The International Journal of Biochemistry & Cell Biology 50 (2014) 64–66
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Organelles in focus
Alternative macroautophagy and mitophagy Shigeomi Shimizu∗ , Shinya Honda, Satoko Arakawa, Hirofumi Yamaguchi Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
a r t i c l e
i n f o
Article history: Received 8 January 2014 Accepted 16 February 2014 Available online 22 February 2014 Keywords: Mitophagy Atg5-independent macroautophagy Erythrocyte maturation Parkin Selective autophagy
a b s t r a c t Mitophagy is a mitochondrial quality control mechanism where damaged and surplus mitochondria are removed by macroautophagy. Mitophagy is associated with various physiological and pathological events such as mitochondrial clearance during terminal differentiation of reticulocytes. There are two different mammalian macroautophagy pathways: the Atg5-dependent conventional pathway and an Atg5-independent alternative pathway; the latter is involved in the erythrocyte mitophagy. © 2014 Elsevier Ltd. All rights reserved.
Facts about organelles • Mammalian macroautophagy can occur via at least two different pathways, which are an Atg5-dependent conventional pathway and an Atg5-independent alternative pathway. • Ulk1-dependent Atg5-independent alternative macroautophagy is the dominant force for elimination of mitochondria from reticulocytes.
was previously considered to be a bulk and non-selective process. But, growing lines of evidence indicate the presence of cargospecific autophagy (selective autophagy) that eliminate specific organelles (Komatsu and Ichimura, 2010; Ding and Yin, 2012), including peroxisomes (pexophagy), pathogens (xenophagy), and mitochondria (mitophagy). In this review, we focus on the mitochondrial autophagy that associates with terminal differentiation of reticulocytes.
1.1. Conventional type of mammalian macroautophagy 1. Introduction Macroautophagy is a catabolic process where cellular contents, including proteins, lipids, and even entire organelles, are digested within lysosomes. The process of macroautophagy begins with the formation and elongation of isolation membranes. These membranes invaginate enclosing various intracellular components inside, resulting in the formation of the double-membrane vesicles called autophagosomes. Subsequently, autophagosomes fuse with lysosomes to generate autolysosomes allowing the degradation of the autophagosomal contents. Macroautophagy occurs constitutively at low levels but is accelerated by various cellular stressors such as DNA or organelle damage and nutrient starvation (Xie and Klionsky, 2007; Mizushima et al., 2008). Macroautophagy
∗ Corresponding author. Tel.: +81 3 5803 4692; fax: +81 3 5803 4821. E-mail address: [email protected] (S. Shimizu). http://dx.doi.org/10.1016/j.biocel.2014.02.016 1357-2725/© 2014 Elsevier Ltd. All rights reserved.
The molecular basis of autophagy was first studied in autophagy-defective mutant yeast (Nakatogawa et al., 2009). Subsequent identification of vertebrate homologs to yeast autophagy proteins has greatly expanded our understanding of the molecular mechanisms of autophagy. It is currently accepted that autophagy is driven by more than 30 autophagy related proteins (Atgs) that are well conserved from yeasts to mammals (Mizushima et al., 2011). Atg1 was the first such protein identified, and it possesses intrinsic serine/threonine kinase activity essential for the initiation of autophagy (Kabeya et al., 2005). Autophagy is regulated by phosphatidylinositol 3-kinase (PI3K) type I and type III. PI3K Type I is activated by growth factors such as insulin, and its activation inhibits autophagy through the regulation of mammalian target of rapamycin (mTOR). Conversely, PI3K type III, which exists in a multiprotein complex including Atg6 (Beclin1), promotes invagination of the membrane at phosphatidylinositol-3-phosphate (PI3P)-rich domains called omegasomes, to generate isolation membranes (Axe et al., 2008). Subsequent expansion and closure of isolation
S. Shimizu et al. / The International Journal of Biochemistry & Cell Biology 50 (2014) 64–66
membranes are mediated by two ubiquitin-like conjugation pathways: the Atg5–Atg12 pathway and the microtubule-associated protein 1 light chain 3 (LC3) pathway (Mizushima et al., 2011). Conjugation of phosphatidylethanolamine (PE) to LC3 facilitates translocation of LC3 from cytosol to the isolation membrane, and this translocation makes this complex a reliable marker of autophagy. The Atg5–Atg12 pathway is essential for macroautophagy because autophagy is largely suppressed in Atg5-deficient cells. 1.2. Discovery of alternative type of mammalian macroautophagy Despite the crucial role of Atg5 in autophagy, Atg5-deficient mouse embryos develop normally until the perinatal period (Kuma et al., 2004), suggesting that an alternative autophagic pathway may exist in such embryos. To study Atg5-independent macroautophagy, we added various drugs into Atg5-deficient mouse embryonic fibroblasts (MEFs) and performed ultrastructural analysis. By adding etoposide, a DNA damaging reagent, we observed typical autophagic structures in Atg5-deficient MEFs (Nishida et al., 2009). The morphology of Atg5-independent autophagic structures was indistinguishable from that of starvation-induced Atg5-dependent autophagic structures. All the stages of autophagosome biogenesis were observed in Atg5-deficient MEFs, including the formation of the isolation membrane (a membrane cisternae curving around part of the cytoplasm), the autophagosome (double-membrane vacuoles generated by sealing of the edges of the isolation membrane), and the autolysosome (single-membrane vacuoles generated by the fusion between the autophagosome and the lysosome) (Nishida et al., 2009). The presence of autolysosomes in Atg5-deficient MEFs was confirmed by immunostaining of the lysosome-associated membrane protein 2 (Lamp2). In general, lysosomes are spread diffusely throughout the cytosol so that Lamp2 immunostaining appears as small well-separated puncta. However, during autophagy, lysosomes fuse with the autophagic vacuoles, thereby increasing the number of large-sized Lamp2 fluorescent puncta. Using these assays, we identified several larger Lamp2 fluorescent puncta in etoposide-treated Atg5-deficient MEFs. The number and size of large Lamp2 fluorescent puncta were equivalent in wild-type and Atg5-deficient MEFs. Thus, mammalian cells possess at least two different autophagic pathways, the Atg5-dependent conventional pathway and an Atg5-independent alternative pathway (alternative macroautophagy, Nishida et al., 2009). We further analyzedWe further analyzed the molecular mechanism of this “alternative macroautophagy” in detail. Gene expression profiles and gene silencing experiments in etoposidetreated and -untreated Atg5-deficient cells show that alternative macroautophagy requires Unc51-like kinase 1 (Ulk1) and the phosphoinositide 3-kinase complexes, both of which act at the initiation steps of conventional macroautophagy. In contrast, other components such as Atg9 and the proteins in the ubiquitin-like protein conjugation system (Atg5, Atg7, and LC3), whose functions lie in the extension of autophagic membranes, are not involved (Fig. 1). Because alternative macroautophagy requires the extension of autophagic membranes, several unidentified molecules should serve these functions. 1.3. Biological roles of mitophagy in mammals Until now, at least two types of mitophagy have been studied in mammals. One is the Parkinson’s disease-associated mitophagy (Narendra et al., 2008) and the other is the mitophagy occurring during erythrocyte maturation (Kent et al., 1966). Parkinson’s disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons.
65
Among the genes associated with familial Parkinson’s disease, Parkin (PARK2) and Pink1 (PARK6) regulate mitophagy for the degradation of damaged mitochondria (Jin et al., 2010). This mechanism is described as follows: first, after mitochondrial depolarization, Pink1 accumulates on the outer mitochondrial membrane. Next, Parkin associates with Pink1 on the outer mitochondrial membrane. Then, the depolarized mitochondria are ubiquitinated by the ubiquitin ligase activity of Parkin. Finally, the ubiquitinated mitochondria are recognized by autophagic molecules and digested by macroautophagy. Thus, genetic mutations of Parkin and Pink1 may cause defects in mitophagy, leading to accumulation of damaged mitochondria, followed by the degeneration of dopaminergic neurons. Because Parkin-mediated mitophagy is largely suppressed by the lack of Atg5, this autophagy should be the Atg5-dependent conventional type (Narendra et al., 2008). 1.4. Involvement of alternative macroautophagy in mitophagy during erythrocyte maturation The representative mitophagy is also observed during erythrocyte maturation. In the terminal stage of erythrocyte maturation, the erythroblasts lose their nuclei to become reticulocytes and reticulocytes are transformed into erythrocytes by elimination of organelles including the mitochondria (Fig. 2A). Because ultrastructural studies have detected autophagic structures engulfing mitochondria, mitochondrial clearance occurs by mitophagy (Kent et al., 1966). In fact, we have observed that mitochondria were engulfed and digested by autophagic vacuoles in wild-type reticulocytes, and surprisingly, an equivalent amount of mitophagy was observed even in Atg5-deficinet reticulocytes (Fig. 2B) (Nishida et al., 2009). Consistently, the number of persisting mitochondria in Atg5-deficinet reticulocytes and erythrocytes was the same as in wild-type cells of each type (Fig. 2C). These data indicated that the Atg5-independent alternative macroautophagy, but not the Atg5-dependent conventional macroautophagy, participates in the elimination of mitochondria by mitophagy. Unlike Atg5, Ulk1 is crucial for mitochondrial clearance in reticulocytes as judged from the failure of mitophagy in Ulk1-deficient mice (Kundu et al., 2008). Because the contribution of Atg5 is small, and Ulk1 initiates both the conventional and alternative macroautophagy, it is likely that the
Fig. 1. Hypothetical model of macroautophagy. There are at least two modes of macroautophagy, i.e. conventional and alternative macroautophagy. Conventional macroautophagy depends on Atg5 and Atg7, is associated with LC3 modification and may originate from ER-mitochondria contact membrane. In contrast, alternative macroautophagy occurs independent of Atg5 or Atg7 expression and LC3 modification. The generation of autophagic vacuoles in this type of macroautophagy may originate from Golgi membrane and late endosomes (LE) in a Rab9-dependent manner. Although both these processes lead to bulk degradation of damaged proteins or organelles by generating autolysosomes, they seem to be activated by different stimuli, in different cell types and have different physiological roles.
66
S. Shimizu et al. / The International Journal of Biochemistry & Cell Biology 50 (2014) 64–66
Fig. 2. Involvement of alternative macroautophagy in mitophagy during erythrocyte maturation. (A) Final stage of red blood cell maturation. During erythrocyte maturation, erythroblasts lose their nuclei to become reticulocytes and reticulocytes are transformed into erythrocytes by elimination of mitochondria. Macroautophagy is involved in the latter process. (B) Electron micrographs of Atg5-deficient erythrocytes. Mitophagy can be observed in the Atg5-deficient erythrocyte. Arrowhead indicates the isolation membrane-autophagosomal structure. Arrow indicates the autophagosome containing mitochondria (*). Mt: non-engulfed mitochondria. G: Golgi-deribed membranes. (C) The number of remained mitochondria per cell and autophagic vacuoles per cell in wild-type and Atg5-deficient erythrocytes are indicated. Lines indicate the mean and SD.
Ulk1-dependent Atg5-independent alternative macroautophagy is the dominant force for mitophagy in reticulocytesFigs. 1 and 2. Mice with organ-specific Atg5 and Atg7 deficiency show various abnormalities, indicating that conventional macroautophagy is crucial for various biological events in many organs. The question is then, why does alternative macroautophagy, but not conventional macroautophagy, have an essential role in erythrocyte maturation? It is considered that the machineries for conventional and alternative macroautophagy are used in a cell type- and stimulusdependent manner. Further studies will be required to better define the relationship between the conventional and alternative macroautophagy. 2. Closing remark In this review, we describe two distinct autophagic pathways, conventional and alternative. The presence of at least two mechanistically distinct forms of autophagy in mammalian cells underscores autophagy as a highly adaptable cellular stress response. Further elucidation of the biological roles of autophagy will require a more complete understanding of (1) the molecular mechanisms of alternative autophagy, (2) the unique functional roles of these two pathways in vivo, and (3) the contribution of each pathway to pathology. Acknowledgements This review is written by the support in part by the Grant-inAid for Scientific Research on Innovative Areas, Grant-in-Aid for Scientific Research (S), Grant-in-Aid for challenging Exploratory Research from the MEXT of Japan, and a grant for the Program
for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO). References Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 2008;182:685–701. Ding WX, Yin XM. Mitophagy: mechanisms, pathophysiological roles, and analysis. Biol Chem 2012;393:547–64. Jin SM, Lazarou M, Wang C, Kane LA, Narendra DP, Youle RJ. Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL. J Cell Biol 2010;191:933–42. Kabeya Y, Kamada Y, Baba M, Takikawa H, Sasaki M, Ohsumi Y. Atg17 functions in cooperation with Atg1 and Atg13 in yeast autophagy. Mol Biol Cell 2005;16:2544–53. Kent G, Minick OT, Volini FI, Orfei E. Autophagic vacuoles in human red cells. Am J Pathol 1966;48:831–57. Komatsu M, Ichimura Y. Selective autophagy regulates various cellular functions. Genes Cells 2010;15:923–33. Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, et al. The role of autophagy during the early neonatal starvation period. Nature 2004;432:1032–6. Kundu M, Lindsten T, Yang CY, Wu J, Zhao F, Zhang J, et al. Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation. Blood 2008;112:1493–502. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature 2008;451:1069–75. Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 2011;27:107–32. Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat Rev Mol Cell Biol 2009;10:458–67. Narendra D, Tanaka A, Suen DF, Youle RJ. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol 2008;183:795–803. Nishida Y, Arakawa S, Fujitani K, Yamaguchi H, Mizuta T, et al. Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 2009;461:654–8. Xie Z, Klionsky DJ. Autophagosome formation: core machinery and adaptations. Nat Cell Biol 2007;9:1102–9.
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Organelles in focus
Alternative macroautophagy and mitophagy Shigeomi Shimizu∗ , Shinya Honda, Satoko Arakawa, Hirofumi Yamaguchi Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
a r t i c l e
i n f o
Article history: Received 8 January 2014 Accepted 16 February 2014 Available online 22 February 2014 Keywords: Mitophagy Atg5-independent macroautophagy Erythrocyte maturation Parkin Selective autophagy
a b s t r a c t Mitophagy is a mitochondrial quality control mechanism where damaged and surplus mitochondria are removed by macroautophagy. Mitophagy is associated with various physiological and pathological events such as mitochondrial clearance during terminal differentiation of reticulocytes. There are two different mammalian macroautophagy pathways: the Atg5-dependent conventional pathway and an Atg5-independent alternative pathway; the latter is involved in the erythrocyte mitophagy. © 2014 Elsevier Ltd. All rights reserved.
Facts about organelles • Mammalian macroautophagy can occur via at least two different pathways, which are an Atg5-dependent conventional pathway and an Atg5-independent alternative pathway. • Ulk1-dependent Atg5-independent alternative macroautophagy is the dominant force for elimination of mitochondria from reticulocytes.
was previously considered to be a bulk and non-selective process. But, growing lines of evidence indicate the presence of cargospecific autophagy (selective autophagy) that eliminate specific organelles (Komatsu and Ichimura, 2010; Ding and Yin, 2012), including peroxisomes (pexophagy), pathogens (xenophagy), and mitochondria (mitophagy). In this review, we focus on the mitochondrial autophagy that associates with terminal differentiation of reticulocytes.
1.1. Conventional type of mammalian macroautophagy 1. Introduction Macroautophagy is a catabolic process where cellular contents, including proteins, lipids, and even entire organelles, are digested within lysosomes. The process of macroautophagy begins with the formation and elongation of isolation membranes. These membranes invaginate enclosing various intracellular components inside, resulting in the formation of the double-membrane vesicles called autophagosomes. Subsequently, autophagosomes fuse with lysosomes to generate autolysosomes allowing the degradation of the autophagosomal contents. Macroautophagy occurs constitutively at low levels but is accelerated by various cellular stressors such as DNA or organelle damage and nutrient starvation (Xie and Klionsky, 2007; Mizushima et al., 2008). Macroautophagy
∗ Corresponding author. Tel.: +81 3 5803 4692; fax: +81 3 5803 4821. E-mail address: [email protected] (S. Shimizu). http://dx.doi.org/10.1016/j.biocel.2014.02.016 1357-2725/© 2014 Elsevier Ltd. All rights reserved.
The molecular basis of autophagy was first studied in autophagy-defective mutant yeast (Nakatogawa et al., 2009). Subsequent identification of vertebrate homologs to yeast autophagy proteins has greatly expanded our understanding of the molecular mechanisms of autophagy. It is currently accepted that autophagy is driven by more than 30 autophagy related proteins (Atgs) that are well conserved from yeasts to mammals (Mizushima et al., 2011). Atg1 was the first such protein identified, and it possesses intrinsic serine/threonine kinase activity essential for the initiation of autophagy (Kabeya et al., 2005). Autophagy is regulated by phosphatidylinositol 3-kinase (PI3K) type I and type III. PI3K Type I is activated by growth factors such as insulin, and its activation inhibits autophagy through the regulation of mammalian target of rapamycin (mTOR). Conversely, PI3K type III, which exists in a multiprotein complex including Atg6 (Beclin1), promotes invagination of the membrane at phosphatidylinositol-3-phosphate (PI3P)-rich domains called omegasomes, to generate isolation membranes (Axe et al., 2008). Subsequent expansion and closure of isolation
S. Shimizu et al. / The International Journal of Biochemistry & Cell Biology 50 (2014) 64–66
membranes are mediated by two ubiquitin-like conjugation pathways: the Atg5–Atg12 pathway and the microtubule-associated protein 1 light chain 3 (LC3) pathway (Mizushima et al., 2011). Conjugation of phosphatidylethanolamine (PE) to LC3 facilitates translocation of LC3 from cytosol to the isolation membrane, and this translocation makes this complex a reliable marker of autophagy. The Atg5–Atg12 pathway is essential for macroautophagy because autophagy is largely suppressed in Atg5-deficient cells. 1.2. Discovery of alternative type of mammalian macroautophagy Despite the crucial role of Atg5 in autophagy, Atg5-deficient mouse embryos develop normally until the perinatal period (Kuma et al., 2004), suggesting that an alternative autophagic pathway may exist in such embryos. To study Atg5-independent macroautophagy, we added various drugs into Atg5-deficient mouse embryonic fibroblasts (MEFs) and performed ultrastructural analysis. By adding etoposide, a DNA damaging reagent, we observed typical autophagic structures in Atg5-deficient MEFs (Nishida et al., 2009). The morphology of Atg5-independent autophagic structures was indistinguishable from that of starvation-induced Atg5-dependent autophagic structures. All the stages of autophagosome biogenesis were observed in Atg5-deficient MEFs, including the formation of the isolation membrane (a membrane cisternae curving around part of the cytoplasm), the autophagosome (double-membrane vacuoles generated by sealing of the edges of the isolation membrane), and the autolysosome (single-membrane vacuoles generated by the fusion between the autophagosome and the lysosome) (Nishida et al., 2009). The presence of autolysosomes in Atg5-deficient MEFs was confirmed by immunostaining of the lysosome-associated membrane protein 2 (Lamp2). In general, lysosomes are spread diffusely throughout the cytosol so that Lamp2 immunostaining appears as small well-separated puncta. However, during autophagy, lysosomes fuse with the autophagic vacuoles, thereby increasing the number of large-sized Lamp2 fluorescent puncta. Using these assays, we identified several larger Lamp2 fluorescent puncta in etoposide-treated Atg5-deficient MEFs. The number and size of large Lamp2 fluorescent puncta were equivalent in wild-type and Atg5-deficient MEFs. Thus, mammalian cells possess at least two different autophagic pathways, the Atg5-dependent conventional pathway and an Atg5-independent alternative pathway (alternative macroautophagy, Nishida et al., 2009). We further analyzedWe further analyzed the molecular mechanism of this “alternative macroautophagy” in detail. Gene expression profiles and gene silencing experiments in etoposidetreated and -untreated Atg5-deficient cells show that alternative macroautophagy requires Unc51-like kinase 1 (Ulk1) and the phosphoinositide 3-kinase complexes, both of which act at the initiation steps of conventional macroautophagy. In contrast, other components such as Atg9 and the proteins in the ubiquitin-like protein conjugation system (Atg5, Atg7, and LC3), whose functions lie in the extension of autophagic membranes, are not involved (Fig. 1). Because alternative macroautophagy requires the extension of autophagic membranes, several unidentified molecules should serve these functions. 1.3. Biological roles of mitophagy in mammals Until now, at least two types of mitophagy have been studied in mammals. One is the Parkinson’s disease-associated mitophagy (Narendra et al., 2008) and the other is the mitophagy occurring during erythrocyte maturation (Kent et al., 1966). Parkinson’s disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons.
65
Among the genes associated with familial Parkinson’s disease, Parkin (PARK2) and Pink1 (PARK6) regulate mitophagy for the degradation of damaged mitochondria (Jin et al., 2010). This mechanism is described as follows: first, after mitochondrial depolarization, Pink1 accumulates on the outer mitochondrial membrane. Next, Parkin associates with Pink1 on the outer mitochondrial membrane. Then, the depolarized mitochondria are ubiquitinated by the ubiquitin ligase activity of Parkin. Finally, the ubiquitinated mitochondria are recognized by autophagic molecules and digested by macroautophagy. Thus, genetic mutations of Parkin and Pink1 may cause defects in mitophagy, leading to accumulation of damaged mitochondria, followed by the degeneration of dopaminergic neurons. Because Parkin-mediated mitophagy is largely suppressed by the lack of Atg5, this autophagy should be the Atg5-dependent conventional type (Narendra et al., 2008). 1.4. Involvement of alternative macroautophagy in mitophagy during erythrocyte maturation The representative mitophagy is also observed during erythrocyte maturation. In the terminal stage of erythrocyte maturation, the erythroblasts lose their nuclei to become reticulocytes and reticulocytes are transformed into erythrocytes by elimination of organelles including the mitochondria (Fig. 2A). Because ultrastructural studies have detected autophagic structures engulfing mitochondria, mitochondrial clearance occurs by mitophagy (Kent et al., 1966). In fact, we have observed that mitochondria were engulfed and digested by autophagic vacuoles in wild-type reticulocytes, and surprisingly, an equivalent amount of mitophagy was observed even in Atg5-deficinet reticulocytes (Fig. 2B) (Nishida et al., 2009). Consistently, the number of persisting mitochondria in Atg5-deficinet reticulocytes and erythrocytes was the same as in wild-type cells of each type (Fig. 2C). These data indicated that the Atg5-independent alternative macroautophagy, but not the Atg5-dependent conventional macroautophagy, participates in the elimination of mitochondria by mitophagy. Unlike Atg5, Ulk1 is crucial for mitochondrial clearance in reticulocytes as judged from the failure of mitophagy in Ulk1-deficient mice (Kundu et al., 2008). Because the contribution of Atg5 is small, and Ulk1 initiates both the conventional and alternative macroautophagy, it is likely that the
Fig. 1. Hypothetical model of macroautophagy. There are at least two modes of macroautophagy, i.e. conventional and alternative macroautophagy. Conventional macroautophagy depends on Atg5 and Atg7, is associated with LC3 modification and may originate from ER-mitochondria contact membrane. In contrast, alternative macroautophagy occurs independent of Atg5 or Atg7 expression and LC3 modification. The generation of autophagic vacuoles in this type of macroautophagy may originate from Golgi membrane and late endosomes (LE) in a Rab9-dependent manner. Although both these processes lead to bulk degradation of damaged proteins or organelles by generating autolysosomes, they seem to be activated by different stimuli, in different cell types and have different physiological roles.
66
S. Shimizu et al. / The International Journal of Biochemistry & Cell Biology 50 (2014) 64–66
Fig. 2. Involvement of alternative macroautophagy in mitophagy during erythrocyte maturation. (A) Final stage of red blood cell maturation. During erythrocyte maturation, erythroblasts lose their nuclei to become reticulocytes and reticulocytes are transformed into erythrocytes by elimination of mitochondria. Macroautophagy is involved in the latter process. (B) Electron micrographs of Atg5-deficient erythrocytes. Mitophagy can be observed in the Atg5-deficient erythrocyte. Arrowhead indicates the isolation membrane-autophagosomal structure. Arrow indicates the autophagosome containing mitochondria (*). Mt: non-engulfed mitochondria. G: Golgi-deribed membranes. (C) The number of remained mitochondria per cell and autophagic vacuoles per cell in wild-type and Atg5-deficient erythrocytes are indicated. Lines indicate the mean and SD.
Ulk1-dependent Atg5-independent alternative macroautophagy is the dominant force for mitophagy in reticulocytesFigs. 1 and 2. Mice with organ-specific Atg5 and Atg7 deficiency show various abnormalities, indicating that conventional macroautophagy is crucial for various biological events in many organs. The question is then, why does alternative macroautophagy, but not conventional macroautophagy, have an essential role in erythrocyte maturation? It is considered that the machineries for conventional and alternative macroautophagy are used in a cell type- and stimulusdependent manner. Further studies will be required to better define the relationship between the conventional and alternative macroautophagy. 2. Closing remark In this review, we describe two distinct autophagic pathways, conventional and alternative. The presence of at least two mechanistically distinct forms of autophagy in mammalian cells underscores autophagy as a highly adaptable cellular stress response. Further elucidation of the biological roles of autophagy will require a more complete understanding of (1) the molecular mechanisms of alternative autophagy, (2) the unique functional roles of these two pathways in vivo, and (3) the contribution of each pathway to pathology. Acknowledgements This review is written by the support in part by the Grant-inAid for Scientific Research on Innovative Areas, Grant-in-Aid for Scientific Research (S), Grant-in-Aid for challenging Exploratory Research from the MEXT of Japan, and a grant for the Program
for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO). References Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 2008;182:685–701. Ding WX, Yin XM. Mitophagy: mechanisms, pathophysiological roles, and analysis. Biol Chem 2012;393:547–64. Jin SM, Lazarou M, Wang C, Kane LA, Narendra DP, Youle RJ. Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL. J Cell Biol 2010;191:933–42. Kabeya Y, Kamada Y, Baba M, Takikawa H, Sasaki M, Ohsumi Y. Atg17 functions in cooperation with Atg1 and Atg13 in yeast autophagy. Mol Biol Cell 2005;16:2544–53. Kent G, Minick OT, Volini FI, Orfei E. Autophagic vacuoles in human red cells. Am J Pathol 1966;48:831–57. Komatsu M, Ichimura Y. Selective autophagy regulates various cellular functions. Genes Cells 2010;15:923–33. Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, et al. The role of autophagy during the early neonatal starvation period. Nature 2004;432:1032–6. Kundu M, Lindsten T, Yang CY, Wu J, Zhao F, Zhang J, et al. Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation. Blood 2008;112:1493–502. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature 2008;451:1069–75. Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 2011;27:107–32. Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat Rev Mol Cell Biol 2009;10:458–67. Narendra D, Tanaka A, Suen DF, Youle RJ. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol 2008;183:795–803. Nishida Y, Arakawa S, Fujitani K, Yamaguchi H, Mizuta T, et al. Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 2009;461:654–8. Xie Z, Klionsky DJ. Autophagosome formation: core machinery and adaptations. Nat Cell Biol 2007;9:1102–9.
