Dissecting the function of Atg1 complex in Dictyostelium autophagy reveals a connection with the pentose phosphate pathway enzyme transketolase

The network of protein–protein interactions of the Dictyostelium discoideum autophagy pathway was investigated by yeast two-hybrid screening of the conserved autophagic proteins Atg1 and Atg8. These analyses confirmed expected interactions described in other organisms and also identified novel interactors that highlight the complexity of autophagy regulation. The Atg1 kinase complex, an essential regulator of autophagy, was investigated in detail here. The composition of the Atg1 complex in D. discoideum is more similar to mammalian cells than to Saccharomyces cerevisiae as, besides Atg13, it contains Atg101, a protein not conserved in this yeast. We found that Atg101 interacts with Atg13 and genetic disruption of these proteins in Dictyostelium leads to an early block in autophagy, although the severity of the developmental phenotype and the degree of autophagic block is higher in Atg13-deficient cells. We have also identified a protein containing zinc-finger B-box and FNIP motifs that interacts with Atg101. Disruption of this protein increases autophagic flux, suggesting that it functions as a negative regulator of Atg101. We also describe the interaction of Atg1 kinase with the pentose phosphate pathway enzyme transketolase (TKT). We found changes in the activity of endogenous TKT activity in strains lacking or overexpressing Atg1, suggesting the presence of an unsuspected regulatory pathway between autophagy and the pentose phosphate pathway in Dictyostelium that seems to be conserved in mammalian cells.     

Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo. Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)-inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3-interaction region (LIR)-dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1-Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR-dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.     

Atg1 kinase regulates early and late steps during autophagy

The notion that phosphorylation constitutes a major mechanism to induce autophagy was established 15 years ago when a conserved Atg1/ULK kinase family was identified as an essential component of the autophagy machinery. The key observation was that starved atg1Δ cells lack autophagosomes in the cytosol and fail to accumulate autophagic bodies in the vacuole. Although many studies have revealed important details of Atg1 activation and function, a cohesive model for how Atg1 regulates the autophagic machinery is lacking. Our recent findings identified conserved steps of temporal and spatial regulation of Atg1/ULK1 kinase at both the PAS and autophagosomal membranes, suggesting that Atg1 not only promotes autophagy induction, but may also facilitate late stages of autophagosome biogenesis.     

Atg1 kinase organizes autophagosome formation by phosphorylating Atg9

The conserved Ser/Thr kinase Atg1/ULK1 plays a crucial role in the regulation of autophagy. However, only very few Atg1 targets have been identified, impeding elucidation of the mechanisms by which Atg1 regulates autophagy. In our study, we determined the Saccharomyces cerevisiae Atg1 consensus phosphorylation sequence using a peptide array-based approach. Among proteins containing this sequence we identified Atg9, another essential component of the autophagic machinery. We showed that phosphorylation of Atg9 by Atg1 is required for phagophore elongation, shedding light on the mechanism by which Atg1 regulates early steps of autophagy.     

Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction

Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.     

Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction

Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.     

Passing membranes to autophagy: Unconventional membrane tethering by Atg17

Macroautophagy delivers cytoplasmic material to lysosomal/vacuolar compartments for degradation. Conserved multisubunit complexes, composed of autophagy-related (Atg) proteins, initiate the formation of membrane precursors, termed phagophores. Under physiological conditions these cup-shaped structures can capture cytoplasmic material highly selectively. Starvation or cytotoxic stresses, however, initiate the formation of much larger phagophores to enclose cytoplasm nonselectively. The biogenesis of nonselective autophagosomes is initiated by the hierarchical assembly of the Atg1 kinase complex and the recruitment of Atg9 vesicles at the phagophore assembly site (PAS). In this punctum we summarize our recent findings regarding tethering of Atg9 vesicles by the Atg1 kinase complex. We discuss membrane tethering by and activation of its central subunit Atg17 in the context of other canonical membrane tethering factors. Our results show that Atg17 suffices to bind and tether Atg9 vesicles. The Atg31-Atg29 subcomplex inhibits Atg17 activity, and activation of Atg17 depends on the formation of the Atg1 kinase complex that involves recruiting Atg1-Atg13. Our studies lead to a model of unconventional membrane tethering in autophagy.     

Detection of Saccharomyces cerevisiae Atg13 by western blot

The proteins that comprise the Atg1 kinase complex constitute a key set of components that participate in macroautophagy (hereafter autophagy). Among these proteins, Atg13 plays a particularly important, although as yet undefined role, in that it is critical for the proper localization of Atg1 to the phagophore assembly site (PAS) and its efficient kinase activity. Atg13 is hyperphosphorylated in vegetative conditions when autophagy occurs at a basal level, and is largely dephosphorylated upon the induction of autophagy. Inhibitory phosphorylation of Atg13 reflects the activity of TOR complex 1 (TORC1) and protein kinase A. Accordingly, monitoring the phosphorylation state of Atg13 provides a convenient way to follow early steps of autophagy induction as well as the activity of some of the upstream nutrient-sensing kinases. However, the detection of Atg13 by western blot can be problematic. Here, we present a detailed protocol for sample preparation and detection of the Atg13 protein from yeast.     

Detection of Saccharomyces cerevisiae Atg13 by western blot

The proteins that comprise the Atg1 kinase complex constitute a key set of components that participate in macroautophagy (hereafter autophagy). Among these proteins, Atg13 plays a particularly important, although as yet undefined role, in that it is critical for the proper localization of Atg1 to the phagophore assembly site (PAS) and its efficient kinase activity. Atg13 is hyperphosphorylated in vegetative conditions when autophagy occurs at a basal level, and is largely dephosphorylated upon the induction of autophagy. Inhibitory phosphorylation of Atg13 reflects the activity of TOR complex 1 (TORC1) and protein kinase A. Accordingly, monitoring the phosphorylation state of Atg13 provides a convenient way to follow early steps of autophagy induction as well as the activity of some of the upstream nutrient-sensing kinases. However, the detection of Atg13 by western blot can be problematic. Here, we present a detailed protocol for sample preparation and detection of the Atg13 protein from yeast.     

A missing piece of the puzzle: Atg11 functions as a scaffold to activate Atg1 for selective autophagy

The mechanism regulating Atg1 kinase activity for the initiation of selective macroautophagy (hereafter autophagy) under nutrient-rich conditions has been a long-standing question. Canonically in yeast, nutrient starvation or rapamycin treatment repress TOR complex 1 and stimulate the Atg1 complex (including at least Atg1, Atg13, Atg17, Atg29 and Atg31), which allows the recruitment of downstream autophagy-related (Atg) components to the phagophore assembly site (PAS), culminating in phagophore formation, and, subsequently, autophagosome biogenesis. Atg1 also functions under conditions promoting selective autophagy that do not necessarily require nutrient deprivation for induction. However, there has been some debate as to whether Atg1 catalytic activity plays a more important role under conditions of nutrient starvation-induced autophagy (i.e., bulk autophagy) vs. selective autophagy (e.g., the cytoplasm-to-vacuole targeting [Cvt] pathway). A recent paper by Kamber and colleagues investigates the mechanism regulating Atg1 activity during selective autophagy.     

Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy

Autophagy is a membrane-mediated degradation process of macromolecule recycling. Although the formation of double-membrane degradation vesicles (autophagosomes) is known to have a central role in autophagy, the mechanism underlying this process remains elusive. The serine/threonine kinase Atg1 has a key role in the induction of autophagy. In this study, we show that overexpression of Drosophila Atg1 promotes the phosphorylation-dependent activation of the actin-associated motor protein myosin II. A novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa), was identified as a link between Atg1 and actomyosin activation. Sqa interacts with Atg1 through its kinase domain and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the formation of autophagosomes under starvation conditions. In mammalian cells, we found that the Sqa mammalian homologue zipper-interacting protein kinase (ZIPK) and myosin II had a critical role in the regulation of starvation-induced autophagy and mammalian Atg9 (mAtg9) trafficking when cells were deprived of nutrients. Our findings provide evidence of a link between Atg1 and the control of Atg9-mediated autophagosome formation through the myosin II motor protein.     

Mechanism and functions of membrane binding by the Atg5–Atg12/Atg16 complex during autophagosome formation

Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane‐bound vesicles termed autophagosomes. The conserved Atg5–Atg12/Atg16 complex is essential for autophagosome formation. Here, we show that the yeast Atg5–Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins, we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5–Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the pre‐autophagosomal structure but is essential for autophagy and cytoplasm‐to‐vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5–Atg12/Atg16 complex during autophagosome formation.     

Activation of Atg1 kinase in autophagy by regulated phosphorylation

Autophagy is a highly regulated trafficking pathway that leads to selective degradation of cellular constituents such as protein aggregates and excessive and damaged organelles. Atg1 is an essential part of the core autophagic machinery, which triggers induction of autophagy and the Cvt pathway. Although changes in Atg1 phosphorylation and complex formation are thought to regulate its function, the mechanism of Atg1 kinase activation remains unclear. Using a quantitative mass spectrometry approach, we identified 29 phosphorylation sites, of which five are either upregulated or downregulated by rapamycin treatment. Two phosphorylation sites, threonine 226 and serine 230, are evolutionarily conserved and located in the activation loop of the amino terminal kinase domain of Atg1. These phosphorylation events are not required for Atg1 localization to the phagosome assembly site (PAS), or the proper assembly of the multisubunit Atg1 kinase complex and binding to its activator Atg13. However, mutation of either one of these sites results in a loss of Atg1 kinase activity and its function in autophagy and the Cvt pathway. Taken together, our data suggest that phosphorylation of Atg1 on multiple sites provides critical mechanisms to regulate Atg1 function in autophagy and the Cvt pathway.     

Pex3 and Atg37 compete to regulate the interaction between the pexophagy receptor,Atg30, and the Hrr25 kinase

Macroautophagy/autophagy is a highly conserved process in which subcellular components destined for degradation are sequestered within autophagosomes. The selectivity of autophagy is determined by autophagy receptors, such as Pichia pastoris Atg30 (autophagy-related 30), which controls the selective degradation of peroxisomes (pexophagy) through the assembly of a receptor-protein complex (RPC). Previously, we proved that the peroxisomal acyl-CoA-binding protein, Atg37, and the highly conserved peroxin, Pex3, are required for RPC formation and efficient pexophagy. Here, we describe how Atg37 and Pex3 regulate the assembly and activation of the pexophagic RPC. We demonstrate that Atg30 requires both Atg37 and Pex3 to recruit Atg8 and Atg11 to the pexophagic RPC, because Atg37 depends on Pex3 for its localization at the peroxisomal membrane. We establish that due to close proximity of Atg37- and Pex3-binding sites in the middle domain of Atg30, the binding of these proteins to Atg30 is mutually exclusive within this region. We also show that direct binding of Pex3 and Atg37 to Atg30 regulates its phosphorylation by the Hrr25 kinase, negatively and positively, respectively. Based on these results we present a model that clarifies the assembly and activation of the pexophagic RPC through the phosphoregulation of Atg30.     

The identification and analysis of phosphorylation sites on the Atg1 protein kinase

Autophagy is a conserved, degradative process that has been implicated in a number of human diseases and is a potential target for therapeutic intervention. It is therefore important that we develop a thorough understanding of the mechanisms regulating this trafficking pathway. The Atg1 protein kinase is a key element of this control as a number of signaling pathways target this enzyme and its associated protein partners. These studies have established that Atg1 activities are controlled, at least in part, by protein phosphorylation. To further this understanding, we used a combined mass spectrometry and molecular biology approach to identify and characterize additional sites of phosphorylation in the Saccharomyces cerevisiae Atg1. Fifteen candidate sites of phosphorylation were identified, including nine that had not been noted previously. Interestingly, our data suggest that the phosphorylation at one of these sites, Ser-34, is inhibitory for both Atg1 kinase activity and autophagy. This site is located within a glycine-rich loop that is highly conserved in protein kinases. Phosphorylation at this position in several cyclin-dependent kinases has also been shown to result in diminished enzymatic activity. In addition, these studies identified Ser-390 as the site of autophosphorylation responsible for the anomalous migration exhibited by Atg1 on SDS-polyacrylamide gels. Finally, a mutational analysis suggested that a number of the sites identified here are important for full autophagy activity in vivo. In all, these studies identified a number of potential sites of regulation within Atg1 and will serve as a framework for future work with this enzyme.     

The identification and analysis of phosphorylation sites on the Atg1 protein kinase

Autophagy is a conserved, degradative process that has been implicated in a number of human diseases and is a potential target for therapeutic intervention. It is therefore important that we develop a thorough understanding of the mechanisms regulating this trafficking pathway. The Atg1 protein kinase is a key element of this control as a number of signaling pathways target this enzyme and its associated protein partners. These studies have established that Atg1 activities are controlled, at least in part, by protein phosphorylation. To further this understanding, we used a combined mass spectrometry and molecular biology approach to identify and characterize additional sites of phosphorylation in the Saccharomyces cerevisiae Atg1. Fifteen candidate sites of phosphorylation were identified, including nine that had not been noted previously. Interestingly, our data suggest that the phosphorylation at one of these sites, Ser-34, is inhibitory for both Atg1 kinase activity and autophagy. This site is located within a glycine-rich loop that is highly conserved in protein kinases. Phosphorylation at this position in several cyclin-dependent kinases has also been shown to result in diminished enzymatic activity. In addition, these studies identified Ser-390 as the site of autophosphorylation responsible for the anomalous migration exhibited by Atg1 on SDS-polyacrylamide gels. Finally, a mutational analysis suggested that a number of the sites identified here are important for full autophagy activity in vivo. In all, these studies identified a number of potential sites of regulation within Atg1 and will serve as a framework for future work with this enzyme.     

Identifying Atg1 Substrates: Four Means to an End

Autophagy is essential for normal development and the response to a variety of stress conditions, including nutrient deprivation. The Atg1 serine/threonine-specific protein kinase appears to be a key regulator of many forms of autophagy that occur in eukaryotic cells. Therefore, to fully understand the regulation of autophagy, it is essential that we identify the signaling pathways regulating Atg1 and the physiologically-relevant targets of Atg1 kinase activity. Although some progress has been made on the former question, no Atg1 substrates important for autophagy have yet been identified. In this review, we discuss four different experimental strategies that should facilitate the search for Atg1 substrates.     

Identifying Atg1 substrates: four means to an end

Autophagy is essential for normal development and the response to a variety of stress conditions, including nutrient deprivation. The Atg1 serine/threonine-specific protein kinase appears to be a key regulator of many forms of autophagy that occur in eukaryotic cells. Therefore, to fully understand the regulation of autophagy, it is essential that we identify the signaling pathways regulating Atg1 and the physiologically-relevant targets of Atg1 kinase activity. Although some progress has been made on the former question, no Atg1 substrates important for autophagy have yet been identified. In this review, we discuss four different experimental strategies that should facilitate the search for Atg1 substrates.     

Activation of Atg1 kinase in autophagy by regulated phosphorylation

Autophagy is a highly regulated trafficking pathway that leads to selective degradation of cellular constituents such as protein aggregates and excessive and damaged organelles. Atg1 is an essential part of the core autophagic machinery, which triggers induction of autophagy and the Cvt pathway. Although changes in Atg1 phosphorylation and complex formation are thought to regulate its function, the mechanism of Atg1 kinase activation remains unclear. Using a quantitative mass spectrometry approach, we identified 29 phosphorylation sites, of which five are either upregulated or downregulated by rapamycin treatment. Two phosphorylation sites, threonine 226 and serine 230, are evolutionarily conserved and located in the activation loop of the amino terminal kinase domain of Atg1. These phosphorylation events are not required for Atg1 localization to the phagosome assembly site (PAS), or the proper assembly of the multisubunit Atg1 kinase complex and binding to its activator Atg13. However, mutation of either one of these sites results in a loss of Atg1 kinase activity and its function in autophagy and the Cvt pathway. Taken together, our data suggest that phosphorylation of Atg1 on multiple sites provides critical mechanisms to regulate Atg1 function in autophagy and the Cvt pathway.     

Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila

Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. Here we characterized Atg17/FIP200, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and we showed that it binds to the other subunits including Atg1, Atg13, and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, we found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. We propose a model according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila.