Molecular mechanisms and physiological functions of mitophagy

Review13 January 2021Open Access Molecular mechanisms and physiological functions of mitophagy Mashun Onishi Mashun Onishi orcid.org/0000-0003-1511-4097 Laboratory of Mitochondrial Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, JapanThese authors contributed equally to this work Search for more papers by this author Koji Yamano Koji Yamano orcid.org/0000-0002-4692-161X The Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, JapanThese authors contributed equally to this work Search for more papers by this author Miyuki Sato Corresponding Author Miyuki Sato [email protected] orcid.org/0000-0002-1944-4918 Laboratory of Molecular Membrane Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan Search for more papers by this author Noriyuki Matsuda Corresponding Author Noriyuki Matsuda [email protected] orcid.org/0000-0001-8199-952X The Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Koji Okamoto Corresponding Author Koji Okamoto [email protected] orcid.org/0000-0003-4730-4522 Laboratory of Mitochondrial Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Japan Search for more papers by this author Mashun Onishi Mashun Onishi orcid.org/0000-0003-1511-4097 Laboratory of Mitochondrial Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, JapanThese authors contributed equally to this work Search for more papers by this author Koji Yamano Koji Yamano orcid.org/0000-0002-4692-161X The Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, JapanThese authors contributed equally to this work Search for more papers by this author Miyuki Sato Corresponding Author Miyuki Sato [email protected] orcid.org/0000-0002-1944-4918 Laboratory of Molecular Membrane Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan Search for more papers by this author Noriyuki Matsuda Corresponding Author Noriyuki Matsuda [email protected] orcid.org/0000-0001-8199-952X The Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Koji Okamoto Corresponding Author Koji Okamoto [email protected] orcid.org/0000-0003-4730-4522 Laboratory of Mitochondrial Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Japan Search for more papers by this author Author Information Mashun Onishi1, Koji Yamano2, Miyuki Sato *,3, Noriyuki Matsuda *,2 and Koji Okamoto *,1 1Laboratory of Mitochondrial Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Japan 2The Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan 3Laboratory of Molecular Membrane Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan *Corresponding author. Tel: +81 27 220 8842; E-mail: [email protected] *Corresponding author. Tel: +81 3 5316 3244; E-mail: [email protected] *Corresponding author. Tel: +81 6 6879 7970; E-mail: [email protected] The EMBO Journal (2021)40:e104705https://doi.org/10.15252/embj.2020104705 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Degradation of mitochondria via a selective form of autophagy, named mitophagy, is a fundamental mechanism conserved from yeast to humans that regulates mitochondrial quality and quantity control. Mitophagy is promoted via specific mitochondrial outer membrane receptors, or ubiquitin molecules conjugated to proteins on the mitochondrial surface leading to the formation of autophagosomes surrounding mitochondria. Mitophagy-mediated elimination of mitochondria plays an important role in many processes including early embryonic development, cell differentiation, inflammation, and apoptosis. Recent advances in analyzing mitophagy in vivo also reveal high rates of steady-state mitochondrial turnover in diverse cell types, highlighting the intracellular housekeeping role of mitophagy. Defects in mitophagy are associated with various pathological conditions such as neurodegeneration, heart failure, cancer, and aging, further underscoring the biological relevance. Here, we review our current molecular understanding of mitophagy, and its physiological implications, and discuss how multiple mitophagy pathways coordinately modulate mitochondrial fitness and populations. Glossary ALLO-1 Allophagy-1 ATG Autophagy-related protein BCL2L1/BCL-XL BCL2 like 1 BCL2L13 B-cell lymphoma 2-like 13 BNIP3 BCL2 and adenovirus E1B 19-kDa-interacting protein 3 BNIP3L Nip3-like protein X (NIX)/BNIP3-like protein CCCP Carbonyl cyanide m-chlorophenylhydrazone cGAS Cyclic GMP-AMP synthase CK2 Casein kinase 2 CPS-6 Mitochondrial endonuclease G DFCP1/ZFYVE1 DFCP1/zinc finger FYVE-type containing 1 FIP200/RB1CC1 FIP200/RB1-inducible coiled-coil protein 1 Fis1 Fission, mitochondrial 1 FKBP8/FKBP38 FK506-binding protein 8 FOXO1 Forkhead box O1 FUNDC1 FUN14 domain-containing protein 1 GABARAP GABA type A receptor-associated protein GABARAPL1/2 GABA type A receptor-associated protein-like 1/2 GFP Green fluorescent protein HOPS Homotypic fusion and vacuole protein sorting IGF-1 Insulin-like growth factor 1 Keap1 Kelch-like ECH-associated protein 1 LC3A/B/C Microtubule-associated protein 1 light chain 3 alpha/beta/gamma LIR LC3-interacting region MARCH5/MITOL Membrane-associated ring-CH-type finger 5 MBP Maltose-binding protein Miro Mitochondrial Rho mTORC1 Mechanistic target of rapamycin complex 1 MUL1 mitochondrial E3 ubiquitin protein ligase 1 NBR1 NBR1 autophagy cargo receptor NDP52/CALCOCO2 NDP52/calcium binding and coiled-coil domain 2 NLRP3 NLR family pyrin domain-containing 3 NOD Nucleotide-binding oligomerization domain NRF2 Nuclear factor, erythroid 2-like 2 OPTN Optineurin p62/SQSTM1 p62/Sequestosome 1 PARL Presenilin-associated rhomboid-like protein PC Phosphatidylcholine PE Phosphatidylethanolamine PGAM5 PGAM family member 5, mitochondrial serine/threonine protein phosphatase PI Phosphatidylinositol PI3K Phosphatidylinositol 3-kinase PI3P Phosphatidylinositol-3-phosphate PINK1 PTEN induced kinase 1 PLEKHM1 Pleckstrin homology and RUN domain-containing M1 RABGEF1 RAB guanine nucleotide exchange factor 1 Rheb Ras homolog, mTORC1 binding SNARE Soluble N-ethylmaleimide-sensitive factor attachment protein receptor Src SRC proto-oncogene, non-receptor tyrosine kinase STING Stimulator of interferon genes TAX1BP1 Tax1 binding protein 1 TBC1D15 TBC1 domain family member 15 TBC1D17 TBC1 domain family member 17 TBK1 TANK-binding kinase 1 TOMM/TOM Translocase of the outer mitochondrial membrane TORC1 Target of rapamycin complex 1 UBAN Ubiquitin-binding domain in ABIN proteins and NEMO ULK1 Unc-51-like autophagy activating kinase 1 USP Ubiquitin specific protease VDAC Voltage-dependent anion channel VPS Vacuolar protein sorting WIPI WD repeat domain, phosphoinositide interacting Introduction Mitochondria are double-membrane-bound subcellular compartments that function in fundamental processes such as ATP production, phospholipid biosynthesis/transport, calcium signaling, and iron homeostasis (Raffaello et al, 2016; Tamura & Endo, 2017; Spinelli & Haigis, 2018). These organelles act as platforms for various events including apoptosis, innate immune response, and cell differentiation (Mehta et al, 2017; Kalkavan & Green, 2018; Lisowski et al, 2018). Since mitochondria generate reactive oxygen species (ROS) from the electron transport chain, they are constantly challenged with oxidative stress that ultimately may lead to their structural and functional failure (Wong et al, 2017). Therefore, cells need sophisticated systems for maintaining mitochondrial fitness. Mitochondrial quality control relies on diverse pathways: ROS scavenging, DNA repair, and protein refolding/degradation (Scheibye-Knudsen et al, 2015). In addition to these processes, mitochondrial fusion and fission play key roles in mitochondrial quality control (Eisner et al, 2018). While fusion promotes content mixing between healthy and partially dysfunctional mitochondria, fission separates damaged mitochondrial components from the mitochondrial pool. The autophagic system targets impaired mitochondria and delivers them to lysosomes for degradation. This catabolic process, called mitophagy, contributes to maintaining mitochondrial quality control (Pickles et al, 2018) and mitochondrial quantity in multiple cell types. In tissues consuming a large amount of ATP such as brain, skeletal muscle, heart, liver, and kidney, mitochondria are highly developed in order to maintain the proper balance between energy demand and supply. When these cells are shifted from normoxia to hypoxia, mitophagy is induced to decrease mitochondrial quantity, thereby adapting cellular metabolism to anaerobic conditions (Wu & Chen, 2015). Thus, mitochondrial biogenesis and degradation are two opposing processes that determine mitochondrial quantity (Ploumi et al, 2017). In addition, mitochondria are almost completely eliminated during erythrocyte maturation (Ney, 2015). Furthermore, accumulating evidence reveals that maternal inheritance of mitochondrial DNA (mtDNA) depends on selective clearance of sperm-derived paternal mitochondria during early embryogenesis (Sato & Sato, 2017). Although autophagy is generally recognized as a bulk degradation process that non-selectively transports cytoplasmic components such as nucleic acids, proteins, and organelles to lysosomes (Nakatogawa, 2020), it also acts as a selective system to mediate clearance of particular organelles (Gatica et al, 2018). Mitophagy is one of the organelle-specific autophagy pathways that serves to maintain cell structure and function (Okamoto, 2014) (Fig 1). The term "mitophagy" was first coined in 2005 (Lemasters, 2005; Priault et al, 2005), and within a few years, major breakthroughs led to the discovery of key proteins that selectively mediate mitochondrial degradation in yeast (Okamoto et al, 2009; Kanki et al, 2009b) and mammalian cells (Schweers et al, 2007; Narendra et al, 2008; Sandoval et al, 2008). In this review, we will describe the molecular mechanisms underlying mitophagy in yeast, worms, Drosophila, and mammalian cells and cover its physiological and pathophysiological functions. Figure 1. Overview of mitophagy (1) Intra- and extracellular cues promote isolation of excess or damaged mitochondria via fragmentation of tubular networks. (2) Mitophagy receptors or ubiquitin–autophagy adaptors that confer selectivity for degradation are recruited and/or activated on the surface of mitochondria. (3) Core autophagy-related proteins target to mitochondria and generate the isolation membrane/phagophore surrounding mitochondria. (4) Targeted mitochondria are enclosed and sequestrated by autophagosomes. (5) Autophagosomes are transported and fused with lytic compartments such as vacuoles in yeast or lysosomes in mammals. (6) Lysosomal or vacuolar acidic hydrolases flow into autophagosomes to degrade mitochondria, and the contents will be recycled. Download figure Download PowerPoint Receptor-mediated mitophagy in yeast Regulation of mitophagy by Atg32 Mitophagy in the budding yeast Saccharomyces cerevisiae is mostly mediated by Atg32, a single-pass transmembrane protein in the outer mitochondrial membrane (OMM) (Okamoto et al, 2009; Kanki et al, 2009) (Fig 2A). In this unicellular eukaryote, mitophagy is induced when cells are grown to stationary phase or upon nitrogen starvation (Tal et al, 2007; Kanki & Klionsky, 2008; Okamoto et al, 2009). Under such conditions, Atg32 expression is induced at the transcriptional level and accumulates on the OMM, forming a complex with Atg8 and Atg11 on the surface of mitochondria. Atg8 is localized to autophagosomes, and Atg11 acts as a scaffold for other Atg proteins to promote autophagosome formation. Loss of Atg32 almost completely abolishes mitophagy while its overexpression increases mitophagy activity, suggesting that this molecule is a rate-limiting factor for regulating the number of mitochondria to be degraded. Atg32 is specifically important to degrade mitochondria and is dispensable for other types of autophagy-related processes including bulk autophagy, the cytoplasm-to-vacuole targeting pathway, ER-phagy, and pexophagy. Figure 2. Receptor-mediated mitophagy (A) Schematic representation of the domain structures of mitophagy receptors in yeast and mammals. AIM/LIR, Atg8-family protein-interacting motif/LC3-interacting region (pink); TM, transmembrane domain (light blue); BH1-4, Bcl-2 homology 1-4 domain (green and light green); PPlase, peptidyl-prolyl cis-trans isomerase domain (orange); TPR, tetratricopeptide repeat domain (purple); CaM, calmodulin-binding domain (dark red). The protein size is indicated as the number of amino acids. (B-D) Models for mitophagy receptor activation and protein recruitment on the mitochondrial surface. The yeast mitophagy receptor Atg32 (B), and the mammalian mitophagy receptors BNIP3, BCL2L13, FKBP8 (C), FUNDC1, and NIX (D) bind to ATG8 family proteins and then target the autophagy machinery to mitochondria. Phosphorylation and dephosphorylation serve as regulatory mechanisms to modulate the activity of mitophagy receptors. For details, see text. Download figure Download PowerPoint Several lines of evidence reveal that phosphorylation is a key event for Atg32-mediated mitophagy (Fig 2B). During respiration or upon a shift from respiration to starvation, Atg32 is phosphorylated in a manner dependent on its Atg11-interacting motif containing Ser114 and Ser119 (Aoki et al, 2011; Kondo-Okamoto et al, 2012). Importantly, this post-translational modification is mediated by CK2, an evolutionarily conserved serine/threonine kinase that regulates a variety of cellular processes (Kanki et al, 2013). CK2 interacts with Atg32 in vivo and directly phosphorylates Atg32 in vitro (Kanki et al, 2013). Mutagenesis of Atg32 Ser114, Ser119, and other conserved residues in the Atg11-interacting motif or impairment of CK2 function destabilizes Atg32-Atg11 interactions and strongly suppresses mitophagy (Aoki et al, 2011; Kondo-Okamoto et al, 2012; Kanki et al, 2013), suggesting that CK2-dependent phosphorylation could act as a regulatory step to activate Atg32 for recruiting Atg11 to mitochondria. A recent study has demonstrated that the protein phosphatase 2A (PP2A)-like protein Ppg1 is critical for dephosphorylation of Atg32 and negatively regulates mitophagy (Furukawa et al, 2018) (Fig 2B). In cells lacking Ppg1, Atg32 is phosphorylated even at the respiratory log phase (stage prior to mitophagy induction), likely resulting in increased Atg32-Atg11 interactions that accelerate mitochondrial degradation (Furukawa et al, 2018). Ppg1-dependent mitophagy suppression also requires its binding partners Far proteins that have previously been suggested to form a complex critical for pheromone-induced cell cycle arrest (Pracheil & Liu, 2013). These findings raise the possibility that the Ppg1-Far complex dephosphorylates Atg32, competing against CK2-mediated phosphorylation under mitophagy non-inducing conditions. Atg32 has been known to be proteolytically cleaved by Yme1, a catalytic subunit of metalloprotease in the inner mitochondrial membrane (IMM) that belongs to the ATPases associated with diverse cellular activities (AAA) protein family (Leonhard et al, 1996). Upon mitophagy, Atg32 is proteolytically processed at its C-terminal portion in a Yme1-dependent manner (Wang et al, 2013) (Fig 2B). Loss of Yme1 leads to a strong decrease in Atg32-Atg11 interactions and mitophagy under nitrogen starvation (Wang et al, 2013). These findings support the idea that Yme1-mediated proteolysis is required for efficient mitophagy. However, other studies suggest minor or no mitophagy deficiencies in cells lacking Yme1 (Welter et al, 2013; Gaspard & McMaster, 2015), raising the possibility that Yme1-dependent processing may be relevant to Atg32-mediated mitophagy in some specific strains and/or under some specific conditions. Regulation of mitophagy via ER factors In yeast, mitochondria and the ER are connected at contact sites via the ER–mitochondria encounter structure (ERMES) complex that facilitates phospholipid transfer between these two organelles (Kornmann et al, 2009). The ERMES complex is localized at discrete foci where the ER and mitochondria are closely positioned, and loss of ERMES leads to severe defects in starvation-induced mitophagy (Bockler & Westermann, 2014). Under starvation conditions, the ERMES component Mmm1 forms foci that partially co-localize with Atg8 dot-like structures, suggesting that autophagosomes are associated with the ER–mitochondria contact sites (Bockler & Westermann, 2014). Ubiquitylation of the ERMES component Mdm12/34 by the E3 ligase Rsp5 has also been linked to mitophagy (Belgareh-Touze et al, 2017). Atg32-mediated mitophagy is also regulated via Get1/2 complex and Opi3, two factors associated with the ER (Sakakibara et al, 2015; Onishi et al, 2018). The Get1/2 complex is important for insertion of tail-anchored proteins into the ER membrane (Schuldiner et al, 2005; Schuldiner et al, 2008; Wang et al, 2014). Loss of Get1/2 causes defects in mitophagy under respiratory conditions, while other types of autophagy-related pathways are slightly or hardly affected (Onishi et al, 2018). How Get1/2 acts in trans to promote mitochondrial clearance remains unclear. Surprisingly, loss of Opi3, a phospholipid methyltransferase localized in the ER, leads to suppression of Atg32 induction during respiration (Sakakibara et al, 2015). Opi3 acts in the phospholipid biosynthesis pathway for conversion of PE into PC. Depletion of Opi3 causes aberrant elevation of glutathione levels that reduces cellular oxidative stress and thus negatively affects induction of Atg32 and mitophagy (Deffieu et al, 2009; Okamoto et al, 2009; Sakakibara et al, 2015). These findings raise the possibility that respiring yeast cells coordinate phospholipid methylation and mitophagy through unknown mechanisms. Receptor-mediated mitophagy in mammals In mammals, mitophagy is mechanistically more complex than in yeast and is induced by different cellular stress signals and developmental changes. Disruption of mitochondrial membrane potential is a potent trigger of mitophagy (Elmore et al, 2001). CCCP, a proton-selective ionophore, and antimycin A (an inhibitor of the respiratory complex III) are commonly used to impair mitochondria and activate mitophagy. Because CCCP is highly toxic and induces non-physiological levels of mitochondrial damage especially in neurons, antimycin A is often used to induce mitophagy in neuronal cells (Cai et al, 2012; Ashrafi et al, 2014). Both reagents trigger mitochondrial depolarization and promote accumulation of mitophagy receptors on the OMM. These receptors are integral membrane proteins that promote specific binding to mammalian Atg8 family members (LC3A/B/C, GABARAP, GABARAP-L1/2) through a conserved LC3-interacting regions (LIRs) and regulate the formation of isolation membranes enclosing mitochondria. Two major types of receptors have been suggested to mediate elimination of mitochondria under physiological and pathological conditions in mammals (Fig 2A). One group includes BNIP3 and BNIP3L (also known as NIX) (Boyd et al, 1994; Matsushima et al, 1998; Chen et al, 1999; Vande Velde et al, 2000; Regula et al, 2002; Kubli et al, 2007; Schweers et al, 2007; Sandoval et al, 2008; Hanna et al, 2012), and the other group includes FUNDC1 (Liu et al, 2012). In addition, BCL2L13 is the mammalian functional counterpart of yeast receptor Atg32 (Murakawa et al, 2015) (Fig 2A). In the following part, we will discuss the molecular functions of mitophagy receptors in mammalian cells and the role of a family of receptors, namely FKBP proteins (Bhujabal et al, 2017). BNIP3 and NIX BNIP3 is required for efficient turnover of mitochondria under hypoxic conditions (Zhang et al, 2008). In response to hypoxia, BNIP3 is upregulated and anchored to the OMM via its C-terminal transmembrane (TM) domain, exposing the N-terminal domain to the cytosol (Hanna et al, 2012). BNIP3 is usually expressed as an inactive monomer in the cytosol, but following stress signals, it forms a stable homodimer via its C-terminal TM domain and is integrated into the OMM (Chen et al, 1997; Ray et al, 2000; Kubli et al, 2008). BNIP3 mutations, which disrupt homodimerization but do not affect mitochondrial localization, cause a mitophagy defect, supporting the idea that homodimerization of BNIP3 is important for efficient degradation of mitochondria (Hanna et al, 2012). Similar to other mitophagy receptors, BNIP3 has a LIR motif at its N-terminal region (Fig 2A) and mutations in this region block the interaction with LC3, leading to mitophagy defects. Phosphorylation of BNIP3 at Ser17 and Ser24 near the LIR motif is important for BNIP3-LC3 interactions (Zhu et al, 2013) (Fig 2C). NIX shows homology to BNIP3 (53–56% amino acid sequence identity) (Matsushima et al, 1998; Chen et al, 1999) and promotes selective degradation of mitochondria during reticulocyte maturation (Schweers et al, 2007; Sandoval et al, 2008). During erythroid differentiation, cell nucleus, mitochondria, and other intracellular organelles are eliminated, so that erythrocytes can keep maximum space for hemoglobin that delivers oxygen (Koury et al, 2005; Yoshida et al, 2005; Fader & Colombo, 2006). With the high sequence similarity between these two proteins, expression of BNIP3 can restore mitochondrial clearance in reticulocytes lacking NIX (Zhang et al, 2012). NIX contains an LIR motif that promotes binding to LC3A, LC3B, GABARAP, GABARAP-L1, and GABARAP-L2 (Novak et al, 2010) (Fig 2A). In CCCP-treated cells, NIX recruits GABARAP-L1 to damaged mitochondria and promotes mitophagy in a manner dependent on its LIR motif (Novak et al, 2010). Phosphorylation of Ser34 and Ser35, two tandem serine residues near the LIR motif, stabilizes NIX-LC3 interactions and promotes mitophagy (Rogov et al, 2017) (Fig 2D). Similar to BNIP3, dimerization of NIX, which is regulated by phosphorylation of its C-terminal region, is important for efficient recruitment of the autophagic machinery to mitochondria (Marinkovic et al, 2020). Accumulation of ROS (triggered by oxidative phosphorylation) promotes NIX-mediated mitophagy via a recruitment of LC3 to mitochondria (Melser et al, 2013). Under conditions of oxidative phosphorylation, Rheb, a small GTPase of the Ras superfamily, translocates to mitochondria and forms a complex with NIX and LC3 to promote mitophagosome formation (Melser et al, 2013). Expression of Rheb in HeLa cells increases mitochondrial respiration, and loss of Rheb decreases the oxygen consumption capacity (Melser et al, 2013). Whether these phenotypes depend on Rheb-induced mitophagy remains to be addressed. BNIP3 has also been shown to bind and inhibit Rheb, which is crucial for mTORC1 activation (Li et al, 2007). As mTORC1 negatively regulates bulk autophagy and mitophagy (Bartolome et al, 2017), BNIP3-dependent mTORC1 inhibition might facilitate mitophagy induction or take part in a positive feedback loop to amplify the initiation signal of mitophagy. Several studies have reported that BNIP3 and NIX act in PINK1/Parkin-mediated mitophagy. NIX is ubiquitylated by Parkin, which in turn promotes targeting of the selective autophagy adaptor NBR1 that binds both ubiquitin and LC3/GABARAP to promote formation of autophagosomes surrounding mitochondria (Gao et al, 2015). In addition, BNIP3 interacts with PINK1 and facilitates accumulation of PINK1 on the OMM, resulting in Parkin translocation to mitochondria (Zhang et al, 2016a). NIX also contributes to CCCP-induced mitochondrial depolarization, and accumulation of Parkin on damaged mitochondria (Ding et al, 2010b). Pathophysiological relevance of BNIP3 and NIX in Parkinson's disease remains unknown. FUNDC1 FUNDC1 is an integral OMM protein that functions as a receptor for hypoxia-induced mitophagy. It contains a typical LIR motif near the N-terminal region and three TM domains (Liu et al, 2012) (Fig 2A). Mutations in the LIR motif disrupt FUNDC1-LC3 interactions and mitophagy induction (Liu et al, 2012). FUNDC1 protein levels are regulated in part by OMM-anchored MARCH5/MITOL (Chen et al, 2017), an E3 ubiquitin ligase that is known to ubiquitylate several proteins acting in mitochondrial dynamics (Yonashiro et al, 2006; Sugiura et al, 2013; Park et al, 2014). FUNDC1 expression is decreased during hypoxia in a ubiquitin–proteasome-dependent manner due to MARCH5-mediated ubiquitylation of FUNDC1 at Lys119 (Chen et al, 2017). Knockdown of endogenous MARCH5 or overexpression of a MARCH5 catalytic mutant impairs ubiquitylation and degradation of FUNDC1, thereby enhancing hypoxia-induced mitophagy (Chen et al, 2017). Similar to Atg32 in yeast cells, FUNDC1 is regulated via phosphorylation and dephosphorylation during mitophagy on residues Ser13 and Tyr18 that are located near the LIR motif. Under normoxia conditions, Ser13 is phosphorylated by CK2, while the Src tyrosine kinase mediates phosphorylation of Tyr18 to negatively regulate FUNDC1-LC3 interactions (Liu et al, 2012; Chen et al, 2014) (Fig 2D). Upon hypoxia, Src becomes inactivated, causing decreased phosphorylation of Tyr18, stabilization of the interaction between FUNDC1 and LC3, and promotion of mitophagosome formation (Liu et al, 2012). The mitochondrial serine/threonine phosphatase PGAM5 dephosphorylates Ser13 and enhances FUNDC1-LC3 interactions to promote mitophagy (Chen et al, 2014). Hypoxia or mitochondrial depolarization induces ULK1 expression and its targeting to mitochondria, leading to FUNDC1 phosphorylation at Ser17 (near the LIR motif) and stabilization of its interaction with LC3 (Wu et al, 2014b). Expression of a FUNDC1 variant defective in ULK1 binding inhibits targeting of ULK1 to mitochondria and mitophagy, suggesting that FUNDC1 also acts as a receptor for ULK1 (Wu et al, 2014b). Under normoxic conditions, BCL2L1/Bcl-xL, an antiapoptotic BH3 domain-containing molecule, binds PGAM5 and inhibits PGAM5-FUNDC1 interactions to prevent dephosphorylation of FUNDC1 Ser13 and mitophagy (Wu et al, 2014a). BCL2L13 Atg32 homologs have so far not been identified in mammalian cells, but findings from yeast reveal that BCL2L13 can induce mitophagy in cells lacking Atg32, raising the possibility that BCL2L13 acts as a mammalian Atg32 functional counterpart (Murakawa et al, 2015). BCL2L13 is an OMM-anchored single-pass membrane protein containing two LIR motifs (Fig 2A). BCL2L13 also regulates mitochondrial morphology and its overexpression induces mitochondrial fragmentation, while its silencing causes mitochondrial elongation (Murakawa et al, 2015). BCL2L13-dependent mitophagy in yeast cells lacking Atg32 is likely mediated via the conventional autophagy machinery as it requires Atg7, a core protein essential for Atg8 lipidation (Murakawa et al, 2015). In addition, mutations in the second LIR motif reduce BCL2L13-dependent mitochondrial degradation in the absence of Atg32, supporting the notion that BCL2L13 promotes mitophagy via Atg8 in yeast (Murakawa et al, 2015). BCL2L13 phosphorylation also seems to contribute to regulation of BCL2L13-LC3 interactions as the mutation at Ser272 near the second LIR motif reduces mitophagy (Murakawa et al, 2015) (Fig 2C). BCL2L13 also interacts with ULK1 to localize the autophagy initiation complex to mitochondria (Murakawa et al, 2019). However, under which physiological conditions BCL2L13 is induced and activated remains to be elucidated. FKBP8 The immunosuppressant drug FK506 (also known as tacrolimus) binds to a conserved family of proteins called FKBP that functions in different cellular processes including transcription, protein folding/trafficking, signaling, and apoptosis (Bonner & Boulianne, 2017). Co-overexpression of FKBP8 and LC3A promotes degradation of depolarized mitochondria in CCCP-treated, Parkin-depleted HeLa cells (Bhujabal et al, 2017). FKBP8 is an integral OMM protein containing a canonical LIR motif near the N-terminus and a TM domain at the C-terminus (Fig 2A). FKBP8 preferentially interacts with LC3A over other Atg8 family proteins in vivo, and this is critical for its mitophagy activity (Bhujabal et al, 2017) (Fig 2C). Moreover, FKBP8 can escape from degradation-prone mitochondria and localizes to the ER via unknown mechanisms (Saita et al, 2013; Bhujabal et al, 2017). Given the complexity due to its versatile functions (Bonner & Boulianne, 2017), further studies are needed to clarify whether endogenous FKBP8 is directly involved in mitophagy. Ubiquitin-mediated mitophagy PINK1 and Parkin Parkinson's disease (PD) is a major neurodegenerative disease characterized by cell death of dopaminergic neurons (Lotharius & Brundin, 2002). PD occurs sporadically in 1–2% of people above 65 years of age but can also arise earlier mostly due to genetic mutations. Common disease phenotypes observed in PD patients are motor symptoms (tremor, bradykinesia, rigidity, and postural instability) that result from dopaminergic neuronal loss in substantia nigra. Non-motor symptoms such as autono