A PCR analysis of the ubiquitin-like conjugation systems in macroautophagy

A central part of the core macroauto-phagy (hereafter autophagy) machinery includes the two ubiquitin-like (Ubl) conjugation systems that involve the Ubl proteins Atg8 and Atg12.¹ Although the functions of these proteins have not been fully elucidated, they play critical roles in autophagosome formation. For example, Atg8 is involved in cargo recognition,^2,3 and the amount of Atg8 in part determines the size of the autophagosome,⁴ whereas Atg12 is part of a trimer that may function as an E3 ligase to facilitate Atg8 conjugation to phosphatidylethanolamine and determine, in part, the site of the conjugation reaction.⁵ Thus, fully functional autophagy requires both the Atg8 and Atg12 conjugation systems. Dysfunctional autophagy is associated with various human pathophysiologies including cancer, neurodegeneration, gastrointestinal disorders and heart disease. So, if you are wondering whether autophagy is operating properly in your own body, what can you do? The problem is that there are relatively few methods for analyzing autophagy in vivo.^6-11 Minimally, you might want to find out if the relevant genes are intact and have the correct sequence. Considering the rapid advances being made in DNA sequencing technology, it is likely only a matter of time before people can submit a DNA sample and obtain a rapid readout of particular genes, or their entire genome. Thus, anticipating the future, we decided to analyze a select set of autophagy-related (ATG) genes, with a focus on those encoding components of the Ubl conjugation systems, by a polymerase chain reaction (PCR)-based method that combines science with art.     

Evolution of Atg1 function and regulation

The serine/threonine kinase Atg1 plays an essential role downstream of TOR for the regulation of autophagy. In yeast, where Atg1 was first identified, a complex regulatory mechanism has been described that includes at least seven other interacting proteins and a phosphorylation-dependent switch. Recent findings confirm that the mammalian Atg1 homologues ULK1 and 2 have autophagy regulatory roles. However, we and others have also demonstrated mechanistic differences with the yeast model and between these two Atg1 family members. Here, we elaborate on our growing understanding of Atg1 function, incorporating findings from yeast, C. elegans, D. melanogaster and mammalian cells. We propose that through evolution, Atg1 proteins have adopted additional cellular functions and regulatory mechanisms, which could involve multiple gene family isoforms working within multi-functional protein complexes. The gene family expansion observed in higher eukaryotes might reflect an increased functional diversity of Atg1 proteins in cell growth, differentiation and survival.     

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.     

A PCR analysis of the ubiquitin-like conjugation systems in macroautophagy

A central part of the core macroauto-phagy (hereafter autophagy) machinery includes the two ubiquitin-like (Ubl) conjugation systems that involve the Ubl proteins Atg8 and Atg12.¹ Although the functions of these proteins have not been fully elucidated, they play critical roles in autophagosome formation. For example, Atg8 is involved in cargo recognition,^2,3 and the amount of Atg8 in part determines the size of the autophagosome,⁴ whereas Atg12 is part of a trimer that may function as an E3 ligase to facilitate Atg8 conjugation to phosphatidylethanolamine and determine, in part, the site of the conjugation reaction.⁵ Thus, fully functional autophagy requires both the Atg8 and Atg12 conjugation systems. Dysfunctional autophagy is associated with various human pathophysiologies including cancer, neurodegeneration, gastrointestinal disorders and heart disease. So, if you are wondering whether autophagy is operating properly in your own body, what can you do? The problem is that there are relatively few methods for analyzing autophagy in vivo.^6-11 Minimally, you might want to find out if the relevant genes are intact and have the correct sequence. Considering the rapid advances being made in DNA sequencing technology, it is likely only a matter of time before people can submit a DNA sample and obtain a rapid readout of particular genes, or their entire genome. Thus, anticipating the future, we decided to analyze a select set of autophagy-related (ATG) genes, with a focus on those encoding components of the Ubl conjugation systems, by a polymerase chain reaction (PCR)-based method that combines science with art.Key words: autophagy, collaboration, gel electrophoresis, membrane, primer     

Autophagy,proteases and the sense of balance

The knowledge of the molecular mechanisms underlying autophagy has considerably improved after the isolation and characterization of autophagy-defective mutants in the yeast Saccharomyces cerevisiae. Two ubiquitin-like conjugation systems are required for yeast autophagy. One of them requires the participation of Atg8 synthesized as a precursor protein, which is cleaved after a Gly residue by a cysteine proteinase called Atg4. The new Gly-terminal residue from Atg8 is activated by Atg7 (an E1-like enzyme) then transferred to Atg3 (an E2-like enzyme) and finally conjugated with membrane-bound phosphatidylethanolamine (PE) through an amide bond. The complex Atg8–PE is also deconjugated by the protease Atg4, facilitating the release of Atg8 from membranes. This modification system, which is essential for the membrane rearrangement dynamics that accompany the initiation and execution of autophagy, is conserved in higher eukaryotes including mammals. We have previously identified and cloned the four human orthologues of the yeast proteinase Atg4, whereas parallel studies have revealed that there are at least six orthologues of yeast Atg8 in mammals (LC3A, LC3B, LC3C, GABARAP, ATG8L/GABARAPL1 and GATE-16/GABARAPL2). Thus, in mammals, the Atg4-Atg8 proteolytic system is composed of four proteinases (autophagins) that may target at least six distinct substrates, contrasting with the simplified yeast system in which one single protease cleaves a sole substrate. Currently, it is unclear why mammals have developed this array of closely related enzymes, as other essential autophagy genes such as Atg3, Atg5 or Atg7 are represented in mammalian cells by a single orthologue. It has been suggested that the multiplication of Atg4 orthologues may reflect a regulatory heterogeneity of functionally redundant proteins or, alternatively, derive from the acquisition of new functions that are not related to autophagy. Our first approach to elucidate this question was based on the generation of autophagin-3/Atg4C-deficient mice, which however presented a minor phenotype. With the generation of autophagin-1/Atg4B-deficient mice, recently reported, we have progressed in our attempt to identify the in vivo physiological and pathological roles of autophagins.     

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.     

The Crystal Structure of Plant ATG12 and its Biological Implication in Autophagy

Atg12 is a post-translational modifier that is activated and conjugated to its single target, Atg5, by a ubiquitin-like conjugation system. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Here, we demonstrate that the Atg12 conjugation system exists in Arabidopsis and is essential for plant autophagy as well as in yeast and mammals. We also report the crystal structure of Arabidopsis thaliana (At) ATG12 at 1.8 Å resolution. Despite no obvious sequence homology with ubiquitin, the structure of AtATG12 shows a ubiquitin fold strikingly similar to those of mammalian homologs of Atg8, the other ubiquitin-like modifier essential for autophagy, which is conjugated to phosphatidylethanolamine. Two types of hydrophobic patches are present on the surface of AtATG12: one is conserved in both Atg12 and Atg8 orthologs, while the other is unique to Atg12 orthologs. Considering that they share Atg7 as an E1-like enzyme, we suggest that the first hydrophobic patch is responsible for the conjugation reaction, while the latter is involved in Atg12-specific functions.     

Dual roles of Atg8-PE deconjugation by Atg4 in autophagy

Modification of target molecules by ubiquitin or ubiquitin-like (Ubl) proteins is generally reversible. Little is known, however, about the physiological function of the reverse reaction, deconjugation. Atg8 is a unique Ubl protein whose conjugation target is the lipid phosphatidylethanolamine (PE). Atg8 functions in the formation of double-membrane autophagosomes, a central step in the well-conserved intracellular degradation pathway of macroautophagy (hereafter autophagy). Here we show that the deconjugation of Atg8-PE by the cysteine protease Atg4 plays dual roles in the formation of autophagosomes. During the early stage of autophagosome formation, deconjugation releases Atg8 from non-autophagosomal membranes to maintain a proper supply of Atg8. At a later stage, the release of Atg8 from intermediate autophagosomal membranes facilitates the maturation of these structures into fusion-capable autophagosomes. These results provide new insights into the functions of Atg8-PE and its deconjugation.     

Atg8: an autophagy-related ubiquitin-like protein family

Autophagy-related (Atg) proteins are eukaryotic factors participating in various stages of the autophagic process. Thus far 34 Atgs have been identified in yeast, including the key autophagic protein Atg8. The Atg8 gene family encodes ubiquitin-like proteins that share a similar structure consisting of two amino-terminal α helices and a ubiquitin-like core. Atg8 family members are expressed in various tissues, where they participate in multiple cellular processes, such as intracellular membrane trafficking and autophagy. Their role in autophagy has been intensively studied. Atg8 proteins undergo a unique ubiquitin-like conjugation to phosphatidylethanolamine on the autophagic membrane, a process essential for autophagosome formation. Whereas yeast has a single Atg8 gene, many other eukaryotes contain multiple Atg8 orthologs. Atg8 genes of multicellular animals can be divided, by sequence similarities, into three subfamilies: microtubule-associated protein 1 light chain 3 (MAP1LC3 or LC3), γ-aminobutyric acid receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE-16), which are present in sponges, cnidarians (such as sea anemones, corals and hydras) and bilateral animals. Although genes from all three subfamilies are found in vertebrates, some invertebrate lineages have lost the genes from one or two subfamilies. The amino terminus of Atg8 proteins varies between the subfamilies and has a regulatory role in their various functions. Here we discuss the evolution of Atg8 proteins and summarize the current view of their function in intracellular trafficking and autophagy from a structural perspective.     

Dual roles of Atg8−PE deconjugation by Atg4 in autophagy

Modification of target molecules by ubiquitin or ubiquitin-like (Ubl) proteins is generally reversible. Little is known, however, about the physiological function of the reverse reaction, deconjugation. Atg8 is a unique Ubl protein whose conjugation target is the lipid phosphatidylethanolamine (PE). Atg8 functions in the formation of double-membrane autophagosomes, a central step in the well-conserved intracellular degradation pathway of macroautophagy (hereafter autophagy). Here we show that the deconjugation of Atg8?PE by the cysteine protease Atg4 plays dual roles in the formation of autophagosomes. During the early stage of autophagosome formation, deconjugation releases Atg8 from non-autophagosomal membranes to maintain a proper supply of Atg8. At a later stage, the release of Atg8 from intermediate autophagosomal membranes facilitates the maturation of these structures into fusion-capable autophagosomes. These results provide new insights into the functions of Atg8?PE and its deconjugation.     

The coiled-coil domain protein EPG-8 plays an essential role in the autophagy pathway in C. elegans

Macroautophagy (hereafter referred to as autophagy) involves the formation of a closed, double membrane structure, called the autophagosome. Most of the Atg proteins that are essential for autophagosome formation are evolutionarily conserved between yeast and higher eukaryotes. The functions of some Atg proteins, however, are mediated by highly divergent proteins in mammalian cells. In this study, we identified a novel coiled-coil domain protein, EPG-8, that plays an essential role in the autophagy pathway in C. elegans. Mutations in epg-8 cause defects in degradation of various autophagy substrates and also compromise survival of animals under nutrient-depletion conditions. In epg-8 mutants, lipidated LGG-1 (the C. elegans Atg8 homolog) accumulates but does not form distinct punctate structures. EPG-8 directly interacts with the C. elegans Beclin 1 homolog, BEC-1. Our study demonstrates that epg-8 may function as a highly divergent homolog of the yeast autophagy gene Atg14.     

Structure-function relationship of Atg12, a ubiquitin-like modifier essential for autophagy

Atg12, a post-translational modifier, is activated and conjugated to Atg5 by a ubiquitin-like conjugation system, though it has no obvious sequence homology to ubiquitin. The Atg12-Atg5 conjugate is essential for autophagy, an intracellular bulk degradation process. Here, we show that the carboxyl-terminal region of Atg12 that is predicted to fold into a ubiquitin-like structure is necessary and sufficient for both conjugation and autophagy, which indicates that the domain essential for autophagy resides in the ubiquitin-fold region. We further show that two hydrophobic residues within the ubiquitin-fold region are important for autophagy: mutation at Y149 affects conjugate formation catalyzed by Atg10, an E2-like enzyme, while mutation at F154 has no effect on Atg12-Atg5 conjugate formation but its hydrophobic nature is essential for autophagy. In response to the F154 mutation, Atg8-PE conjugation, the other ubiquitin-like conjugation in autophagy, is severely reduced and autophagosome formation fails. Gel filtration analysis suggests that F154 plays a critical role in the assembly of a functional Atg12-Atg5.Atg16 complex that is requisite for autophagosome formation.     

The crystal structure of plant ATG12 and its biological implication in autophagy

Atg12 is a post-translational modifier that is activated and conjugated to its single target, Atg5, by a ubiquitin-like conjugation system. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Here, we demonstrate that the Atg12 conjugation system exists in Arabidopsis and is essential for plant autophagy as well as in yeast and mammals. We also report the crystal structure of Arabidopsis thaliana (At) ATG12 at 1.8 A resolution. Despite no obvious sequence homology with ubiquitin, the structure of AtATG12 shows a ubiquitin fold strikingly similar to those of mammalian homologs of Atg8, the other ubiquitin-like modifier essential for autophagy, which is conjugated to phosphatidylethanolamine. Two types of hydrophobic patches are present on the surface of AtATG12: one is conserved in both Atg12 and Atg8 orthologs, while the other is unique to Atg12 orthologs. Considering that they share Atg7 as an E1-like enzyme, we suggest that the first hydrophobic patch is responsible for the conjugation reaction, while the latter is involved in Atg12-specific functions.     

Autophagy-related Protein 8 (Atg8) Family Interacting Motif in Atg3 Mediates the Atg3-Atg8 Interaction and Is Crucial for the Cytoplasm-to-Vacuole Targeting Pathway

The autophagy-related protein 8 (Atg8) conjugation system is essential for the formation of double-membrane vesicles called autophagosomes during autophagy, a bulk degradation process conserved among most eukaryotes. It is also important in yeast for recognizing target vacuolar enzymes through the receptor protein Atg19 during the cytoplasm-to-vacuole targeting (Cvt) pathway, a selective type of autophagy. Atg3 is an E2-like enzyme that conjugates Atg8 with phosphatidylethanolamine. Here, we show that Atg3 directly interacts with Atg8 through the WEDL sequence, which is distinct from canonical interaction between E2 and ubiquitin-like modifiers. Moreover, NMR experiments suggest that the mode of interaction between Atg8 and Atg3 is quite similar to that between Atg8/LC3 and the Atg8 family interacting motif (AIM) conserved in autophagic receptors, such as Atg19 and p62. Thus, the WEDL sequence in Atg3 is a canonical AIM. In vitro analyses showed that Atg3 AIM is crucial for the transfer of Atg8 from the Atg8∼Atg3 thioester intermediate to phosphatidylethanolamine but not for the formation of the intermediate. Intriguingly, in vivo experiments showed that it is necessary for the Cvt pathway but not for starvation-induced autophagy. Atg3 AIM attenuated the inhibitory effect of Atg19 on Atg8 lipidation in vitro, suggesting that Atg3 AIM may be important for the lipidation of Atg19-bound Atg8 during the Cvt pathway.     

Beyond Atg8 binding: The role of AIM/LIR motifs in autophagy

Selective macroautophagy/autophagy mediates the selective delivery of cytoplasmic cargo material via autophagosomes into the lytic compartment for degradation. This selectivity is mediated by cargo receptor molecules that link the cargo to the phagophore (the precursor of the autophagosome) membrane via their simultaneous interaction with the cargo and Atg8 proteins on the membrane. Atg8 proteins are attached to membrane in a conjugation reaction and the cargo receptors bind them via short peptide motifs called Atg8-interacting motifs/LC3-interacting regions (AIMs/LIRs). We have recently shown for the yeast Atg19 cargo receptor that the AIM/LIR motifs also serve to recruit the Atg12–Atg5-Atg16 complex, which stimulates Atg8 conjugation, to the cargo. We could further show in a reconstituted system that the recruitment of the Atg12–Atg5-Atg16 complex is sufficient for cargo-directed Atg8 conjugation. Our results suggest that AIM/LIR motifs could have more general roles in autophagy.     

A novel,human Atg13 binding protein,Atg101, interacts with ULK1 and is essential for macroautophagy

Macroautophagy is an intracellular, vesicle-mediated mechanism for the sequestration and ultimate lysosomal degradation of cytoplasmic proteins, organelles and macromolecules. The macroautophagy process and many of the autophagy specific (Atg) proteins are remarkably well conserved in higher eukaryotes. In yeast, the Atg1 kinase complex includes Atg1, Atg13, Atg17, and at least four other interacting proteins, some of which are phosphorylated in a TOR-dependent manner, placing the Atg1 signaling complex downstream of a major nutrient-sensing pathway. Atg1 orthologs, including mammalian unc-51-like kinase 1 (ULK1), have been identified in higher eukaryotes and have been functionally linked to autophagy. This suggests that other components of the Atg1 complex exist in higher eukaryotes. Recently, a putative human Atg13 ortholog, FLJ20698, was identified by gapped-BLAST analysis. We show here that FLJ20698 (Atg13) is a ULK1-interacting phosphoprotein that is essential for macroautophagy. Furthermore, we identify a novel, human Atg13-interacting protein, FLJ11773, which we have termed Atg101. Atg101 is essential for autophagy and interacts with ULK1 in an Atg13-dependent manner. Additionally, we present evidence that intracellular localization of the ULK1 complex is regulated by nutrient conditions. Finally, we demonstrate that Atg101 stabilizes the expression of Atg13 in the cell, suggesting that Atg101 contributes to Atg13 function by protecting Atg13 from proteasomal degradation. Therefore, the identification of the novel protein, Atg101, and the validation of Atg13 and Atg101 as ULK1-interacting proteins, suggests an Atg1 complex is involved in the induction of macroautophagy in mammalian cells.     

Identification and characterization of two rice autophagy associated genes, OsAtg8 and OsAtg4

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. The molecular machinery responsible for yeast and mammalian autophagy has begun to be elucidated at the cellular level. A genome-wide search revealed significant conservation among autophagy genes in yeast and Arabidopsis. Up till now, however, there is no report about rice autophagy associated genes. Here we cloned OsAtg8 and OsAtg4 from Oryza sativa and detected their expression patterns in various tissues. Immunoblotting analysis showed that carboxyl terminus of OsAtg8 can be cleaved in yeast cell. Mutation analysis revealed that the conserved Gly117 residue of OsAtg8 was essential for its characteristic C-terminal cleavage as similar to that found in mammalian and yeast Atg8. We further proved that OsAtg8 interacted with OsAtg4, and this interaction was not affected by the conserved Gly117 mutation. Our results demonstrate that Atg8 conjugation pathway is conserved in rice and may play important roles in rice autophagy.     

Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors

Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.