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.     

Expression of a ULK1/2 binding-deficient ATG13 variant can partially restore autophagic activity in ATG13-deficient cells

Autophagy describes an intracellular process responsible for the lysosome-dependent degradation of cytosolic components. The ULK1/2 complex comprising the kinase ULK1/2 and the accessory proteins ATG13, RB1CC1, and ATG101 has been identified as a central player in the autophagy network, and it represents the main entry point for autophagy-regulating kinases such as MTOR and AMPK. It is generally accepted that the ULK1 complex is constitutively assembled independent of nutrient supply. Here we report the characterization of the ATG13 region required for the binding of ULK1/2. This binding site is established by an extremely short peptide motif at the C terminus of ATG13. This motif is mandatory for the recruitment of ULK1 into the autophagy-initiating high-molecular mass complex. Expression of a ULK1/2 binding-deficient ATG13 variant in ATG13-deficient cells resulted in diminished but not completely abolished autophagic activity. Collectively, we propose that autophagy can be executed by mechanisms that are dependent or independent of the ULK1/2-ATG13 interaction.     

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.     

Poliovirus induces autophagic signaling independent of the ULK1 complex

Poliovirus (PV), like many positive-strand RNA viruses, subverts the macroautophagy/autophagy pathway to promote its own replication. Here, we investigate whether the virus uses the canonical autophagic signaling complex, consisting of the ULK1/2 kinases, ATG13, RB1CC1, and ATG101, to activate autophagy. We find that the virus sends autophagic signals independent of the ULK1 complex, and that the members of the autophagic complex are not required for normal levels of viral replication. We also show that the SQSTM1/p62 receptor protein is not degraded in a conventional manner during infection, but is likely cleaved in a manner similar to that shown for coxsackievirus B3. This means that SQSTM1, normally used to monitor autophagic degradation, cannot be used to accurately monitor degradation during poliovirus infection. In fact, autophagic degradation may be affected by the loss of SQSTM1 at the same time as autophagic signals are being sent. Finally, we demonstrate that ULK1 and ULK2 protein levels are greatly reduced during PV infection, and ATG13, RB1CC1, and ATG101 protein levels are reduced as well. Surprisingly, autophagic signaling appears to increase as ULK1 levels decrease. Overexpression of wild-type or dominant-negative ULK1 constructs does not affect virus replication, indicating that ULK1 degradation may be a side effect of the ULK1-independent signaling mechanism used by PV, inducing complex instability. This demonstration of ULK1-independent autophagic signaling is novel and leads to a model by which the virus is signaling to generate autophagosomes downstream of ULK1, while at the same time, cleaving cargo receptors, which may affect cargo loading and autophagic degradative flux. Our data suggest that PV has a finely-tuned relationship with the autophagic machinery, generating autophagosomes without using the primary autophagy signaling pathway.

Abbreviations: ACTB - actin beta; ATG13 - autophagy related 13; ATG14 - autophagy related 14; ATG101 - autophagy related 101; BECN1 - beclin 1; CVB3 - coxsackievirus B3; DMV - double-membraned vesicles; EM - electron microscopy; EMCV - encephalomyocarditis virus; EV-71 - enterovirus 71; FMDV - foot and mouth disease virus; GFP - green fluorescent protein; MAP1LC3B/LC3B - microtubule associated protein 1 light chain 3 beta; MOI - multiplicity of infection; MTOR - mechanistic target of rapamycin kinase; PIK3C3 - phosphatidylinositol 3-kinase catalytic subunit type 3; PRKAA2 - protein kinase AMP-activated catalytic subunit alpha 2; PSMG1 - proteasome assembly chaperone 1; PSMG2 - proteasome assembly chaperone 2PV - poliovirus; RB1CC1 - RB1 inducible coiled-coil 1; SQSTM1 - sequestosome 1; ULK1 - unc-51 like autophagy activating kinase 1; ULK2 - unc-51 like autophagy activating kinase 2; WIPI1 - WD repeat domain, phosphoinositide interacting 1     

Arabidopsis ATG11, a scaffold that links the ATG1-ATG13 kinase complex to general autophagy and selective mitophagy

Autophagy is essential for nutrient recycling and intracellular housekeeping in plants by removing unwanted cytoplasmic constituents, aggregated polypeptides, and damaged organelles. The autophagy-related (ATG)1-ATG13 kinase complex is an upstream regulator that integrates metabolic and environmental cues into a coherent autophagic response directed by other ATG components. Our recent studies with Arabidopsis thaliana revealed that ATG11, an accessory protein of the ATG1-ATG13 complex, acts as a scaffold that connects the complex to autophagic membranes. We showed that ATG11 encourages proper behavior of the ATG1-ATG13 complex and faithful delivery of autophagic vesicles to the vacuole, likely through its interaction with ATG8. In addition, we demonstrated that Arabidopsis mitochondria are degraded during senescence via an autophagic route that requires ATG11 and other ATG components. Together, ATG11 appears to be an important modulator of the ATG1-ATG13 complex and a multifunctional scaffold required for bulk autophagy and the selective clearance of mitochondria.     

The mammalian autophagy initiator complex contains 2 HORMA domain proteins

ATG101 is an essential component of the ULK complex responsible for initiating cellular autophagy in mammalian cells; its 3-dimensional structure and molecular function, however, are currently unclear. Here we present the X-ray structure of human ATG101. The protein displays an open HORMA domain fold. Both structural properties and biophysical evidence indicate that ATG101 is locked in this conformation, in contrast to the prototypical HORMA domain protein MAD2. Moreover, we discuss a potential mode of dimerization with ATG13 as a fundamental aspect of ATG101 function.     

The ULK complex initiates autophagosome formation at phosphatidylinositol synthase-enriched ER subdomains

In our recent paper, we biochemically analyzed autophagosome-related membranes at the initiation stage of macroautophagy/autophagy using atg knockout (KO) cells and demonstrated that the ULK complex is recruited to 2 distinct membranes: the ER membrane and ATG9A-positive autophagosome precursors. We have also identified phosphatidylinositol synthase (PIS)-enriched ER subdomains as the initiation site of autophagosome formation. Based on these findings, we propose that the ULK complex, the PIS-enriched ER subdomain, and ATG9A vesicles together initiate autophagosome formation.     

The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14

ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.     

ULK1 phosphorylates Ser30 of BECN1 in association with ATG14 to stimulate autophagy induction

ULK1 (unc51-like autophagy activating kinase 1) is a serine/threonine kinase that plays a key role in regulating macroautophagy/autophagy induction in response to amino acid starvation. Despite the recent progress in understanding ULK1 functions, the molecular mechanism by which ULK1 regulates the induction of autophagy remains elusive. In this study, we determined that ULK1 phosphorylates Ser30 of BECN1 (Beclin 1) in association with ATG14 (autophagy-related 14) but not with UVRAG (UV radiation resistance associated). The Ser30 phosphorylation was induced by deprivation of amino acids or treatments with Torin 1 or rapamycin, the conditions that inhibit MTORC1 (mechanistic target of rapamycin complex 1), and requires ATG13 and RB1CC1 (RB1 inducible coiled-coil 1), proteins that interact with ULK1. Hypoxia or glutamine deprivation, which inhibit MTORC1, was also able to increase the phosphorylation in a manner dependent upon ULK1 and ULK2. Blocking the BECN1 phosphorylation by replacing Ser30 with alanine suppressed the amino acid starvation-induced activation of the ATG14-containing PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) kinase, and reduced autophagy flux and the formation of phagophores and autophagosomes. The Ser30-to-Ala mutation did not affect the ULK1-mediated phosphorylations of BECN1 Ser15 or ATG14 Ser29, indicating that the BECN1 Ser30 phosphorylation might regulate autophagy independently of those 2 sites. Taken together, these results demonstrate that BECN1 Ser30 is a ULK1 target site whose phosphorylation activates the ATG14-containing PIK3C3 complex and stimulates autophagosome formation in response to amino acid starvation, hypoxia, and MTORC1 inhibition.     

Arabidopsis ATG11, a scaffold that links the ATG1-ATG13 kinase complex to general autophagy and selective mitophagy

Autophagy is essential for nutrient recycling and intracellular housekeeping in plants by removing unwanted cytoplasmic constituents, aggregated polypeptides, and damaged organelles. The autophagy-related (ATG)1-ATG13 kinase complex is an upstream regulator that integrates metabolic and environmental cues into a coherent autophagic response directed by other ATG components. Our recent studies with Arabidopsis thaliana revealed that ATG11, an accessory protein of the ATG1-ATG13 complex, acts as a scaffold that connects the complex to autophagic membranes. We showed that ATG11 encourages proper behavior of the ATG1-ATG13 complex and faithful delivery of autophagic vesicles to the vacuole, likely through its interaction with ATG8. In addition, we demonstrated that Arabidopsis mitochondria are degraded during senescence via an autophagic route that requires ATG11 and other ATG components. Together, ATG11 appears to be an important modulator of the ATG1-ATG13 complex and a multifunctional scaffold required for bulk autophagy and the selective clearance of mitochondria.     

The autophagy-inducing kinases,ULK1 and ULK2, regulate axon guidance in the developing mouse forebrain via a noncanonical pathway

Mammalian ULK1 (unc-51 like kinase 1) and ULK2, Caenorhabditis elegans UNC-51, and Drosophila melanogaster Atg1 are serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. C. elegans UNC-51 and D. melanogaster Atg1 also promote axonal growth and defasciculation; disruption of these genes results in defective axon guidance in invertebrates. Although disrupting ULK1/2 function impairs normal neurite outgrowth in vitro, the role of ULK1 and ULK2 in the developing brain remains poorly characterized. Here, we show that ULK1 and ULK2 are required for proper projection of axons in the forebrain. Mice lacking Ulk1 and Ulk2 in their central nervous systems showed defects in axonal pathfinding and defasciculation affecting the corpus callosum, anterior commissure, corticothalamic axons and thalamocortical axons. These defects impaired the midline crossing of callosal axons and caused hypoplasia of the anterior commissure and disorganization of the somatosensory cortex. The axon guidance defects observed in ulk1/2 double-knockout mice and central nervous system-specific (Nes-Cre) Ulk1/2-conditional double-knockout mice were not recapitulated in mice lacking other autophagy genes (i.e., Atg7 or Rb1cc1 [RB1-inducible coiled-coil 1]). The brains of Ulk1/2-deficient mice did not show stem cell defects previously attributed to defective autophagy in ambra1 (autophagy/Beclin 1 regulator 1)- and Rb1cc1-deficient mice or accumulation of SQSTM1 (sequestosome 1)⁺ or ubiquitin⁺ deposits. Together, these data demonstrate that ULK1 and ULK2 regulate axon guidance during mammalian brain development via a noncanonical (i.e., autophagy-independent) pathway.     

The C. elegans ATG101 homolog EPG-9 directly interacts with EPG-1/Atg13 and is essential for autophagy

Autophagy is an evolutionarily conserved catabolic process that involves the engulfment of cytoplasmic contents in a closed double-membrane structure, called the autophagosome, and their subsequent delivery to the vacuole/lysosomes for degradation. Genetic screens in Saccharomyces cerevisiae have identified more than 30 autophagy-related (Atg) genes that are essential for autophagosome formation. Here we isolated a novel autophagy gene, epg-9, whose loss of function causes defective autophagic degradation of a variety of protein aggregates during C. elegans embryogenesis. Mutations in epg-9 also reduce survival of animals under food depletion conditions. epg-9 mutants exhibit autophagy phenotypes characteristic of those associated with loss of function of unc-51/Atg1 and epg-1/Atg13. epg-9 encodes a protein with significant homology to mammalian ATG101. EPG-9 directly interacts with EPG-1/Atg13. Our study indicates that EPG-9 forms a complex with EPG-1 in the aggrephagy pathway in C. elegans.     

Oxidative stress impairs autophagy through oxidation of ATG3 and ATG7

Dysfunctional macroautophagy/autophagy has been causatively linked to aging and the pathogenesis of many diseases, which are also broadly characterized by dysregulated cellular redox. As the autophagy-related (ATG) conjugation systems that mediate autophagosome maturation are cysteine dependent, their oxidation may account for loss in this catabolic process under conditions of oxidative stress. During active autophagy, LC3 is transferred from the catalytic thiol of ATG7 to the active site thiol of ATG3, where it is conjugated to phosphatidylethanolamine. In our recent study, we show LC3 is bound to the catalytic thiols of inactive ATG3 and ATG7 through a stable thioester, which becomes transient upon autophagy stimulation. Transient interaction with LC3 exposes the catalytic thiols on ATG3 and ATG7, which under pro-oxidizing conditions undergo inhibitory oxidation. This process was found to be upregulated in aged mouse tissue and therefore may account, at least in part, for impaired autophagy observed during aging.     

Phosphorylation of ULK1 serine 746 dictates ATG5-independent autophagy

ABSTRACT

There is a type of noncanonical autophagy, which is independent of ATG5 (autophagy related 5), also referred to as alternative autophagy. Both canonical and ATG5-independent alternative autophagy require the initiator ULK1 (unc-51 like kinase 1), but how ULK1 regulates these two types of autophagy differently remains unclear. A recent paper from Torii et al. demonstrates that phosphorylation of ULK1 at Ser746 by RIPK3 (receptor interacting serine/threonine kinase 3) is the key difference between these two types of autophagy; this phosphorylation is exclusively found during alternative autophagy.     

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

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Open and closed HORMAs regulate autophagy initiation

The Atg1/ULK complex functions as the most upstream factor among Atg proteins to initiate autophagy. ATG101 is a constitutive component of the Atg1/ULK complex in most eukaryotes except for budding yeast, and plays an essential role in autophagy; however, the structure and functions of ATG101 were largely unknown. Recently, we determined the crystal structure of fission yeast Atg101 in complex with the closed HORMA domain of Atg13, revealing that Atg101 is also a HORMA protein with an open conformation. These 2 HORMA proteins play essential roles in autophagy initiation through recruiting downstream factors to the autophagosome formation site.