Selective removal of dishevelled by autophagy: A role of p62

Autophagy has long been viewed as a process to remove long-lived or misfolded protein aggregates and aging and damaged organelles. Our study identified a previously unknown function of autophagy in suppression of Wnt signaling. A signaling protein, Dishevelled (Dvl), can be degraded via the autophagy pathway upon starvation. In this selective degradation, p62/sequestosome-1 binds to ubiquitinated Dvl proteins and promotes Dvl aggregation. Intriguingly, LC3 can also directly interact with Dvl. The studies on the mechanism for autophagic clearance of Dishevelled led to several interesting findings.     

Different effects of Atg2 and Atg18 mutations on Atg8a and Atg9 trafficking during starvation in Drosophila

The Atg2–Atg18 complex acts in parallel to Atg8 and regulates Atg9 recycling from phagophore assembly site (PAS) during autophagy in yeast. Here we show that in Drosophila, both Atg9 and Atg18 are required for Atg8a puncta formation, unlike Atg2. Selective autophagic degradation of ubiquitinated proteins is mediated by Ref(2)P/p62. The transmembrane protein Atg9 accumulates on refractory to Sigma P (Ref(2)P) aggregates in Atg7, Atg8a and Atg2 mutants. No accumulation of Atg9 is seen on Ref(2)P in cells lacking Atg18 or Vps34 lipid kinase function, while the Atg1 complex subunit FIP200 is recruited. The simultaneous interaction of Atg18 with both Atg9 and Ref(2)P raises the possibility that Atg18 may facilitate selective degradation of ubiquitinated protein aggregates by autophagy.     

Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation

Autophagy is an intracellular degradation process to clear up aggregated proteins or aged and damaged organelles. The Beclin1-Vps34-Atg14L complex is essential for autophagosome formation. However, how the complex formation is regulated is unclear. Here, we show that Dapper1 (Dpr1) acts as a critical regulator of the Beclin1-Vps34-Atg14L complex to promote autophagy. Dpr1 ablation in the central nervous system results in motor coordination defect and accumulation of p62 and ubiquitinated proteins. Dpr1 increases autophagosome formation as indicated by elevated puncta formation of LC3, Atg14L and DFCP1 (Double FYVE-containing protein 1). Conversely, loss of Dpr1 impairs LC3 lipidation and causes p62/SQSTM1 accumulation. Dpr1 directly interacts with Beclin1 and Atg14L and enhances the Beclin1-Vps34 interaction and Vps34 activity. Together, our findings suggest that Dpr1 enhances the Atg14L-Beclin1-Vps34 complex formation to drive autophagy.     

The scaffold protein EPG-7 links cargo–receptor complexes with the autophagic assembly machinery

The mechanism by which protein aggregates are selectively degraded by autophagy is poorly understood. Previous studies show that a family of Atg8-interacting proteins function as receptors linking specific cargoes to the autophagic machinery. Here we demonstrate that during Caenorhabditis elegans embryogenesis, epg-7 functions as a scaffold protein mediating autophagic degradation of several protein aggregates, including aggregates of the p62 homologue SQST-1, but has little effect on other autophagy-regulated processes. EPG-7 self-oligomerizes and is degraded by autophagy independently of SQST-1. SQST-1 directly interacts with EPG-7 and colocalizes with EPG-7 aggregates in autophagy mutants. Mutations in epg-7 impair association of SQST-1 aggregates with LGG-1/Atg8 puncta. EPG-7 interacts with multiple ATG proteins and colocalizes with ATG-9 puncta in various autophagy mutants. Unlike core autophagy genes, epg-7 is dispensable for starvation-induced autophagic degradation of substrate aggregates. Our results indicate that under physiological conditions a scaffold protein endows cargo specificity and also elevates degradation efficiency by linking the cargo–receptor complex with the autophagic machinery.     

Dapper 1 antagonizes Wnt signaling by promoting dishevelled degradation

Wnt signaling plays pivotal roles in the regulation of embryogenesis and cancer development. Xenopus Dapper (Dpr) was identified as an interacting protein for Dishevelled (Dvl), a Wnt signaling mediator, and modulates Wnt signaling. However, it is largely unclear how Dpr regulates Wnt signaling. Here, we present evidence that human Dpr1, the ortholog of Xenopus Dpr, inhibits Wnt signaling. We have identified the regions responsible for the Dpr-Dvl interaction in both proteins and found that the interaction interface is formed between the DEP (Dishevelled, Egl-10, and pleckstrin) domain of Dvl and the central and the C-terminal regions of Dpr1. The inhibitory function of human Dpr1 requires both its N and C terminus. Overexpression of the C-terminal region corresponding to the last 225 amino acids of Dpr1, in contrast to wild-type Dpr1, enhances Wnt signaling, suggesting a dominant negative function of this region. Furthermore, we have shown that Dpr1 induces Dvl degradation via a lysosome inhibitor-sensitive and proteasome inhibitor-insensitive mechanism. Knockdown of Dpr1 by RNA interference up-regulates endogenous Dvl2 protein. Taken together, our data indicate that the inhibitory activity of Dpr on Wnt signaling is conserved from Xenopus to human and that Dpr1 antagonizes Wnt signaling by inducing Dvl degradation.     

Pex3 confines pexophagy receptor activity of Atg36 to peroxisomes by regulating Hrr25-mediated phosphorylation and proteasomal degradation

In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36–Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36–Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.     

Proteases in autophagy

Autophagy is a process involved in the proteolytic degradation of cellular macromolecules in lysosomes, which requires the activity of proteases, enzymes that hydrolyse peptide bonds and play a critical role in the initiation and execution of autophagy. Importantly, proteases also inhibit autophagy in certain cases. The initial steps of macroautophagy depend on the proteolytic processing of a particular protein, Atg8, by a cysteine protease, Atg4. This processing step is essential for conjugation of Atg8 with phosphatidylethanolamine and, subsequently, autophagosome formation. Lysosomal hydrolases, known as cathepsins, can be divided into several groups based on the catalitic residue in the active site, namely, cysteine, serine and aspartic cathepsins, which catalyse the cleavage of peptide bonds of autophagy substrates and, together with other factors, dispose of the autophagic flux. Whilst most cathepsins degrade autophagosomal content, some, such as cathepsin L, also degrade lysosomal membrane components, GABARAP-II and LC3-II. In contrast, cathepsin A, a serine protease, is involved in inhibition of chaperon-mediated autophagy through proteolytic processing of LAMP-2A. In addition, other families of calcium-dependent non-lysosomal cysteine proteases, such as calpains, and cysteine aspartate-specific proteases, such as caspases, may cleave autophagy-related proteins, negatively influencing the execution of autophagic processes. Here we discuss the current state of knowledge concerning protein degradation by autophagy and outline the role of proteases in autophagic processes. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Protein kinase A-mediated 14-3-3 association impedes human Dapper1 to promote dishevelled degradation

Wnt signaling regulates embryo development and tissue homeostasis, and its deregulation leads to an array of diseases, including cancer. Dapper1 has been shown to be a key negative regulator of Wnt signaling. However, its function and regulation remain poorly understood. In this study, we report that 14-3-3β interacts with human Dapper1 (hDpr1). The interaction is dependent on protein kinase A (PKA)-mediated phosphorylation of hDpr1 at Ser-237 and Ser-827. 14-3-3β binding attenuates the ability of hDpr1 to promote Dishevelled (Dvl) degradation, thus enhancing Wnt signaling. We further provide evidence that PKA-mediated Dpr1 phosphorylation may contribute to growth and tumor formation of colon cancer Caco2 cells. Finally, we show that cyclooxygenase-2 expression and PKA activation are positively correlated with Dvl protein levels in colon cancer samples. Together, our findings establish a novel layer of regulation of Wnt signaling by PKA via the 14-3-3-Dpr1-Dvl axis.     

Hrr25 phosphorylates the autophagic receptor Atg34 to promote vacuolar transport of α-mannosidase under nitrogen starvation conditions

In Saccharomyces cerevisiae, under nitrogen-starvation conditions, the α-mannosidase Ams1 is recognized by the autophagic receptor Atg34 and transported into the vacuole, where it functions as an active enzyme. In this study, we identified Hrr25 as the kinase that phosphorylates Atg34 under these conditions. Hrr25-mediated phosphorylation does not affect the interaction of Atg34 with Ams1, but instead promotes Atg34 binding to the adaptor protein Atg11, which recruits the autophagy machinery to the Ams1–Atg34 complex, resulting in activation of the vacuolar transport of Ams1. Our findings reveal the regulatory mechanism of a biosynthetic pathway mediated by the autophagy machinery.     

Nrbf2 Protein Suppresses Autophagy by Modulating Atg14L Protein-containing Beclin 1-Vps34 Complex Architecture and Reducing Intracellular Phosphatidylinositol-3 Phosphate Levels

Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1^−/−;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.     

USP19 modulates autophagy and antiviral immune responses by deubiquitinating Beclin‐1

Autophagy, mediated by a number of autophagy‐related (ATG) proteins, plays an important role in the bulk degradation of cellular constituents. Beclin‐1 (also known as Atg6 in yeast) is a core protein essential for autophagic initiation and other biological processes. The activity of Beclin‐1 is tightly regulated by multiple post‐translational modifications, including ubiquitination, yet the molecular mechanism underpinning its reversible deubiquitination remains poorly defined. Here, we identified ubiquitin‐specific protease 19 (USP19) as a positive regulator of autophagy, but a negative regulator of type I interferon (IFN) signaling. USP19 stabilizes Beclin‐1 by removing the K11‐linked ubiquitin chains of Beclin‐1 at lysine 437. Moreover, we found that USP19 negatively regulates type I IFN signaling pathway, by blocking RIG‐I‐MAVS interaction in a Beclin‐1‐dependent manner. Depletion of either USP19 or Beclin‐1 inhibits autophagic flux and promotes type I IFN signaling as well as cellular antiviral immunity. Our findings reveal novel dual functions of the USP19‐Beclin‐1 axis by balancing autophagy and the production of type I IFNs.     

The BECN1-USP19 axis plays a role in the crosstalk between autophagy and antiviral immune responses

Macroautophagy/autophagy is a conserved intracellular degradation system that traffics substrates including protein aggregates, defunct or disused organelles and invading pathogens to lysosomes via double-membrane vesicles called autophagosomes. BECN1/Beclin 1 functions as a key protein in autophagy initiation and progression; however, the role of BECN1 in innate immunity has not been fully investigated. Recently, we have found that USP19 affects the ubiquitination of BECN1, hence promoting the formation of autophagosomes and inhibiting DDX58/RIG-I-mediated type I interferon signaling.     

The membrane peroxin PEX3 induces peroxisome-ubiquitination-linked pexophagy

Peroxisomes are degraded by a selective type of autophagy known as pexophagy. Several different types of pexophagy have been reported in mammalian cells. However, the mechanisms underlying how peroxisomes are recognized by autophagy-related machinery remain elusive. PEX3 is a peroxisomal membrane protein (PMP) that functions in the import of PMPs into the peroxisomal membrane and has been shown to interact with pexophagic receptor proteins during pexophagy in yeast. Thus, PEX3 is important not only for peroxisome biogenesis, but also for peroxisome degradation. However, whether PEX3 is involved in the degradation of peroxisomes in mammalian cells is unclear. Here, we report that high levels of PEX3 expression induce pexophagy. In PEX3-loaded cells, peroxisomes are ubiquitinated, clustered, and degraded in lysosomes. Peroxisome targeting of PEX3 is essential for the initial step of this degradation pathway. The degradation of peroxisomes is inhibited by treatment with autophagy inhibitors or siRNA against NBR1, which encodes an autophagic receptor protein. These results indicate that ubiquitin- and NBR1-mediated pexophagy is induced by increased expression of PEX3 in mammalian cells. In addition, another autophagic receptor protein, SQSTM1/p62, is required only for the clustering of peroxisomes. Expression of a PEX3 mutant with substitution of all lysine and cysteine residues by arginine and alanine, respectively, also induces peroxisome ubiquitination and degradation, hence suggesting that ubiquitination of PEX3 is dispensable for pexophagy and an endogenous, unidentified peroxisomal protein is ubiquitinated on the peroxisomal membrane.     

Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis

Autophagy is an essential process for eliminating ubiquitinated protein aggregates and dysfunctional organelles. Defective autophagy is associated with various degenerative diseases such as Parkinson disease. Through a genetic screening in Drosophila, we identified CG11148, whose product is orthologous to GIGYF1 (GRB10-interacting GYF protein 1) and GIGYF2 in mammals, as a new autophagy regulator; we hereafter refer to this gene as Gyf. Silencing of Gyf completely suppressed the effect of Atg1-Atg13 activation in stimulating autophagic flux and inducing autophagic eye degeneration. Although Gyf silencing did not affect Atg1-induced Atg13 phosphorylation or Atg6-Pi3K59F (class III PtdIns3K)-dependent Fyve puncta formation, it inhibited formation of Atg13 puncta, suggesting that Gyf controls autophagy through regulating subcellular localization of the Atg1-Atg13 complex. Gyf silencing also inhibited Atg1-Atg13-induced formation of Atg9 puncta, which is accumulated upon active membrane trafficking into autophagosomes. Gyf-null mutants also exhibited substantial defects in developmental or starvation-induced accumulation of autophagosomes and autolysosomes in the larval fat body. Furthermore, heads and thoraxes from Gyf-null adults exhibited strongly reduced expression of autophagosome-associated Atg8a-II compared to wild-type (WT) tissues. The decrease in Atg8a-II was directly correlated with an increased accumulation of ubiquitinated proteins and dysfunctional mitochondria in neuron and muscle, which together led to severe locomotor defects and early mortality. These results suggest that Gyf-mediated autophagy regulation is important for maintaining neuromuscular homeostasis and preventing degenerative pathologies of the tissues. Since human mutations in the GIGYF2 locus were reported to be associated with a type of familial Parkinson disease, the homeostatic role of Gyf-family proteins is likely to be evolutionarily conserved.     

The composition of a protein aggregate modulates the specificity and efficiency of its autophagic degradation

The mechanism underlying autophagic degradation of a protein aggregate remains largely unknown. A family of receptor proteins that simultaneously bind to the cargo and the Atg8 family of autophagy proteins (such as the MAP1LC3/LC3 subfamily) has been shown to confer cargo selectivity. The selectivity and efficiency of protein aggregate removal is also modulated by scaffold proteins that interact with receptor proteins and ATG proteins. During C. elegans embryogenesis, autophagic clearance of the cargoes PGL-1 and PGL-3 requires the receptor protein SEPA-1 and the scaffold protein EPG-2. SEPA-1 and EPG-2 also form aggregates that are degraded by autophagy. Here we investigated the effect of composition and organization of PGL granules on their autophagic degradation. We found that depletion of PGL-1 or PGL-3 facilitates the degradation of SEPA-1 and EPG-2. Removal of EPG-2 is also promoted when SEPA-1 is absent. Depletion of PGL-1 or PGL-3 renders the degradation of SEPA-1 independent of EPG-2. We further showed that overexpression of SEPA-1 or EPG-2 as well as SQST-1 or EPG-7 (scaffold protein), which belong to different classes of aggregate, has no evident effect on the degradation of the other type. Our results indicate that the composition and organization of protein aggregates provide another layer of regulation to modulate degradation efficiency.     

AMPK-independent induction of autophagy by cytosolic Ca2+ increase

Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca²⁺ can induce autophagy by inhibition of mTOR via the CaMKK-α/β-mediated activation of AMPK. Here we demonstrate that Ca²⁺ signaling can additionally induce autophagy independently of the Ca²⁺-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca²⁺ induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca²⁺-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca²⁺ by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca²⁺-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca²⁺ flux modifications. Importantly, using AMPK-α1^?/?α2^?/? MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-α/β inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca²⁺-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.     

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux.     

ATG13

During the past 20 years, autophagy signaling has entered the main stage of the cell biological theater. Autophagy represents an intracellular degradation process that is involved in both the bulk recycling of cytoplasmic components and the selective removal of organelles, protein aggregates, or intracellular pathogens. The understanding of autophagy has been greatly facilitated by the characterization of the molecular machinery governing this process. In yeast, initiation of autophagy is controlled by the Atg1 kinase complex, which is composed of the Ser/Thr kinase Atg1, the adaptor protein Atg13, and the ternary complex of Atg17-Atg31-Atg29. In vertebrates, the orthologous ULK1 kinase complex contains the Ser/Thr kinase ULK1 and the accessory proteins ATG13, RB1CC1, and ATG101. Among these components, Atg1/ULK1 have gained major attention in the past, i.e., for the identification of upstream regulatory kinases, the characterization of downstream substrates controlling the autophagic flux, or as a druggable target for the modulation of autophagy. However, accumulating data indicate that the function of Atg13/ATG13 has been likely underestimated so far. In addition to ensuring proper Atg1/ULK1 recruitment and activity, this adaptor molecule has been implicated in ULK1-independent autophagy processes. Furthermore, recent data have identified additional binding partners of Atg13/ATG13 besides the components of the Atg1/ULK1 complex, e.g., Atg8 family proteins or acidic phospholipids. Therefore, in this review we will center the spotlight on Atg13/ATG13 and summarize the role that Atg13/ATG13 assumes in the autophagy stage play.     

ATG13: Just a companion,or an executor of the autophagic program?

During the past 20 years, autophagy signaling has entered the main stage of the cell biological theater. Autophagy represents an intracellular degradation process that is involved in both the bulk recycling of cytoplasmic components and the selective removal of organelles, protein aggregates, or intracellular pathogens. The understanding of autophagy has been greatly facilitated by the characterization of the molecular machinery governing this process. In yeast, initiation of autophagy is controlled by the Atg1 kinase complex, which is composed of the Ser/Thr kinase Atg1, the adaptor protein Atg13, and the ternary complex of Atg17-Atg31-Atg29. In vertebrates, the orthologous ULK1 kinase complex contains the Ser/Thr kinase ULK1 and the accessory proteins ATG13, RB1CC1, and ATG101. Among these components, Atg1/ULK1 have gained major attention in the past, i.e., for the identification of upstream regulatory kinases, the characterization of downstream substrates controlling the autophagic flux, or as a druggable target for the modulation of autophagy. However, accumulating data indicate that the function of Atg13/ATG13 has been likely underestimated so far. In addition to ensuring proper Atg1/ULK1 recruitment and activity, this adaptor molecule has been implicated in ULK1-independent autophagy processes. Furthermore, recent data have identified additional binding partners of Atg13/ATG13 besides the components of the Atg1/ULK1 complex, e.g., Atg8 family proteins or acidic phospholipids. Therefore, in this review we will center the spotlight on Atg13/ATG13 and summarize the role that Atg13/ATG13 assumes in the autophagy stage play.