The mitochondrial intermembrane space protein mitofissin drives mitochondrial fission required for mitophagy" @default.
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