Mol. Cells 2018; 41(1): 35-44
Published online January 23, 2018
https://doi.org/10.14348/molcells.2018.2214
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: tfukuda@med.niigata-u.ac.jp (TF); kanki@med.niigata-u.ac.jp (TK)
Mitochondria are responsible for supplying of most of the cell’s energy via oxidative phosphorylation. However, mitochondria also can be deleterious for a cell because they are the primary source of reactive oxygen species, which are generated as a byproduct of respiration. Accumulation of mitochondrial and cellular oxidative damage leads to diverse pathologies. Thus, it is important to maintain a population of healthy and functional mitochondria for normal cellular metabolism. Eukaryotes have developed defense mechanisms to cope with aberrant mitochondria. Mitochondria autophagy (known as mitophagy) is thought to be one such process that selectively sequesters dysfunctional or excess mitochondria within double-membrane autophagosomes and carries them into lysosomes/vacuoles for degradation. The power of genetics and conservation of fundamental cellular processes among eukaryotes make yeast an excellent model for understanding the general mechanisms, regulation, and function of mitophagy. In budding yeast, a mitochondrial surface protein, Atg32, serves as a mitochondrial receptor for selective autophagy that interacts with Atg11, an adaptor protein for selective types of autophagy, and Atg8, a ubiquitin-like protein localized to the isolation membrane. Atg32 is regulated transcriptionally and post-translationally to control mitophagy. Moreover, because Atg32 is a mitophagy-specific protein, analysis of its deficient mutant enables investigation of the physiological roles of mitophagy. Here, we review recent progress in the understanding of the molecular mechanisms and functional importance of mitophagy in yeast at multiple levels.
Keywords Atg32, autophagy, mitochondria, mitophagy, yeast
Mitochondria, double-membrane-enclosed organelles in eukaryotic cells, are involved in a wide range of cell signaling that controls cell death, immune responses, and calcium homeostasis. Mitochondria also play various roles in numerous metabolic pathways, such as amino acid metabolism, the TCA cycle, oxidative phosphorylation, fatty acid oxidation, and biosynthesis of heme and iron–sulfur clusters. In particular, oxidative phosphorylation in mitochondria produces a large amount of the energy required for cellular activities. In contrast, mitochondria generate reactive oxygen species (ROS) as a byproduct of the respiratory chain, which can cause severe oxidative damage to mitochondrial materials, such as proteins, nucleic acids, and lipids, leading to the production of dysfunctional mitochondria. Mitochondrial dysfunction has been associated with aging and various human diseases, such as neurodegeneration, metabolic disorders, diabetes, and cancer (Wallace, 2005). Thus, eliminating excess or dysfunctional mitochondria is crucial for protecting cells from the potential harm associated with disordered mitochondrial metabolism. One important pathway that contributes to mitochondrial quantity and quality control is the selective removal of mitochondria by autophagy, which is called mitophagy (Lemasters, 2005). In humans, mitophagy deficiency is associated with mitochondrial dysfunction and disorders, such as neuronal degeneration (Youle and Narendra, 2011).
Autophagy is a ubiquitous catabolic process that is highly conserved among eukaryotes (Fig. 1A). Double-membranous structures called isolation membranes emerge at the preautophagosomal structure or phagophore assembly site (PAS), and they extend to sequester cytoplasmic constituents as cargos into double-membrane vesicles called autophagosomes (Wen and Klionsky, 2016). Autophagosomes are then transported and fused to the lysosome/vacuole where the cargos are digested by hydrolytic enzymes for recycling. Studies of autophagy using the budding yeast
Important breakthroughs in the understating of mitophagy in yeast have been provided by identification of genes involved in mitophagy. These have made it possible to study whether mitochondria are selectively delivered to the vacuole, how induction of mitophagy is regulated, and how mitochondria are targeted to the autophagy machinery.
In early studies, mitophagy was detected by electron microscopy as mitochondrial fragments in the vacuole (Kissova et al., 2007; Takeshige et al., 1992). Methods to monitor mitophagy have been developed using GFP-tagged mitochondrial proteins, whose localization to the vacuole, degradation, and release of free GFP indicate the occurrence of mitophagy (Kanki and Klionsky, 2008; Kissova et al., 2004). Mitophagy can be induced by long-term cultivation in respiratory medium that contains a nonfermentable carbon source, such as lactate and glycerol, or by a shift from respiratory medium to nitrogen-free fermentation medium that contains glucose (Kanki and Klionsky, 2008; Kissova et al., 2004; Tal et al., 2007). These methods have revealed that most of the basic Atg proteins are essential for mitophagy (Kanki and Klionsky, 2008; Kanki et al., 2009b; Okamoto et al., 2009). Moreover, mitophagy requires Atg11, Atg20, and At24, which are important for selective autophagy but are dispensable for bulk autophagy (Kanki and Klionsky, 2008; Okamoto et al., 2009), indicating that mitophagy is a selective type of autophagy. Atg11 is a scaffold for assembling the PAS and serves as an adaptor protein for selective autophagy to recruit cargos to the PAS (Kim et al., 2001). The Atg17–Atg29–Atg31 protein complex, another autophagy scaffold specific to bulk autophagy (Kawamata et al., 2008), is dispensable for mitophagy, supporting the notion that mitophagy proceeds as a selective autophagy (Kanki and Klionsky, 2008; Okamoto et al., 2009). A precise role for Atg20 and Atg24 in selective autophagy remains unclear (Nice et al., 2002). Recently, Atg20 and Atg24 have been shown to be involved in autophagic degradation of mitochondria in the fission yeast
Selective autophagy requires specific receptor proteins that tether cargos to the site of autophagosome formation. In yeast, specific receptors have been described for the Cvt pathway (Atg19 and Atg34), pexophagy (Atg30 in the methylotrophic yeast
Expression of Atg32 is induced upon mitophagy induction. When Atg32 is analyzed by immunoblotting, multiple sizes of Atg32 are detected, suggesting that Atg32 undergoes post-translational modifications. Phosphorylation of Atg32 by casein kinase 2 (CK2) is one of the well-studied modifications of Atg32. Modifications other than phosphorylation also have been revealed.
Efficient induction of mitophagy in
The induction of Atg32 is affected by N-terminal acetyltransferase A (NatA), which co-translationally catalyzes acetylation of nascent peptide chains (Eiyama and Okamoto, 2015; Polevoda and Sherman, 2003). The enzymatic activity and ribosomal association of NatA are important for yeast mitophagy (Eiyama and Okamoto, 2015). Transcription of
Under mitophagy-inducing conditions, Atg32 is phosphory-lated at Ser114 and Ser119 (Fig. 1D) (Aoki et al., 2011). Specifically, phosphorylation of Ser114 is crucial for mitophagy and for the Atg32–Atg11 interaction, while phosphorylation at Ser114 and Ser119 are dispensable for Atg32–Atg8 binding (Aoki et al., 2011; Kondo-Okamoto et al., 2012). CK2 was identified as a kinase that catalyzes the phosphorylation of Atg32 at Ser114 and Ser119 (Kanki et al., 2013). CK2-deficient cells exhibit a reduction in Atg32 phosphorylation, Atg32–Atg11 interaction, and mitophagy. Since CK2 is active in nutrient-rich conditions, it remains unclear how CK2-dependent phosphorylation takes place and promotes mitophagy during starvation. In addition to the phosphorylation required for Atg11 binding, Thr119 near the AIM of PpAtg32 in
Although the N-terminal cytosolic region of Atg32 is phosphorylated and important for mitophagy, its C-terminus in the intermembrane space is dispensable for mitophagy (Aoki et al., 2011; Kondo-Okamoto et al., 2012). Atg32 was found to be proteolytically processed at its C-terminal inter-membrane space domain upon induction of mitophagy (Wang et al., 2013). This proteolytic processing is mediated by the inner membrane i-AAA (ATPases associated with various cellular activities) protease Yme1. Blocking the processing by C-terminal tagging of Atg32 or by the
Post-translational modification of Atg32 that increases the molecular weight of Atg32 by approximately 20 kDa has been detected (Levchenko et al., 2016). This modification takes place after mitophagy induction upon starvation or rapamycin treatment, and it is detectable only in the absence of the vacuolar proteinase Pep4, suggesting that the modified form of Atg32 is efficiently degraded in vacuoles. The modification requires the autophagy machinery, including the Atg1–Atg13 complex, Atg8 and its conjugation proteins, and Atg11. A role for this modification remains to be elucidated, but exploring the temporal order and interrelationship among this modification, Yme1-mediated C-terminal processing, and CK2-mediated N-terminal phosphorylation would be an interesting future direction to pursue.
Although Atg32 is induced by mitophagy-inducing conditions, its ectopic expression under nutrient-rich conditions does not cause mitophagy, indicating that other factors are necessary for mitophagy induction. Several factors have been identified, but the molecular mechanisms of mitophagy induction are still unknown.
Treatment of cells with
Accumulation of dysfunctional mitochondria can trigger mitophagy. In mammals, mitophagy can be induced by drugs that impair mitochondrial oxidative phosphorylation, including the uncoupler carbonyl cyanide
In addition to Atg32, multiple factors have been shown to be involved in mitophagy. In most cases, their roles are specifically crucial for mitophagy but not for other types of autophagy, suggesting that mitophagy is facilitated and regulated by these specific mechanisms.
The mitochondrial protein phosphatase homolog Aup1 is required for mitophagy during long-term cultivation in respiration medium (Tal et al., 2007). Aup1 appears to regulate mitophagy through the transcription factor Rtg3 by an unknown mechanism (Journo et al., 2009).
The mitochondrial outer membrane protein Atg33 was identified through a genome-wide screen for mitophagy-deficient mutants (Kanki et al., 2009a). Atg33 facilitates mitophagy, but its role remains unknown. Whi2, which functions in the general stress response and Ras–protein kinase A (PKA) signaling pathway, is required for efficient mitophagy induction by rapamycin treatment (Mendl et al., 2011). In contrast, Whi2 is dispensable for mitophagy induction by nitrogen starvation (Mao et al., 2013). Cells lacking Whi2 exhibit aberrant mitochondrial morphology, mitochondrial dysfunction, and ROS accumulation, raising the possibility that damaged mitochondria are not eliminated effectively (Leadsham et al., 2009). Whi2 might regulate mitophagy through nutrient signaling pathways that involve the stress response transcription factor Msn2 and/or Ras–PKA (Muller and Reichert, 2011).
In mammalian cells, autophagosomes are generated at ER-mitochondria contact sites where lipids were supplied for growth of isolation membrane (Hamasaki et al., 2013). In yeast, ER-mitochondria contact is mediated by the ER-mitochondria encounter structure (ERMES) complex (Klecker et al., 2014). Lack of ERMES subunits causes severe mitophagy defects (Bockler and Westermann, 2014). Artificial tethering between mitochondria and ER in the ERMES mutants suppresses mitophagy defects, indicating that mitophagy is promoted by the contact between mitochondria and the ER
Sphingolipids are involved in the regulation of mitochondrial function. In yeast, Isc1, which generates ceramides from complex sphingolipids, translocates to the mitochondria when respiration is induced (Vaena de Avalos et al., 2004). Yeast cells lacking Isc1 exhibit mitochondrial dysfunction and hyperactivation of mitophagy (Teixeira et al., 2015). Mitophagy deficiency exacerbates the growth defect and shortened chronological lifespan of the
Cardiolipin, a unique dimeric phospholipid, is synthesized and localized in the mitochondrial inner membrane. Yeast cells lacking the cardiolipin synthase Crd1 exhibit a mitophagy defect (Shen et al., 2017). In mammalian neuronal cells, cardiolipin directly regulates mitophagy. Upon mitophagy induction, cardiolipin is redistributed to the outer from the inner mitochondrial membrane (Chu et al., 2013). LC3, a homolog of Atg8, can bind to cardiolipin, and this binding is important for mitophagy.
Maturation of cardiolipin requires the cardiolipin remodeling enzyme, tafazzin (TAZ). Mutations in
The roles of ubiquitination in mitophagy have been revealed, especially in mammals. The E3 ubiquitin ligase Parkin translocates to the mitochondria upon induction of mitophagy and promotes ubiquitination of mitochondrial proteins (Yamano et al., 2016; Youle and Narendra, 2011). Reichert and coworkers developed a method that enables a biochemical high-throughput screen for mitophagy regulators in a genome-wide manner (Muller et al., 2015). They identified 86 positive and 10 negative regulators of mitophagy. Among them, the Ubp3–Bre5 deubiquitination complex, which translocates to mitochondria upon rapamycin-induced starvation, suppresses mitophagy, whereas it promotes other types of selective autophagy, such as ribophagy and the Cvt pathway (Kraft et al., 2008; Muller et al., 2015). Identifying the targets of Upb3–Bre5 and the mechanism of opposing effects of Ubp3–Bre5 on mitophagy and ribophagy/Cvt would be intriguing.
Mitochondrial fusion and fission determine the morphology and size of mitochondria in response to various intra- and extracellular stimuli (Okamoto and Shaw, 2005). As the size of mitochondria is much larger than that of autophagosomes, mitochondrial fission has been thought to facilitate mitophagy by dividing mitochondria into small fragments that can be engulfed by isolation membranes. Indeed, mitophagy is reduced in yeast cells lacking Dnm1, a dynamin-related GTPase that assembles at fission sites and promotes mitochondrial fission (Abeliovich et al., 2013; Kanki et al., 2009a; Mao et al., 2013). Moreover, when mitophagy is induced, Dnm1 is recruited to the sites of the mitochondria that are destined for degradation (Mao et al., 2013). Dnm1 can bind to Atg11, and this binding is required for the recruitment of Dnm1 to degrading mitochondria and efficient mitophagy. It is thus proposed that Atg11 recruits the fission machinery to the sites of mitophagosome formation.
Although mitophagy is reduced to some extent, substantial mitophagy takes place in the absence of the fission machinery (Abeliovich et al., 2013; Bernhardt et al., 2015; Mao et al., 2013; Mendl et al., 2011; Yamashita et al., 2016). Moreover, mitophagy can be induced in mammalian cells by hypoxia or the iron-chelating drug DFP in Drp1 (mammalian homolog of Dnm1)-deficient cells (Yamashita et al., 2016). Mitophagosomes can be detected in both mammalian and yeast cells in the absence of Drp1/Dnm1 (Yamashita et al., 2016). These insights raise the possibility that Dnm1-independent mitochondrial division occurs during mitophagy. Indeed, a small fragment of mammalian mitochondria is divided and released from parental mitochondria during mitophagosome formation. This process is independent of Drp1 and depends on elongation of the isolation membrane. Molecular mechanisms of the Drp1/Dnm1-independent mitochondrial division during mitophagy are of particular interest for further study.
In mammals, mitophagy is thought to be related to physiological events. Recently, the physiological importance of mitophagy in yeast has begun to be clarified. As Atg32 is a mitophagy-specific protein, its deficient mutant can be utilized to investigate the biological significance of mitophagy in yeast.
It has been shown that autophagy-deficient yeast cells accumulate ROS and dysfunctional mitochondria during starvation (Suzuki et al., 2011; Zhang et al., 2007). In
Loss of Atg32 shortens the chronological lifespan of cells grown under a caloric restriction condition (Richard et al., 2013). Under the condition, the
Yeast cells usually carry multiple copies of identical mtDNA (homoplasmy), but cells also can contain mtDNA with different sequences (heteroplasmy) derived from replication error. The fraction of mtDNA variants changes among generations due to competition (Stewart and Chinnery, 2015). Yeast zygotes inherit mtDNA from both gametes by mating, producing heteroplasmic diploids. Mutant mtDNA with large deletions (
The budding yeast
Mitophagy is proposed to be an important target for improving ethanol fermentation. During alcohol brewing, mitophagy is induced (Shiroma et al., 2014). Production of CO2 and ethanol is enhanced in mitophagy-deficient yeast cells that lack Atg32. Interestingly, fermentation is reduced in the
The molecular mechanism of mitophagy in mammals and yeasts is greatly different. The mammalian homolog of yeast Atg32 has not been identified, whereas several functional counterparts of Atg32, such as BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), BNIP3L/Nix, BCL2-like 13 (BCL2L13), and FUN14 domain-containing protein 1 (FUNDC1), have been reported as mitophagy receptors in mammals (Murakawa et al., 2015; Novak et al., 2010; Sandoval et al., 2008; Schweers et al., 2007; Thomas et al., 2011). All of these receptors are integrated into the outer mitochondrial membrane and have LIRs, which can interact with LC3, a mammalian homolog of Atg8. Thus, the interaction between the LIR of mitophagy receptors and LC3 is thought to be an important step in selecting mitochondria as cargo.
BNIP3 and its homolog BNIP3L/NIX are BH3-only proteins and members of the pro-apoptotic BCL2 family. BNIP3 and BNIP3L/NIX have one transmembrane region and an LIR on its N-terminal region exposed to the cytoplasm. BNIP3L/Nix-related mitophagy is reported to be important for the elimination of mitochondria during the maturation of reticulocytes to erythrocytes. Thus, the peripheral blood of BNIP3L/NIX knockout mice has been reported to show decreased mature erythrocytes and increased reticulocytes (Sandoval et al., 2008; Schweers et al., 2007). The interaction between BNIP3 and LC3 is affected by the phosphorylation status of BNIP3. When Ser17 and Ser24 adjacent to the LIR are phosphorylated, the interaction between BNIP3 and LC3 is enhanced; however, the regulation of the phosphorylation of BNIP3 remains unclear (Zhu et al., 2013).
BCL2L13 has one transmembrane region and an LIR on its N-terminal region exposed to the cytoplasm. BCL2L13 was identified as one of the functional counterparts of Atg32 because the exogenous expression of BCL2L13 can partially recover a mitophagy defect in the
FUNDC1 has three transmembrane regions and an LIR on its N-terminal region exposed to the cytoplasm. When cells are cultured in hypoxic conditions or when cellular mitochondria are depolarized under stress conditions, FUNDC1 is dephosphorylated at its Ser13 and can interact with LC3 (Chen et al., 2014; Liu et al., 2012).
PTEN-induced putative kinase 1 (PINK1) and Parkin are causative genes of young onset familial Parkinson’s disease. PINK1 and Parkin accumulate on depolarized mitochondria and induce selective autophagic degradation of mitochondria (Narendra et al., 2008; 2010). PINK1 has a mitochondrial targeting signal (MTS) and is constitutively transported into the mitochondrial inner membrane where mitochondrial processing peptidase (MPP) cleaves MTS from PINK1. The cleaved form of PINK1 is further cleaved by rhomboid protease presenilin-associated rhomboid-like (PARL) and then completely degraded by the ubiquitin proteasome system (Greene et al., 2012; Jin et al., 2010; Yamano and Youle, 2013). When mitochondria are depolarized, PINK1 cannot translocate to the mitochondrial inner membrane and is accumulated on the mitochondrial outer membrane. Then, the accumulated PINK1 recruits Parkin from the cytoplasm to the mitochondria, and the Parkin ubiquitinates mitochondrial proteins. Autophagy adaptor proteins, such as the neighbor of BRCA1 gene 1 (NBR1), optineurin (OPTN), calcium binding and coiled-coil domain 2 (CALCOCO2/NDP52), and TAX1-binding protein 1 (TAX1BP1), have a ubiquitin-binding domain and interact with ubiquitinated mitochondrial proteins (Lazarou et al., 2015). These autophagy adaptor proteins also have LIRs and recruit an isolation membrane via interaction with LC3 for the selective autophagic degradation of the mitochondria.
Although both mitophagy receptor-mediated mitophagy and PINK1/Parkin-mediated mitophagy have been well studied recently, it remains unknown how these two types of mitophagy differ in expression among different tissues or in output of mitochondrial degradation.
In eukaryotic cells, mitophagy degrades excess or dysfunctional mitochondria to ensure a healthy population of the multitasking organelles. Atg32 was identified as a mitophagy receptor in yeast, which interacts with Atg11 and Atg8, to recruit mitochondria to the site of mitophagosome formation. Atg32 is regulated at multiple steps, including transcription, post-translational modifications, and proteolysis. Several mitophagy receptors have been identified and analyzed in mammalian cells. Thus, Atg32 is a good example for understanding the complex regulatory network controlling mitophagy through receptors. Mechanisms that induce mitophagy or that prevent excessive mitophagy have remained obscure. Through the study of nutrient signaling pathways, ubiquitin systems, and lipid metabolisms, it will be revealed how various signaling cascades and molecules positively and negatively regulate mitophagy. Furthermore, dissecting the molecular steps for selective recognition and removal of dysfunctional mitochondria is an important issue that should be addressed.
Using the
Mol. Cells 2018; 41(1): 35-44
Published online January 31, 2018 https://doi.org/10.14348/molcells.2018.2214
Copyright © The Korean Society for Molecular and Cellular Biology.
Tomoyuki Fukuda*, and Tomotake Kanki*
Department of Cellular Physiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan
Correspondence to:*Correspondence: tfukuda@med.niigata-u.ac.jp (TF); kanki@med.niigata-u.ac.jp (TK)
Mitochondria are responsible for supplying of most of the cell’s energy via oxidative phosphorylation. However, mitochondria also can be deleterious for a cell because they are the primary source of reactive oxygen species, which are generated as a byproduct of respiration. Accumulation of mitochondrial and cellular oxidative damage leads to diverse pathologies. Thus, it is important to maintain a population of healthy and functional mitochondria for normal cellular metabolism. Eukaryotes have developed defense mechanisms to cope with aberrant mitochondria. Mitochondria autophagy (known as mitophagy) is thought to be one such process that selectively sequesters dysfunctional or excess mitochondria within double-membrane autophagosomes and carries them into lysosomes/vacuoles for degradation. The power of genetics and conservation of fundamental cellular processes among eukaryotes make yeast an excellent model for understanding the general mechanisms, regulation, and function of mitophagy. In budding yeast, a mitochondrial surface protein, Atg32, serves as a mitochondrial receptor for selective autophagy that interacts with Atg11, an adaptor protein for selective types of autophagy, and Atg8, a ubiquitin-like protein localized to the isolation membrane. Atg32 is regulated transcriptionally and post-translationally to control mitophagy. Moreover, because Atg32 is a mitophagy-specific protein, analysis of its deficient mutant enables investigation of the physiological roles of mitophagy. Here, we review recent progress in the understanding of the molecular mechanisms and functional importance of mitophagy in yeast at multiple levels.
Keywords: Atg32, autophagy, mitochondria, mitophagy, yeast
Mitochondria, double-membrane-enclosed organelles in eukaryotic cells, are involved in a wide range of cell signaling that controls cell death, immune responses, and calcium homeostasis. Mitochondria also play various roles in numerous metabolic pathways, such as amino acid metabolism, the TCA cycle, oxidative phosphorylation, fatty acid oxidation, and biosynthesis of heme and iron–sulfur clusters. In particular, oxidative phosphorylation in mitochondria produces a large amount of the energy required for cellular activities. In contrast, mitochondria generate reactive oxygen species (ROS) as a byproduct of the respiratory chain, which can cause severe oxidative damage to mitochondrial materials, such as proteins, nucleic acids, and lipids, leading to the production of dysfunctional mitochondria. Mitochondrial dysfunction has been associated with aging and various human diseases, such as neurodegeneration, metabolic disorders, diabetes, and cancer (Wallace, 2005). Thus, eliminating excess or dysfunctional mitochondria is crucial for protecting cells from the potential harm associated with disordered mitochondrial metabolism. One important pathway that contributes to mitochondrial quantity and quality control is the selective removal of mitochondria by autophagy, which is called mitophagy (Lemasters, 2005). In humans, mitophagy deficiency is associated with mitochondrial dysfunction and disorders, such as neuronal degeneration (Youle and Narendra, 2011).
Autophagy is a ubiquitous catabolic process that is highly conserved among eukaryotes (Fig. 1A). Double-membranous structures called isolation membranes emerge at the preautophagosomal structure or phagophore assembly site (PAS), and they extend to sequester cytoplasmic constituents as cargos into double-membrane vesicles called autophagosomes (Wen and Klionsky, 2016). Autophagosomes are then transported and fused to the lysosome/vacuole where the cargos are digested by hydrolytic enzymes for recycling. Studies of autophagy using the budding yeast
Important breakthroughs in the understating of mitophagy in yeast have been provided by identification of genes involved in mitophagy. These have made it possible to study whether mitochondria are selectively delivered to the vacuole, how induction of mitophagy is regulated, and how mitochondria are targeted to the autophagy machinery.
In early studies, mitophagy was detected by electron microscopy as mitochondrial fragments in the vacuole (Kissova et al., 2007; Takeshige et al., 1992). Methods to monitor mitophagy have been developed using GFP-tagged mitochondrial proteins, whose localization to the vacuole, degradation, and release of free GFP indicate the occurrence of mitophagy (Kanki and Klionsky, 2008; Kissova et al., 2004). Mitophagy can be induced by long-term cultivation in respiratory medium that contains a nonfermentable carbon source, such as lactate and glycerol, or by a shift from respiratory medium to nitrogen-free fermentation medium that contains glucose (Kanki and Klionsky, 2008; Kissova et al., 2004; Tal et al., 2007). These methods have revealed that most of the basic Atg proteins are essential for mitophagy (Kanki and Klionsky, 2008; Kanki et al., 2009b; Okamoto et al., 2009). Moreover, mitophagy requires Atg11, Atg20, and At24, which are important for selective autophagy but are dispensable for bulk autophagy (Kanki and Klionsky, 2008; Okamoto et al., 2009), indicating that mitophagy is a selective type of autophagy. Atg11 is a scaffold for assembling the PAS and serves as an adaptor protein for selective autophagy to recruit cargos to the PAS (Kim et al., 2001). The Atg17–Atg29–Atg31 protein complex, another autophagy scaffold specific to bulk autophagy (Kawamata et al., 2008), is dispensable for mitophagy, supporting the notion that mitophagy proceeds as a selective autophagy (Kanki and Klionsky, 2008; Okamoto et al., 2009). A precise role for Atg20 and Atg24 in selective autophagy remains unclear (Nice et al., 2002). Recently, Atg20 and Atg24 have been shown to be involved in autophagic degradation of mitochondria in the fission yeast
Selective autophagy requires specific receptor proteins that tether cargos to the site of autophagosome formation. In yeast, specific receptors have been described for the Cvt pathway (Atg19 and Atg34), pexophagy (Atg30 in the methylotrophic yeast
Expression of Atg32 is induced upon mitophagy induction. When Atg32 is analyzed by immunoblotting, multiple sizes of Atg32 are detected, suggesting that Atg32 undergoes post-translational modifications. Phosphorylation of Atg32 by casein kinase 2 (CK2) is one of the well-studied modifications of Atg32. Modifications other than phosphorylation also have been revealed.
Efficient induction of mitophagy in
The induction of Atg32 is affected by N-terminal acetyltransferase A (NatA), which co-translationally catalyzes acetylation of nascent peptide chains (Eiyama and Okamoto, 2015; Polevoda and Sherman, 2003). The enzymatic activity and ribosomal association of NatA are important for yeast mitophagy (Eiyama and Okamoto, 2015). Transcription of
Under mitophagy-inducing conditions, Atg32 is phosphory-lated at Ser114 and Ser119 (Fig. 1D) (Aoki et al., 2011). Specifically, phosphorylation of Ser114 is crucial for mitophagy and for the Atg32–Atg11 interaction, while phosphorylation at Ser114 and Ser119 are dispensable for Atg32–Atg8 binding (Aoki et al., 2011; Kondo-Okamoto et al., 2012). CK2 was identified as a kinase that catalyzes the phosphorylation of Atg32 at Ser114 and Ser119 (Kanki et al., 2013). CK2-deficient cells exhibit a reduction in Atg32 phosphorylation, Atg32–Atg11 interaction, and mitophagy. Since CK2 is active in nutrient-rich conditions, it remains unclear how CK2-dependent phosphorylation takes place and promotes mitophagy during starvation. In addition to the phosphorylation required for Atg11 binding, Thr119 near the AIM of PpAtg32 in
Although the N-terminal cytosolic region of Atg32 is phosphorylated and important for mitophagy, its C-terminus in the intermembrane space is dispensable for mitophagy (Aoki et al., 2011; Kondo-Okamoto et al., 2012). Atg32 was found to be proteolytically processed at its C-terminal inter-membrane space domain upon induction of mitophagy (Wang et al., 2013). This proteolytic processing is mediated by the inner membrane i-AAA (ATPases associated with various cellular activities) protease Yme1. Blocking the processing by C-terminal tagging of Atg32 or by the
Post-translational modification of Atg32 that increases the molecular weight of Atg32 by approximately 20 kDa has been detected (Levchenko et al., 2016). This modification takes place after mitophagy induction upon starvation or rapamycin treatment, and it is detectable only in the absence of the vacuolar proteinase Pep4, suggesting that the modified form of Atg32 is efficiently degraded in vacuoles. The modification requires the autophagy machinery, including the Atg1–Atg13 complex, Atg8 and its conjugation proteins, and Atg11. A role for this modification remains to be elucidated, but exploring the temporal order and interrelationship among this modification, Yme1-mediated C-terminal processing, and CK2-mediated N-terminal phosphorylation would be an interesting future direction to pursue.
Although Atg32 is induced by mitophagy-inducing conditions, its ectopic expression under nutrient-rich conditions does not cause mitophagy, indicating that other factors are necessary for mitophagy induction. Several factors have been identified, but the molecular mechanisms of mitophagy induction are still unknown.
Treatment of cells with
Accumulation of dysfunctional mitochondria can trigger mitophagy. In mammals, mitophagy can be induced by drugs that impair mitochondrial oxidative phosphorylation, including the uncoupler carbonyl cyanide
In addition to Atg32, multiple factors have been shown to be involved in mitophagy. In most cases, their roles are specifically crucial for mitophagy but not for other types of autophagy, suggesting that mitophagy is facilitated and regulated by these specific mechanisms.
The mitochondrial protein phosphatase homolog Aup1 is required for mitophagy during long-term cultivation in respiration medium (Tal et al., 2007). Aup1 appears to regulate mitophagy through the transcription factor Rtg3 by an unknown mechanism (Journo et al., 2009).
The mitochondrial outer membrane protein Atg33 was identified through a genome-wide screen for mitophagy-deficient mutants (Kanki et al., 2009a). Atg33 facilitates mitophagy, but its role remains unknown. Whi2, which functions in the general stress response and Ras–protein kinase A (PKA) signaling pathway, is required for efficient mitophagy induction by rapamycin treatment (Mendl et al., 2011). In contrast, Whi2 is dispensable for mitophagy induction by nitrogen starvation (Mao et al., 2013). Cells lacking Whi2 exhibit aberrant mitochondrial morphology, mitochondrial dysfunction, and ROS accumulation, raising the possibility that damaged mitochondria are not eliminated effectively (Leadsham et al., 2009). Whi2 might regulate mitophagy through nutrient signaling pathways that involve the stress response transcription factor Msn2 and/or Ras–PKA (Muller and Reichert, 2011).
In mammalian cells, autophagosomes are generated at ER-mitochondria contact sites where lipids were supplied for growth of isolation membrane (Hamasaki et al., 2013). In yeast, ER-mitochondria contact is mediated by the ER-mitochondria encounter structure (ERMES) complex (Klecker et al., 2014). Lack of ERMES subunits causes severe mitophagy defects (Bockler and Westermann, 2014). Artificial tethering between mitochondria and ER in the ERMES mutants suppresses mitophagy defects, indicating that mitophagy is promoted by the contact between mitochondria and the ER
Sphingolipids are involved in the regulation of mitochondrial function. In yeast, Isc1, which generates ceramides from complex sphingolipids, translocates to the mitochondria when respiration is induced (Vaena de Avalos et al., 2004). Yeast cells lacking Isc1 exhibit mitochondrial dysfunction and hyperactivation of mitophagy (Teixeira et al., 2015). Mitophagy deficiency exacerbates the growth defect and shortened chronological lifespan of the
Cardiolipin, a unique dimeric phospholipid, is synthesized and localized in the mitochondrial inner membrane. Yeast cells lacking the cardiolipin synthase Crd1 exhibit a mitophagy defect (Shen et al., 2017). In mammalian neuronal cells, cardiolipin directly regulates mitophagy. Upon mitophagy induction, cardiolipin is redistributed to the outer from the inner mitochondrial membrane (Chu et al., 2013). LC3, a homolog of Atg8, can bind to cardiolipin, and this binding is important for mitophagy.
Maturation of cardiolipin requires the cardiolipin remodeling enzyme, tafazzin (TAZ). Mutations in
The roles of ubiquitination in mitophagy have been revealed, especially in mammals. The E3 ubiquitin ligase Parkin translocates to the mitochondria upon induction of mitophagy and promotes ubiquitination of mitochondrial proteins (Yamano et al., 2016; Youle and Narendra, 2011). Reichert and coworkers developed a method that enables a biochemical high-throughput screen for mitophagy regulators in a genome-wide manner (Muller et al., 2015). They identified 86 positive and 10 negative regulators of mitophagy. Among them, the Ubp3–Bre5 deubiquitination complex, which translocates to mitochondria upon rapamycin-induced starvation, suppresses mitophagy, whereas it promotes other types of selective autophagy, such as ribophagy and the Cvt pathway (Kraft et al., 2008; Muller et al., 2015). Identifying the targets of Upb3–Bre5 and the mechanism of opposing effects of Ubp3–Bre5 on mitophagy and ribophagy/Cvt would be intriguing.
Mitochondrial fusion and fission determine the morphology and size of mitochondria in response to various intra- and extracellular stimuli (Okamoto and Shaw, 2005). As the size of mitochondria is much larger than that of autophagosomes, mitochondrial fission has been thought to facilitate mitophagy by dividing mitochondria into small fragments that can be engulfed by isolation membranes. Indeed, mitophagy is reduced in yeast cells lacking Dnm1, a dynamin-related GTPase that assembles at fission sites and promotes mitochondrial fission (Abeliovich et al., 2013; Kanki et al., 2009a; Mao et al., 2013). Moreover, when mitophagy is induced, Dnm1 is recruited to the sites of the mitochondria that are destined for degradation (Mao et al., 2013). Dnm1 can bind to Atg11, and this binding is required for the recruitment of Dnm1 to degrading mitochondria and efficient mitophagy. It is thus proposed that Atg11 recruits the fission machinery to the sites of mitophagosome formation.
Although mitophagy is reduced to some extent, substantial mitophagy takes place in the absence of the fission machinery (Abeliovich et al., 2013; Bernhardt et al., 2015; Mao et al., 2013; Mendl et al., 2011; Yamashita et al., 2016). Moreover, mitophagy can be induced in mammalian cells by hypoxia or the iron-chelating drug DFP in Drp1 (mammalian homolog of Dnm1)-deficient cells (Yamashita et al., 2016). Mitophagosomes can be detected in both mammalian and yeast cells in the absence of Drp1/Dnm1 (Yamashita et al., 2016). These insights raise the possibility that Dnm1-independent mitochondrial division occurs during mitophagy. Indeed, a small fragment of mammalian mitochondria is divided and released from parental mitochondria during mitophagosome formation. This process is independent of Drp1 and depends on elongation of the isolation membrane. Molecular mechanisms of the Drp1/Dnm1-independent mitochondrial division during mitophagy are of particular interest for further study.
In mammals, mitophagy is thought to be related to physiological events. Recently, the physiological importance of mitophagy in yeast has begun to be clarified. As Atg32 is a mitophagy-specific protein, its deficient mutant can be utilized to investigate the biological significance of mitophagy in yeast.
It has been shown that autophagy-deficient yeast cells accumulate ROS and dysfunctional mitochondria during starvation (Suzuki et al., 2011; Zhang et al., 2007). In
Loss of Atg32 shortens the chronological lifespan of cells grown under a caloric restriction condition (Richard et al., 2013). Under the condition, the
Yeast cells usually carry multiple copies of identical mtDNA (homoplasmy), but cells also can contain mtDNA with different sequences (heteroplasmy) derived from replication error. The fraction of mtDNA variants changes among generations due to competition (Stewart and Chinnery, 2015). Yeast zygotes inherit mtDNA from both gametes by mating, producing heteroplasmic diploids. Mutant mtDNA with large deletions (
The budding yeast
Mitophagy is proposed to be an important target for improving ethanol fermentation. During alcohol brewing, mitophagy is induced (Shiroma et al., 2014). Production of CO2 and ethanol is enhanced in mitophagy-deficient yeast cells that lack Atg32. Interestingly, fermentation is reduced in the
The molecular mechanism of mitophagy in mammals and yeasts is greatly different. The mammalian homolog of yeast Atg32 has not been identified, whereas several functional counterparts of Atg32, such as BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), BNIP3L/Nix, BCL2-like 13 (BCL2L13), and FUN14 domain-containing protein 1 (FUNDC1), have been reported as mitophagy receptors in mammals (Murakawa et al., 2015; Novak et al., 2010; Sandoval et al., 2008; Schweers et al., 2007; Thomas et al., 2011). All of these receptors are integrated into the outer mitochondrial membrane and have LIRs, which can interact with LC3, a mammalian homolog of Atg8. Thus, the interaction between the LIR of mitophagy receptors and LC3 is thought to be an important step in selecting mitochondria as cargo.
BNIP3 and its homolog BNIP3L/NIX are BH3-only proteins and members of the pro-apoptotic BCL2 family. BNIP3 and BNIP3L/NIX have one transmembrane region and an LIR on its N-terminal region exposed to the cytoplasm. BNIP3L/Nix-related mitophagy is reported to be important for the elimination of mitochondria during the maturation of reticulocytes to erythrocytes. Thus, the peripheral blood of BNIP3L/NIX knockout mice has been reported to show decreased mature erythrocytes and increased reticulocytes (Sandoval et al., 2008; Schweers et al., 2007). The interaction between BNIP3 and LC3 is affected by the phosphorylation status of BNIP3. When Ser17 and Ser24 adjacent to the LIR are phosphorylated, the interaction between BNIP3 and LC3 is enhanced; however, the regulation of the phosphorylation of BNIP3 remains unclear (Zhu et al., 2013).
BCL2L13 has one transmembrane region and an LIR on its N-terminal region exposed to the cytoplasm. BCL2L13 was identified as one of the functional counterparts of Atg32 because the exogenous expression of BCL2L13 can partially recover a mitophagy defect in the
FUNDC1 has three transmembrane regions and an LIR on its N-terminal region exposed to the cytoplasm. When cells are cultured in hypoxic conditions or when cellular mitochondria are depolarized under stress conditions, FUNDC1 is dephosphorylated at its Ser13 and can interact with LC3 (Chen et al., 2014; Liu et al., 2012).
PTEN-induced putative kinase 1 (PINK1) and Parkin are causative genes of young onset familial Parkinson’s disease. PINK1 and Parkin accumulate on depolarized mitochondria and induce selective autophagic degradation of mitochondria (Narendra et al., 2008; 2010). PINK1 has a mitochondrial targeting signal (MTS) and is constitutively transported into the mitochondrial inner membrane where mitochondrial processing peptidase (MPP) cleaves MTS from PINK1. The cleaved form of PINK1 is further cleaved by rhomboid protease presenilin-associated rhomboid-like (PARL) and then completely degraded by the ubiquitin proteasome system (Greene et al., 2012; Jin et al., 2010; Yamano and Youle, 2013). When mitochondria are depolarized, PINK1 cannot translocate to the mitochondrial inner membrane and is accumulated on the mitochondrial outer membrane. Then, the accumulated PINK1 recruits Parkin from the cytoplasm to the mitochondria, and the Parkin ubiquitinates mitochondrial proteins. Autophagy adaptor proteins, such as the neighbor of BRCA1 gene 1 (NBR1), optineurin (OPTN), calcium binding and coiled-coil domain 2 (CALCOCO2/NDP52), and TAX1-binding protein 1 (TAX1BP1), have a ubiquitin-binding domain and interact with ubiquitinated mitochondrial proteins (Lazarou et al., 2015). These autophagy adaptor proteins also have LIRs and recruit an isolation membrane via interaction with LC3 for the selective autophagic degradation of the mitochondria.
Although both mitophagy receptor-mediated mitophagy and PINK1/Parkin-mediated mitophagy have been well studied recently, it remains unknown how these two types of mitophagy differ in expression among different tissues or in output of mitochondrial degradation.
In eukaryotic cells, mitophagy degrades excess or dysfunctional mitochondria to ensure a healthy population of the multitasking organelles. Atg32 was identified as a mitophagy receptor in yeast, which interacts with Atg11 and Atg8, to recruit mitochondria to the site of mitophagosome formation. Atg32 is regulated at multiple steps, including transcription, post-translational modifications, and proteolysis. Several mitophagy receptors have been identified and analyzed in mammalian cells. Thus, Atg32 is a good example for understanding the complex regulatory network controlling mitophagy through receptors. Mechanisms that induce mitophagy or that prevent excessive mitophagy have remained obscure. Through the study of nutrient signaling pathways, ubiquitin systems, and lipid metabolisms, it will be revealed how various signaling cascades and molecules positively and negatively regulate mitophagy. Furthermore, dissecting the molecular steps for selective recognition and removal of dysfunctional mitochondria is an important issue that should be addressed.
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