Dynamics of Autophagosome Formation

Autophagy, literally defined as “self-eating,” functions as a degradation process by recycling cytoplasmic contents under stress conditions or during development. Upon activation of autophagy, a membrane structure known as a phagophore forms and expands, finally closing to form a double-membrane vesicle called an autophagosome (Fig. 1; Lamb et al., 2013; Yin et al., 2016). The completed autophagosome, which contains the autophagic cargo, is delivered to the vacuole (plants and yeast) or lysosome (animals). The outer membrane fuses with the vacuolar/lysosomal membrane, and the inner membrane and contents are released into the vacuole/lysosome as an autophagic body and are degraded by hydrolases. The breakdown products are transported back into the cytoplasm for reuse by the cell (Yang and Bassham, 2015). A proposed model for autophagosome formation in plant cells. Once autophagy is induced, a crescent-shaped isolation membrane named a phagophore is assembled on its membrane origin (e.g. ER). During this process, the ATG1 complex is activated, and downstream regulators (question mark) are recruited onto the initiation site. PI3P is generated on the phagophore assembly site, and ATG8 is conjugated onto the membrane to become the ATG8-PE form. Also, ATG5 and SH3P2 have been shown to localize on the phagophore structures. In addition, ATG9 vesicles are required for the efficient budding of the phagophore from the ER platform. In the subsequent steps, more ATG8 lipidation occurs, and the isolation membrane will elongate and close to form the completed double-membrane autophagosome. Finally, the autophagosome will deliver the cargos into the vacuole by fusion with the vacuole. With the help of the acidic environment and hydrolysis enzymes within the vacuole, the cargos will be degraded. Other regulators involved in the later steps after phagophore initiation are not listed. The initial identification of many autophagy-related (ATG) genes in yeast (Tsukada and Ohsumi, 1993; Thumm et al., 1994; Harding et al., 1995) was key in beginning to understand the mechanism by which autophagy occurs. The core machinery for autophagosome formation includes ATG1, which forms a complex with ATG13 for the induction of autophagy (Kamada et al., 2000); two ubiquitin-like conjugates, ATG12-ATG5 and ATG8-PE, which are recruited to the phagophore assembly site and play an important role in autophagosome formation (Yin et al., 2016); and ATG9, which may function in the recruitment of other ATG components and membrane to the forming autophagosome (Reggiori et al., 2005). In plants, autophagy has been well studied as a response to stress conditions, including nutrient deficiency (Doelling et al., 2002; Hanaoka et al., 2002), salt and drought stress (Liu et al., 2009), heat stress (Zhou et al., 2013; Yang et al., 2016), oxidative stress (Xiong et al., 2007), hypoxia (Chen et al., 2015), pathogen attack (Liu et al., 2005; Lai et al., 2011), and endoplasmic reticulum (ER) stress (Liu et al., 2012; Fig. 2). In this review, we summarize recent advances in our understanding of the dynamics of plant autophagy, focusing on regulation of autophagy and mechanisms of autophagosome formation. TOR-dependent and -independent regulatory pathways for autophagy in plants. Autophagy can be activated by abiotic stress, including osmotic, nutrient, salt, oxidative, and ER stress. This activation can be regulated in a TOR-dependent or -independent manner. A, Upon osmotic, nutrient, or salt stress, the SnRK1 complex can inhibit TOR, leading to activation of the ATG1 complex or deactivation of S6K and Tap46, in turn activating autophagy. SnRK1 can also activate the ATG1 complex by phosphorylation of ATG1, leading to activation of autophagy. B, Upon oxidative or ER stress, SnRK1 activates the ATG1 complex, leading to the activation of autophagy. Upon ER stress, unfolded proteins accumulate and activate IRE1b, leading to autophagy. Ovals represent kinase complexes, hexagons represent TOR targets, and octagons represent components of the ER stress response pathway. The autophagy pathway is highly conserved among all eukaryotes. In plants, it is activated during development and in response to stress, and a basal level of autophagy is important for cellular homeostasis (Wang et al., 2017). Appropriate activation of autophagy is critical in balancing growth with stress tolerance, and better understanding and subsequent modification of the pathway could lead to improvements in crop growth and yield. In this section, we will discuss known regulators of the autophagy pathway in plants. Autophagy is induced by nutrient depletion, most likely as a mechanism for nutrient recycling and generation of substrates for energy metabolism (Doelling et al., 2002; Hanaoka et al., 2002). The energy sensor Snf1-related protein kinase 1 (SnRK1) is a heterotrimeric complex that has been suggested to be a master regulator of metabolism in plants in response to nutrient and energy deficiency (Sugden et al., 1999; Baena-González et al., 2007; Crozet et al., 2014). The animal and yeast orthologs of SnRK1, AMP-activated kinase (AMPK) and Suc nonfermenting 1 (Snf1), respectively, are energy and metabolic sensors that maintain cellular homeostasis and activate autophagy under low-energy conditions (Hardie, 2011; Carroll and Dunlop, 2017). AMPK/Snf1 can regulate autophagy by inhibiting the target of rapamycin (TOR) complex (Lee et al., 2010), a negative regulator of autophagy, therefore allowing autophagy to become active, or by direct phosphorylation of ATG1, which also leads to the activation of autophagy (Wang et al., 2001; Egan et al., 2011; Kim et al., 2011). In Arabidopsis (Arabidopsis thaliana), there are two isoforms of the SnRK1 complex catalytic subunit, KIN10 and KIN11, with KIN10 being responsible for most of the SnRK1 activity (Baena-González et al., 2007; Jossier et al., 2009; Crozet et al., 2014). A kin10 kin11 double mutant is lethal, and reduced expression via virus-induced gene silencing leads to decreased activation of stress and starvation genes and to deformed leaves, flowers, and inflorescence (Baena-González et al., 2007), indicating that SnRK1 functions in development and stress responses. Overexpression of the KIN10 gene in Arabidopsis leads to constitutive activation of autophagy (Chen et al., 2017; Soto-Burgos and Bassham, 2017), suggesting a positive role in the regulation of autophagy. KIN10 overexpression led to increased phosphorylation of ATG1 during Suc starvation, suggesting that KIN10 regulates autophagy by affecting the phosphorylation of ATG1 (Chen et al., 2017), as in mammals and yeast. Autophagy is activated during a wide range of abiotic stresses, and a kin10 knockout mutant failed to activate autophagy during most of these stresses (Soto-Burgos and Bassham, 2017). This indicates that KIN10 activates autophagy not just in response to nutrient deficiency or energy depletion as predicted, but also during other abiotic stresses, indicating a wider role for SnRK1 in regulation of autophagy than previously expected. The TOR complex is a key regulator of the balance between growth and autophagy in all eukaryotes tested (Noda and Ohsumi, 1998; Pattingre et al., 2008; Liu and Bassham, 2010). In plants, TOR is activated when nutrients are abundant, in turn enhancing mRNA translation initiation, ribosome biogenesis, cell wall synthesis and growth and inhibiting autophagy (Deprost et al., 2007; Ren et al., 2011; Xiong and Sheen, 2015). Upon nutrient deficiency, TOR is inactivated, reducing growth and allowing the activation of autophagy (Pattingre et al., 2008). The Arabidopsis TOR complex consists of TOR itself, a Ser/Thr kinase (Menand et al., 2002), the Regulatory Associated Protein of TOR, RAPTOR (Anderson et al., 2005; Deprost et al., 2005), which presents substrates to TOR for phosphorylation (Hara et al., 2002), and Lethal with Sec Thirteen 8, which stabilizes the complex (Moreau et al., 2012). A complete knockout of TOR is embryo-lethal (Menand et al., 2002), while knockdown by RNA interference leads to arrest of plant growth and development and constitutive autophagy (Deprost et al., 2007; Liu and Bassham, 2010). Disruption of RAPTOR in Arabidopsis similarly leads to defects in plant growth and development, although less severe than those of a TOR knockout, as well as constitutive autophagy (Anderson et al., 2005; Deprost et al., 2005; Pu et al., 2017). Overexpression of TOR blocks autophagy induced by nutrient starvation, salt, and osmotic stresses, while autophagy induced by oxidative and ER stress is not affected (Pu et al., 2017). Regulation of autophagy can therefore be TOR dependent or independent (Fig. 2) depending on the environmental stress to which the plant is subjected. RAPTOR interacts with ribosomal p70 S6 kinase (S6K) in response to osmotic stress signals, suggesting a role for S6K in the TOR signaling pathway and plant stress responses (Mahfouz et al., 2006). Several other proteins have also been shown to interact with RAPTOR or TOR in vitro, including Arabidopsis Mei2-like1 (Anderson et al., 2005) and transcription factor E2Fa. Tap42/α4 is an effector of TOR in yeast and mammals, and its Arabidopsis homolog, Tap46, has been identified as a downstream target of TOR. Tap46 is phosphorylated by TOR and interacts with protein phosphatase type 2A, a regulator of autophagy in yeast (Ahn et al., 2011). Overexpression or reduction of expression of Tap46 correlates with TOR activity (Ahn et al., 2015), suggesting that Tap46 is a positive regulator of the TOR pathway. Silencing of the TAP46 gene using virus-induced gene silencing in tobacco (Nicotiana tabacum) led to induction of autophagy, as in the TOR RNAi plants (Ahn et al., 2011), indicating that it can negatively regulate autophagy. This suggests that Tap46 acts as a positive effector in the TOR signaling pathway, leading to the regulation of autophagy. Recently, a connection between the TOR signaling pathway and SnRK1 complex has been demonstrated. KIN10 interacts with RAPTOR in vivo and can phosphorylate RAPTOR in vitro, like its mammalian orthologs (Nukarinen et al., 2016). Blocking TOR activity in a kin10 mutant led to activation of autophagy, while inhibition of SnRK1 activity in a raptor1b mutant failed to block the constitutive autophagy observed in this mutant (Soto-Burgos and Bassham, 2017). SnRK1 therefore acts upstream of the TOR signaling pathway in the regulation of autophagy (Soto-Burgos and Bassham, 2017), at least under conditions in which activation of autophagy is TOR dependent. A major regulator of autophagy and a downstream substrate of TOR in yeast and animals is the ATG1/ATG13 kinase complex. ATG1 is the catalytic subunit of the complex and activates autophagy in response to nutrient depletion (Diaz-Troya et al., 2008; Mizushima 2010). In mammals, AMPK promotes autophagy by phosphorylating Ulk1 (ATG1 homolog) upon Glc starvation (Kim et al., 2011). In Arabidopsis, KIN10 overexpression results in an increase in ATG1 phosphorylation, suggesting that this mode of regulation is conserved (Chen et al., 2017). In yeast, TOR phosphorylates ATG13 in nutrient-rich conditions, causing a decrease in its affinity for ATG1, preventing their association and therefore repressing autophagy. Under starvation conditions, TOR becomes inactive, leading to the dephosphorylation of ATG13, allowing ATG1 to associate with ATG13 and activate autophagy (Nakatogawa et al., 2009; Kamada et al., 2010). In mammals, unlike in yeast, ATG1 associates with ATG13 under all conditions, indicating that the regulatory mechanism of ATG1/ATG13 differs between mammals and yeast (Lee et al., 2007). In Arabidopsis, ATG1 and ATG13 are present in multiple copies, which are most likely functionally redundant. During nutrient starvation, ATG1a is hyper-phosphorylated, potentially by SnRK1 (Chen et al., 2017), while ATG13a is hypo-phosphorylated (Suttangkakul et al., 2011), suggesting that the ATG1 complex functions in a manner similar to yeast in terms of autophagy regulation. Disruption of ATG13 leads to phenotypes similar to those of mutants in other core autophagy genes, with hypersensitivity to nutrient starvation and accelerated senescence. atg13 mutants have defects in the formation of autophagic bodies, suggesting that the complex acts upstream of autophagosome formation (Suttangkakul et al., 2011). ATG1a associates with autophagic bodies and is delivered to the vacuole for degradation, indicating that the ATG1 complex is a substrate of autophagy. A negative feedback mechanism is therefore proposed to exist to reduce activated ATG1 complex levels after induction of autophagy by nutrient deficiency (Suttangkakul et al., 2011). This turnover might be an attempt to reset autophagic induction by the of activated ATG1 kinase during of phagophore assembly (Suttangkakul et al., 2011). on this and by with ATG1 complex functions in animals and yeast, we that the ATG1 complex may regulate autophagy in Arabidopsis via its phosphorylation by TOR SnRK1 (Fig. 2). Autophagy is induced by ER stress, in which of unfolded and proteins within the ER activates the unfolded protein response Liu et al., 2012; Liu and 2016). of TOR activity leads to activation of autophagy during abiotic stresses, autophagy induced by ER stress to be independent of TOR (Pu et al., 2017), as are during ER stress in TOR overexpression ER autophagy on an ER stress sensor that activates the and et al., and During ER stress, is activated by and et al., an mRNA a transcription factor et al., 2011). The mRNA is an protein that is into the and genes as and 2005; et al., 2011). genes have been identified in Arabidopsis, and et al., 2001; et al., 2011; et al., and a mutant in is to form after ER stress using or indicating that is required for the induction of autophagy by ER stress (Liu et al., 2012). in or have on autophagy during ER stress, suggesting that is involved in the regulation of autophagy and that its activity is not required (Liu et al., 2012). The of or overexpression of activation of autophagy by or and expression of a protein in the ER was to autophagy via The of unfolded proteins in the by IRE1b, is therefore a key in activating autophagy during ER stress (Yang et al., 2016). heat is a major factor that ER stress, as by the of mRNA by et al., and by of et al., The induction of autophagy by heat stress is also to the of proteins (Yang et al., 2016). Autophagy activation is reduced in an mutant during heat stress, to the indicating that the autophagy response during heat stress is dependent on (Yang et al., and is most likely as an ER stress of the identified regulators of plant autophagy and is known regulation of autophagy-related In the transcription factor has been shown to drought by the activation of ATG genes and the induction of autophagy (Wang et al., The Arabidopsis transcription factor has also been suggested to regulate autophagy. is important for plant to et al., and a yeast that interacts with a core autophagy et al., 2011). a mutant was in of and induction of autophagy upon with et al., 2011). Silencing of in led to reduced ATG gene expression and autophagosome during heat stress (Zhou et al., suggesting that it also functions in abiotic stress responses. Several pathways for regulation of autophagy in plants have been identified the have been shown to negatively regulate autophagy et al., et al., 2015). In Arabidopsis, there are multiple isoforms of including and and and enzymes et al., in the isoforms and have constitutive autophagy, suggesting that can negatively regulate autophagy et al., 2015). In silencing of activated autophagy, overexpression of oxidative autophagy et al., 2015). interact with in vivo and in vitro, but upon oxidative stress this et al., 2015). Disruption of led to et al., et al., 2015), although this is to autophagy is these that negatively regulates autophagy with has been to the regulation of autophagy. is an important signaling in mammalian and that this is also in plants. has been identified as a of the signaling pathway and and has in regulation of (Chen et al., and to et al., et al., 2010), and (Wang et al., stress. is an that is involved in the of and the degradation of et al., 2010). A in the gene generation in the and promotes the of ATG8 and ATG8-PE, indicating activation of autophagy. of to a mutant or of gene the and lipidation of ATG8 proteins et al., 2012). Recently, it was that the negative regulation of autophagy by is independent of and therefore regulates autophagy by an pathway et al., 2016). the plant has been shown to interact with in vivo and in and to regulate autophagy et al., 2017). Silencing of tobacco reduced the autophagy activity induced by or oxidative stress, while overexpression of increased autophagy activity and cell et al., 2017). therefore has and depending on the more becomes autophagy is regulated in plants. have been is to understand the autophagy pathway is under conditions and the regulatory components are to the of autophagy activation autophagy is activated, a conserved autophagosome formation process has been observed in plant which initiation, and degradation (Fig. 1; Liu and Bassham, 2012). may with for to the the vacuole, to including and for degradation et al., 2016). a membrane process to to the membrane, elongate the membrane for and with other like and finally the In the we will an on membrane dynamics during autophagosome and we to be to the on yeast and mammalian have our understanding of the mechanisms of autophagosome and multiple membrane have been et al., A which is by an double membrane with highly is called a phagophore or isolation has been shown that the phagophore from an structure on an ER called the et al., 2008). 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ATG9 vesicles have been as a key to deliver the membrane or other regulators to the phagophore In yeast, autophagy is in the mutant et al., 2012). In deficiency in ATG9 leads to autophagosome upon induction and decreased autophagic suggesting that mammalian ATG9 is not in basal conditions et al., 2012). also that ATG9 is with ATG and regulators for phagophore initiation et al., et al., 2016). In plants, of Arabidopsis mutants that autophagy is less when with other mutants like and et al., 2002). Recently, has into the of ATG9 in the formation of et al., 2017). 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