Tissue-specific autophagy alterations and increased tumorigenesis in mice deficient in Atg4C/autophagin-3

Atg4C/autophagin-3 is a member of a family of cysteine proteinases proposed to be involved in the processing and delipidation of the mammalian orthologues of yeast Atg8, an essential component of an ubiquitin-like modification system required for execution of autophagy. To date, the in vivo role of the different members of this family of proteinases remains unclear. To gain further insights into the functional relevance of Atg4 orthologues, we have generated mutant mice deficient in Atg4C/autophagin-3. These mice are viable and fertile and do not display any obvious abnormalities, indicating that they are able to develop the autophagic response required during the early neonatal period. However, Atg4C-/--starved mice show a decreased autophagic activity in the diaphragm as assessed by immunoblotting studies and by fluorescence microscopic analysis of samples from Atg4C-/- GFP-LC3 transgenic mice. In addition, animals deficient in Atg4C show an increased susceptibility to develop fibrosarcomas induced by chemical carcinogens. Based on these results, we propose that Atg4C is not essential for autophagy development under normal conditions but is required for a proper autophagic response under stressful conditions such as prolonged starvation. We also propose that this enzyme could play an in vivo role in events associated with tumor progression.     

The Atg1 kinase complex is involved in the regulation of protein recruitment to initiate sequestering vesicle formation for nonspecific autophagy in Saccharomyces cerevisiae

Autophagy is the major degradative process for recycling cytoplasmic constituents and eliminating unnecessary organelles in eukaryotic cells. Most autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS), a proposed site for vesicle formation during either nonspecific or specific types of autophagy. Therefore, appropriate recruitment of Atg proteins to this site is critical for their function in autophagy. Atg11 facilitates PAS recruitment for the cytoplasm-to-vacuole targeting pathway, which is a specific, autophagy-like process that occurs under vegetative conditions. In contrast, it is not known how Atg proteins are recruited to the PAS, nor which components are involved in PAS formation under nonspecific autophagy-inducing, starvation conditions. Here, we studied PAS assembly during nonspecific autophagy, using an atg11Delta mutant background to eliminate the PAS formation that occurs during vegetative growth. We found that protein complexes containing the Atg1 kinase have two roles for PAS formation during nonspecific autophagy. The Atg1 C terminus mediates an interaction with Atg13 and Atg17, facilitating a structural role of Atg1 that is needed to efficiently organize an initial step of PAS assembly, whereas Atg1 kinase activity affects the dynamics of protein movement at the PAS involved in Atg protein cycling.     

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.     

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.     

Phosphorylation of Atg31 is required for autophagy

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17- Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.     

Atg22 recycles amino acids to link the degradative and recycling functions of autophagy

In response to stress conditions (such as nutrient limitation or accumulation of damaged organelles) and certain pathological situations, eukaryotic cells use autophagy as a survival mechanism. During nutrient stress the main purpose of autophagy is to degrade cytoplasmic materials within the lysosome/vacuole lumen and generate an internal nutrient pool that is recycled back to the cytosol. This study elucidates a molecular mechanism for linking the degradative and recycling roles of autophagy. We show that in contrast to published studies, Atg22 is not directly required for the breakdown of autophagic bodies within the lysosome/vacuole. Instead, we demonstrate that Atg22, Avt3, and Avt4 are partially redundant vacuolar effluxers, which mediate the efflux of leucine and other amino acids resulting from autophagic degradation. The release of autophagic amino acids allows the maintenance of protein synthesis and viability during nitrogen starvation. We propose a "recycling" model that includes the efflux of macromolecules from the lysosome/vacuole as the final step of autophagy.     

Rab7b modulates autophagic flux by interacting with Atg4B

Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co‐localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B.     

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.     

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.     

Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis

Atg8 is a ubiquitin-like protein required for autophagy in the budding yeast Saccharomyces cerevisiae. A ubiquitin-like system mediates the conjugation of the C terminus of Atg8 to the lipid phosphatidylethanolamine (PE), and this conjugate (Atg8-PE) plays a crucial role in autophagosome formation at the phagophore assembly site/pre-autophagosomal structure (PAS). The cysteine protease Atg4 processes the C terminus of newly synthesized Atg8 and also delipidates Atg8 to release the protein from membranes. While the former is a prerequisite for lipidation of Atg8, the significance of the latter in autophagy has remained unclear. Here, we show that autophagosome formation is significantly retarded in cells deficient for Atg4-mediated delipidation of Atg8. We find that Atg8-PE accumulates on various organelle membranes including the vacuole, the endosome and the ER in these cells, which depletes unlipidated Atg8 and thereby attenuates its localization to the PAS. Our results suggest that the Atg8-PE that accumulates on organelle membranes is erroneously produced by lipidation system components independently of the normal autophagic process. It is also suggested that delipidation of Atg8 by Atg4 on different organelle membranes promotes autophagosome formation. Considered together with other results, we propose that Atg4 acts to compensate for the intrinsic defect in the lipidation system; it recycles Atg8-PE generated on inappropriate membranes to maintain a reservoir of unlipidated Atg8 that is required for autophagosome formation at the PAS.     

Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis

Atg8 is a ubiquitin-like protein required for autophagy in the budding yeast Saccharomyces cerevisiae. A ubiquitin-like system mediates the conjugation of the C terminus of Atg8 to the lipid phosphatidylethanolamine (PE), and this conjugate (Atg8–PE) plays a crucial role in autophagosome formation at the phagophore assembly site/pre-autophagosomal structure (PAS). The cysteine protease Atg4 processes the C terminus of newly synthesized Atg8 and also delipidates Atg8 to release the protein from membranes. While the former is a prerequisite for lipidation of Atg8, the significance of the latter in autophagy has remained unclear. Here, we show that autophagosome formation is significantly retarded in cells deficient for Atg4-mediated delipidation of Atg8. We find that Atg8–PE accumulates on various organelle membranes including the vacuole, the endosome and the ER in these cells, which depletes unlipidated Atg8 and thereby attenuates its localization to the PAS. Our results suggest that the Atg8–PE that accumulates on organelle membranes is erroneously produced by lipidation system components independently of the normal autophagic process. It is also suggested that delipidation of Atg8 by Atg4 on different organelle membranes promotes autophagosome formation. Considered together with other results, we propose that Atg4 acts to compensate for the intrinsic defect in the lipidation system; it recycles Atg8–PE generated on inappropriate membranes to maintain a reservoir of unlipidated Atg8 that is required for autophagosome formation at the PAS.     

A cycling protein complex required for selective autophagy

Survival of environmental stress conditions requires the maintenance of cellular homeostasis. To preserve this balance, cells utilize a degradative mechanism known as autophagy. During this process, in response to starvation or other stresses, bulk cytoplasm is non-specifically sequestered within double-membrane vesicles and delivered to the lysosome/vacuole for subsequent degradation and recycling. The cytoplasm to vacuole targeting (Cvt) pathway is a type of specific autophagy, which occurs constitutively during growing conditions. Here, we examine three autophagy-related (Atg) proteins, Atg9, Atg23 and Atg27, which exhibit a unique localization pattern, residing both at the pre-autophagosomal structure (PAS) and other peripheral sites. These proteins colocalize, interact with one another in vivo, and form a functional complex. Furthermore, all three proteins cycle between the PAS and the other sites, and depend upon one another for this movement. Our data suggest that Atg9, Atg23 and Atg27 play a role in Atg protein retrieval from the PAS. In addition, Atg9 and Atg27 are the only known integral membrane Atg proteins involved in vesicle formation; a better understanding of their function may offer insight into the mechanism of membrane delivery to the PAS, the site of double-membrane vesicle assembly.     

Trs130 Participates in Autophagy Through GTPases Ypt31/32 in Saccharomyces cerevisiae

Trs130 is a specific component of the transport protein particle II complex, which functions as a guanine nucleotide exchange factor (GEF) for Rab GTPases Ypt31/32. Ypt31/32 is known to be involved in autophagy, although the precise mechanism has not been thoroughly studied. In this study, we investigated the potential involvement of Trs130 in autophagy and found that both the cytoplasm‐to‐vacuole targeting (Cvt) pathway and starvation‐induced autophagy were defective in a trs130ts (trs130 temperature‐sensitive) mutant. Mutant cells could not transport Atg8 and Atg9 to the pre‐autophagosomal structure/phagophore assembly site (PAS) properly, resulting in multiple Atg8 dots and Atg9 dots dispersed in the cytoplasm. Some dots were trapped in the trans‐Golgi. Genetic studies showed that the effect of the Trs130 mutation was downstream of Atg5 and upstream of Atg1, Atg13, Atg9 and Atg14 on the autophagic pathway. Furthermore, overexpression of Ypt31 or Ypt32, but not of Ypt1, rescued autophagy defects in trs130ts and trs65ts (Trs130‐HA Trs120‐myc trs65Δ) mutants. Our data provide mechanistic insight into how Trs130 participates in autophagy and suggest that vesicular trafficking regulated by GTPases/GEFs is important in the transport of autophagy proteins from the trans‐Golgi to the PAS.     

A Cycling Protein Complex Required for Selective Autophagy

Survival of environmental stress conditions requires the maintenance of cellular homeostasis. To preserve this balance, cells utilize a degradative mechanism known as autophagy. During this process, in response to starvation or other stresses, bulk cytoplasm is non-specifically sequestered within double-membrane vesicles and delivered to the lysosome/vacuole for subsequent degradation and recycling. The cytoplasm to vacuole targeting (Cvt) pathway is a type of specific autophagy, which occurs constitutively during growing conditions. Here, we examine three autophagy-related (Atg) proteins, Atg9, Atg23 and Atg27, which exhibit a unique localization pattern, residing both at the pre-autophagosomal structure (PAS) and other peripheral sites. These proteins colocalize, interact with one another in vivo, and form a functional complex. Furthermore, all three proteins cycle between the PAS and the other sites, and depend upon one another for this movement. Our data suggest that Atg9, Atg23 and Atg27 play a role in Atg protein retrieval from the PAS. In addition, Atg9 and Atg27 are the only known integral membrane Atg proteins involved in vesicle formation; a better understanding of their function may offer insight into the mechanism of membrane delivery to the PAS, the site of double-membrane vesicle assembly.     

The requirement of uncoordinated 51-like kinase 1 (ULK1) and ULK2 in the regulation of autophagy

Autophagy is an evolutionarily conserved physiological process of self-digestion by a cell to adapt to various stresses, including starvation. Its molecular basis involves the concerted activation of proteins encoded by the family of autophagy-related (Atg) genes. The best characterized is the serine/threonine protein kinase Atg1 in yeast which appears to be essential at the early stage of autophagy. In mammals, five Atg1 homologues have been identified as uncoordinated (UNC) 51-like kinase 1 to 4 and STK36. ULK1 and ULK2 are the most closely related members of the family, sharing 78% homology within their protein kinase domains. However, the specific function of ULK1 and ULK2 in mammalian autophagy is not fully understood. Here, we demonstrate that ULK1 and ULK2 are functionally redundant protein kinases required to mediate autophagy under nutrient-deprived conditions in fibroblasts. In contrast, ULK1, but not ULK2, is critical to induce the autophagic response of cerebellar granule neurons (CGN) to low potassium concentration in serum-free conditions. Furthermore, we found that ULK1 has a cytoprotective function in neurons. Together, these results provide strong genetic evidence that ULK1 is an essential component of the autophagic signaling pathway. The ability of ULK2 to compensate for the loss of ULK1 function is cell-type specific.     

Organization of the pre-autophagosomal structure responsible for autophagosome formation

Autophagy induced by nutrient depletion is involved in survival during starvation conditions. In addition to starvation-induced autophagy, the yeast Saccharomyces cerevisiae also has a constitutive autophagy-like system, the Cvt pathway. Among 31 autophagy-related (Atg) proteins, the function of Atg17, Atg29, and Atg31 is required specifically for autophagy. In this study, we investigated the role of autophagy-specific (i.e., non-Cvt) proteins under autophagy-inducing conditions. For this purpose, we used atg11Delta cells in which the Cvt pathway is abrogated. The autophagy-unique proteins are required for the localization of Atg proteins to the pre-autophagosomal structure (PAS), the putative site for autophagosome formation, under starvation condition. It is likely that these Atg proteins function as a ternary complex, because Atg29 and Atg31 bind to Atg17. The Atg1 kinase complex (Atg1-Atg13) is also essential for recruitment of Atg proteins to the PAS. The assembly of Atg proteins to the PAS is observed only under autophagy-inducing conditions, indicating that this structure is specifically involved in autophagosome formation. Our results suggest that Atg1 complex and the autophagy-unique Atg proteins cooperatively organize the PAS in response to starvation signals.     

Evidence that RME-1, a conserved C. elegans EH-domain protein, functions in endocytic recycling

In genetic screens for new endocytosis genes in Caenorhabditis elegans we identified RME-1, a member of a conserved class of Eps15-homology (EH)-domain proteins. Here we show that RME-1 is associated with the periphery of endocytic organelles, which is consistent with a direct role in endocytic transport. Endocytic defects in rme-1 mutants indicate that the protein is likely to have a function in endocytic recycling. Evidence from studies of mammalian RME-1 also points to a function for RME-1 in recycling, specifically in the exit of membrane proteins from recycling endosomes. These studies show a conserved function in endocytic recycling for the RME-1 family of EH proteins.     

Atg9 trafficking in Mammalian cells

The molecular mechanisms of autophagy have been best characterized in the yeast Saccharomyces cerevisiae, where a number of proteins have been identified to be essential for this degradative pathway. ATG (autophagy-related) proteins(1) localize to a unique compartment, the pre-autophagosomal structure (PAS). Isolation membranes are suggested to originate from the PAS, enwrapping cytoplasmic components to form a double membrane autophagosome, which then fuses with the vacuole. Although many Atg proteins have been identified, the source of the PAS membrane in yeast is unknown. Identification of the source of the PAS in yeast has been hindered due to the transient association of Atg proteins with forming autophagosomes.(2) Likewise, in mammalian cells, it is not known if a PAS equivalent exists or if the formation of autophagosomes occurs from numerous membrane sources. The identification of stably associated markers would allow us to address this question further. Thus, characterization of the only transmembrane autophagy protein so far identified, Atg9, may aid in the search for the source of the PAS. Recent data from our lab suggests that mammalian Atg9 (mAtg9) traffics between the Golgi and endosomes, and suggests an involvement of the Golgi complex in the autophagic pathway.(3) Here we address the implications of our model with regard to membrane trafficking events in mammalian cells after starvation.     

Assortment of phosphatidylinositol 3-kinase complexes--Atg14p directs association of complex I to the pre-autophagosomal structure in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, two similar phosphatidylinositol 3-kinase complexes (complexes I and II) function in distinct biological processes, complex I in autophagy and complex II in the vacuolar protein sorting via endosomes. Atg14p is only integrated into complex I, likely facilitating the function of complex I in autophagy. Deletion analysis of Atg14p revealed that N-terminal region containing the coiled-coil structures was essential and sufficient for autophagy. Atg14p localized to pre-autophagosomal structure (PAS) and vacuolar membranes, whereas Vps38p, a component specific to complex II, localized to endosomes and vacuolar membranes. Vps34p and Vps30p, components shared by the two complexes, localized to the PAS, vacuolar membranes, and several punctate structures that included endosomes. The localization of these components to the PAS was Atg14p dependent but not dependent on Vps38p. Conversely, localization of these proteins to endosomes required Vps38p but not Atg14p. Vps15p, regulatory subunit of the Vps34p complexes, localized to the PAS, vacuolar membranes, and punctate structures independent of both Atg14p and Vps38p. Together, these results indicate that complexes I and II function in distinct biological processes by localizing to specific compartments in a manner mediated by specific components of each complex, Atg14p and Vps38p, respectively.     

Coordinated regulation of autophagy by p38α MAPK through mAtg9 and p38IP

Autophagy, a lysosomal degradation pathway, is essential for homeostasis, development, neurological diseases, and cancer. Regulation of autophagy in human disease is not well understood. Atg9 is a transmembrane protein required for autophagy, and it has been proposed that trafficking of Atg9 may regulate autophagy. Mammalian Atg9 traffics between the TGN and endosomes in basal conditions, and newly formed autophagosomes in response to signals inducing autophagy. We identified p38IP as a new mAtg9 interactor and showed that this interaction is regulated by p38α MAPK. p38IP is required for starvation‐induced mAtg9 trafficking and autophagosome formation. Manipulation of p38IP and p38α alters mAtg9 localization, suggesting p38α regulates, through p38IP, the starvation‐induced mAtg9 trafficking to forming autophagosomes. Furthermore, we show that p38α is a negative regulator of both basal autophagy and starvation‐induced autophagy, and suggest that this regulation may be through a direct competition with mAtg9 for binding to p38IP. Our results provide evidence for a link between the MAPK pathway and the control of autophagy through mAtg9 and p38IP.