The FAP motif within human ATG7, an autophagy-related E1-like enzyme, is essential for the E2-substrate reaction of LC3 lipidation

ATG7 is an autophagy-related E1-like enzyme that is essential for two ubiquitination-like reactions, ATG12-conjugation and LC3-lipidation. The existence of functional sequences at the amino-terminal region of human ATG7 remains uncertain. Mutational analyses of ATG7 revealed that both mutant ATG7ΔFAP lacking the FAP motif and ATG7FAPtoDDD, in which the Phe15-Ala16-Pro17 sequence was changed to Asp-Asp-Asp, could not complement defects in endogenous ATG12-conjugation and LC3-lipidation when expressed in Atg7-deficient mouse embryonic fibroblasts (MEFs). However, wild-type ATG7 complemented the defects in these cells. Overexpression of GFP-ATG10 and GFP-ATG12 rescued a defect in ATG12-conjugation in Atg7-deficient MEFs expressing mutant ATG7ΔFAP and ATG7FAPtoDDD, whereas overexpression of all ATG proteins related to ATG12-conjugation and LC3-lipidation could not rescue a defect in LC3-lipidation in Atg7-deficient MEFs expressing these ATG7 mutants. Both ATG7ΔFAP and ATG7FAPtoDDD mutants showed severe defects in the formation of an E2-substrate intermediate of ATG3 with LC3 in LC3-lipidation, but were able to form an E1-substrate intermediate of ATG7 with LC3 and the E1- and E2-substrate intermediates in ATG12-conjugation with reduced efficiency. These ATG7 mutants could also form the ATG12-ATG3 conjugate. Co-immunoprecipitation experiments revealed that the FAP motif of ATG7 is essential for the interaction of ATG7 with ATG3, but not for ATG7-homodimerization. These results indicated that the FAP motif of ATG7 is indispensable for formation of the ATG3-LC3 E2-substrate intermediate through the interaction of ATG7 with ATG3.     

Phosphatidylserine in addition to phosphatidylethanolamine is an in vitro target of the mammalian Atg8 modifiers, LC3, GABARAP, and GATE-16

In yeast, phosphatidylethanolamine is a target of the Atg8 modifier in ubiquitylation-like reactions essential for autophagy. Three human Atg8 (hAtg8) homologs, LC3, GABARAP, and GATE-16, have been characterized as modifiers in reactions mediated by hAtg7 (an E1-like enzyme) and hAtg3 (an E2-like enzyme) as in yeast Atg8 lipidation, but their final targets have not been identified. The results of a recent study in which COS7 cells were incubated with [14C]ethanolamine for 48 h suggested that phosphatidylethanolamine is a target of LC3. However, these results were not conclusive because of the long incubation time. To identify the phospholipid targets of Atg8 homologs, we reconstituted conjugation systems for mammalian Atg8 homologs in vitro using purified recombinant Atg proteins and liposomes. Each purified mutant Atg8 homolog with an exposed C-terminal Gly formed an E1-substrate intermediate with hAtg7 via a thioester bond in an ATP-dependent manner and formed an E2-substrate intermediate with hAtg3 via a thioester bond dependent on ATP and hAtg7. A conjugated form of each Atg8 homolog was observed in the presence of hAtg7, hAtg3, ATP, and liposomes. In addition to phosphatidylethanolamine, in vitro conjugation experiments using synthetic phospholipid liposomes showed that phosphatidylserine is also a target of LC3, GABARAP, and GATE-16. In contrast, thin layer chromatography of phospholipids released on hAtg4B-digestion from endogenous LC3-phospholipid conjugate revealed that phosphatidylethanolamine, but not phosphatidylserine, is the predominant target phospholipid of LC3 in vivo. The discrepancy between in vitro and in vivo reactions suggested that there may be selective factor(s) involved in the endogenous LC3 conjugation system.     

The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy

Two ubiquitin-like molecules, Atg12 and LC3/Atg8, are involved in autophagosome biogenesis. Atg12 is conjugated to Atg5 and forms an ~800-kDa protein complex with Atg16L (referred to as Atg16L complex). LC3/Atg8 is conjugated to phosphatidylethanolamine and is associated with autophagosome formation, perhaps by enabling membrane elongation. Although the Atg16L complex is required for efficient LC3 lipidation, its role is unknown. Here, we show that overexpression of Atg12 or Atg16L inhibits autophagosome formation. Mechanistically, the site of LC3 lipidation is determined by the membrane localization of the Atg16L complex as well as the interaction of Atg12 with Atg3, the E2 enzyme for the LC3 lipidation process. Forced localization of Atg16L to the plasma membrane enabled ectopic LC3 lipidation at that site. We propose that the Atg16L complex is a new type of E3-like enzyme that functions as a scaffold for LC3 lipidation by dynamically localizing to the putative source membranes for autophagosome formation.     

The structure of Atg4B–LC3 complex reveals the mechanism of LC3 processing and delipidation during autophagy

Atg8 is conjugated to phosphatidylethanolamine (PE) by ubiquitin‐like conjugation reactions. Atg8 has at least two functions in autophagy: membrane biogenesis and target recognition. Regulation of PE conjugation and deconjugation of Atg8 is crucial for these functions in which Atg4 has a critical function by both processing Atg8 precursors and deconjugating Atg8–PE. Here, we report the crystal structures of catalytically inert human Atg4B (HsAtg4B) in complex with processed and unprocessed forms of LC3, a mammalian orthologue of yeast Atg8. On LC3 binding, the regulatory loop and the N‐terminal tail of HsAtg4B undergo large conformational changes. The regulatory loop masking the entrance of the active site of free HsAtg4B is lifted by LC3 Phe119, so that a groove is formed along which the LC3 tail enters the active site. At the same time, the N‐terminal tail masking the exit of the active site of HsAtg4B in the free form is detached from the enzyme core and a large flat surface is exposed, which might enable the enzyme to access the membrane‐bound LC3–PE.     

Molecular cloning and characterization of rat LC3A and LC3B--two novel markers of autophagosome

Rat microtubule-associated protein light chain 3 (LC3) is a homologue of yeast Atg8, an essential component of autophagy. Following synthesis, the C-terminus of rat LC3 is cleaved by a cysteine protease-Atg4, to produce LC3-I, which is located in cytosolic fraction. LC3-I can be converted to LC3-II through the processing by Atg7 (E1-like enzyme) and Atg3 (E2-like enzyme). LC3-II is modified by phosphatidylethanolamine on C-terminus and binds tightly to autophagosomal membrane. Here we reported the cloning of two novel variants of rat LC3, named LC3A and LC3B, respectively, and LC3B is an alternative splicing variant of LC3. LC3A, LC3B, and LC3 showed different expression patterns in rat tissues, suggesting a functional divergence among these proteins. When LC3A and LC3B were overexpressed, both exhibited two forms (18 and 16 kDa, representing types of I and II, separately), which might be due to post-translational modification including the characteristic C-terminal cleavage at these two proteins as similar to that found in rat LC3 and yeast Atg8. Subcellular localization demonstrated that both LC3A and LC3B are colocalized with LC3 and associated with the autophagic membranes. Mutation analysis further revealed that the conserved Gly120 residues of LC3A and LC3B are essential for their characteristic C-terminal cleavage and localization to autophagic membranes. Present data suggested that LC3A and LC3B could also be used as two novel autophagosomal markers.     

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.     

An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure

In the process of autophagy, a ubiquitin-like molecule, LC3/Atg8, is conjugated to phosphatidylethanolamine (PE) and associates with forming autophagosomes. In mammalian cells, the existence of multiple Atg8 homologues (referred to as LC3 paralogues) has hampered genetic analysis of the lipidation of LC3 paralogues. Here, we show that overexpression of an inactive mutant of Atg4B, a protease that processes pro-LC3 paralogues, inhibits autophagic degradation and lipidation of LC3 paralogues. Inhibition was caused by sequestration of free LC3 paralogues in stable complexes with the Atg4B mutant. In mutant overexpressing cells, Atg5- and ULK1-positive intermediate autophagic structures accumulated. The length of these membrane structures was comparable to that in control cells; however, a significant number were not closed. These results show that the lipidation of LC3 paralogues is involved in the completion of autophagosome formation in mammalian cells. This study also provides a powerful tool for a wide variety of studies of autophagy in the future.     

Binding to E1 and E3 is mutually exclusive for the human autophagy E2 Atg3

Ubiquitin‐like proteins (UBLs) are activated, transferred and conjugated by E1‐E2‐E3 enzyme cascades. E2 enzymes for canonical UBLs such as ubiquitin, SUMO, and NEDD8 typically use common surfaces to bind to E1 and E3 enzymes. Thus, canonical E2s are required to disengage from E1 prior to E3‐mediated UBL ligation. However, E1, E2, and E3 enzymes in the autophagy pathway are structurally and functionally distinct from canonical enzymes, and it has not been possible to predict whether autophagy UBL cascades are organized according to the same principles. Here, we address this question for the pathway mediating lipidation of the human autophagy UBL, LC3. We utilized bioinformatic and experimental approaches to identify a distinctive region in the autophagy E2, Atg3, that binds to the autophagy E3, Atg12～Atg5‐Atg16. Short peptides corresponding to this Atg3 sequence inhibit LC3 lipidation in vitro. Notably, the E3‐binding site on Atg3 overlaps with the binding site for the E1, Atg7. Accordingly, the E3 competes with Atg7 for binding to Atg3, implying that Atg3 likely cycles back and forth between binding to Atg7 for loading with the UBL LC3 and binding to E3 to promote LC3 lipidation. The results show that common organizational principles underlie canonical and noncanonical UBL transfer cascades, but are established through distinct structural features.     

Mammalian Atg8s: One is simply not enough

Yeast Atg8, a key factor in the autophagic process, is a ubiquitin-like protein that undergoes a unique conjugation to phosphatidylethanolamine (PE). Atg8 plays a dual role in early stages of autophagosome formation: It was implicated in recruitment of cargo proteins such as Atg19 and Atg32 for Cvt and mitophagy, respectively, and in autophagosome biogenesis, serving as an elongation factor by mediating membrane hemi-fusion. Similarly, the mammalian Atg8 proteins, LC3s and GABARAPs, recruit cargo into autophagosomes by binding to adaptor proteins such as p62, NBR1 and Nix. These functions, however, are not essential for bulk autophagic flux. Other studies in which the activity of the mammalian Atg8s was blocked either by knockout of the E2-like enzyme Atg3 or by using a dominant negative mutant of the promiscuous protease Atg4B revealed, in agreement with the yeast Atg8 data, that the mammalian factors are crucial for the formation of normal and mature autophagosomes. While it seems that the single yeast Atg8 and the mammalian Atg8s share similar roles, it is still unclear why the mammalian system employs several homologs. Recent publications demonstrated that the mammalian Atg8s differ in their cargo specificity, as Nix, for example, binds exclusively to GABARAP-L1. This may suggest that these proteins exhibit distinct activity also in autophagosome biogenesis. In our study we divided the mammalian Atg8s into two subfamilies of homologs based on amino acid similarity, the LC3 and GABARAP/GATE-16 subfamilies, and tested their essentiality and role in autophagy. In agreement with previous studies we found that the mammalian Atg8s are essential for autophagy but, more importantly, that each of these subfamilies has a distinct role in the process of autophagosome biogenesis.     

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.     

LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization

Autophagy is an intracellular bulk degradation system, through which a portion of the cytoplasm is delivered to lysosomes to be degraded. Microtuble-associated protein light chain 3 (LC3), a mammalian homolog of yeast Atg8, has been used as a specific marker to monitor autophagy. Upon induction of autophagy, LC3 is conjugated to phosphatidylethanolamine and targeted to autophagic membranes. Therefore, changes in LC3 localization have been used to measure autophagy. However, this method has some limitations. In this report, we show that LC3 protein tends to aggregate in an autophagy-independent manner when it is transiently overexpressed by transfection. In addition, LC3 is easily incorporated into intracellular protein aggregates, such as inclusion bodies induced by polyQ expression or formed in autophagy-deficient hepatocytes, neurons, or senescent fibroblasts. These findings demonstrate that punctate dots containing LC3 do not always represent autophagic structures. Therefore, LC3 localization should be carefully interpreted, particularly if LC3 is overexpressed by transient transfection or if aggregates are formed within cells.     

Human light chain 3/MAP1LC3B is cleaved at its carboxyl-terminal Met121 to expose Gly120 for lipidation and targeting to autophagosomal membranes

Human light chain 3/MAP1LC3B, an autophagosomal ortholog of yeast Atg8, is conjugated to phospholipid (PL) via ubiquitylation-like reactions mediated by human Atg7 and Atg3. Since human Atg4B was found to cleave the carboxyl terminus of MAP1LC3B in vitro, we hypothesized that this exposes its carboxyl-terminal Gly(120). It was recently reported, however, that when Myc-MAP1LC3B-His is expressed in HEK293 cells, its carboxyl terminus is not cleaved. (Tanida, I., Sou, Y.-s., Ezaki, J., Minematsu-Ikeguchi, N., Ueno, T., and Kominami, E. (2004) J. Biol. Chem. 279, 36268-36276). To clarify this contradiction, we sought to determine whether the carboxyl terminus of MAP1LC3B is cleaved to expose Gly(120) for further ubiquitylation-like reactions. When MAP1LC3B-3xFLAG and Myc-MAP1LC3B-His were expressed in HEK293 cells, their carboxyl termini were cleaved, whereas there was little cleavage of mutant proteins MAP1LC3B(G120A)-3xFLAG and Myc-MAP1LC3B(G120A)-His, containing Ala in place of Gly(120). An in vitro assay showed that Gly(120) is essential for carboxyl-terminal cleavage by human Atg4B as well as for formation of the intermediates Atg7-MAP1LC3B (ubiquitin-activating enzyme-substrate) and Atg3-MAP1LC3B (ubiquitin carrier protein-substrate). Recombinant MAP1LC3B-PL was fractionated into the 100,000 x g pellet in a manner similar to that shown for endogenous MAP1LC3B-PL. RNA interference of MAP1LC3B mRNA resulted in a decrease in both endogenous MAP1LC3B-PL and MAP1LC3B. These results indicate that the carboxyl terminus of MAP1LC3B is cleaved to expose Gly(120) for further ubiquitylation-like reactions.     

The amino-terminal region of Atg3 is essential for association with phosphatidylethanolamine in Atg8 lipidation

Autophagy is a bulk degradation process conserved among eukaryotes. In macro-autophagy, autophagosomes sequester cytoplasmic components and deliver their contents to lysosomes/vacuoles. Autophagosome formation requires the conjugation of Atg8, a ubiquitin-like protein, to phosphatidylethanolamine (PE). Here we report that the amino (N)-terminal region of Atg3, an E2-like enzyme for Atg8, plays a crucial role in Atg8-PE conjugation. The conjugating activities of Atg3 mutants lacking the 7 N-terminal amino acid residues or containing a Leu-to-Asp mutation at position 6 were severely impaired both in vivo and in vitro. In addition, the amino-terminal region is critical for interaction with the substrate, PE.

Structured summary

MINT-7010457: ATG8 (uniprotkb:P38182) and ATG3 (uniprotkb:P40344) bind (MI:0407) by biochemical (MI:0401)     

The crystal structure of Atg3, an autophagy-related ubiquitin carrier protein (E2) enzyme that mediates Atg8 lipidation

Atg3 is an E2-like enzyme that catalyzes the conjugation of Atg8 and phosphatidylethanolamine (PE). The Atg8-PE conjugate is essential for autophagy, which is the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. We report here the crystal structure of Saccharomyces cerevisiae Atg3 at 2.5-A resolution. Atg3 has an alpha/beta-fold, and its core region is topologically similar to canonical E2 enzymes. Atg3 has two regions inserted in the core region, one of which consists of approximately 80 residues and has a random coil structure in solution and another with a long alpha-helical structure that protrudes from the core region as far as 30 A. In vivo and in vitro analyses suggested that the former region is responsible for binding Atg7, an E1-like enzyme, and that the latter is responsible for binding Atg8. A sulfate ion was bound near the catalytic cysteine of Atg3, suggesting a possible binding site for the phosphate moiety of PE. The structure of Atg3 provides a molecular basis for understanding the unique lipidation reaction that Atg3 carries out.     

The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. 'Protein modifications: beyond the usual suspects' review series

As a lysosomal/vacuolar degradative pathway that is conserved in eukaryotic organisms, autophagy mediates the turnover of long-lived proteins and excess or aberrant organelles. The main characteristic of autophagy is the formation of a double-membrane vesicle, the autophagosome, which envelops part of the cytoplasm and delivers it to the lysosome/vacuole for breakdown and eventual recycling of the degradation products. Among the approximately 30 autophagy-related (Atg) genes identified so far, there are two ubiquitin-like proteins, Atg12 and Atg8. Analogous to ubiquitination, Atg12 is conjugated to Atg5 by Atg7--an E1-like protein--and Atg10--an E2-like protein. Similarly, Atg7 and Atg3 are the respective E1-like and E2-like proteins that mediate the conjugation of Atg8 to phosphatidylethanolamine. Both Atg12-Atg5 and Atg8 localize to the developing autophagosome. The Atg12-Atg5 conjugate facilitates the lipidation of Atg8 and directs its correct subcellular localization. Atg8-phosphatidylethanolamine is probably a scaffold protein that supports membrane expansion and the amount present correlates with the size of autophagosomes.     

Structural insights into E2–E3 interaction for LC3 lipidation

The members of the LC3/Atg8 family of proteins are covalently attached to phagophore and autophagosomal membranes. At the last step of the LC3 lipidation cascade, LC3 is transferred from the E2 enzyme ATG3 to phosphatidylethanolamine (PE). This transfer is stimulated by the ATG12–ATG5-ATG16L1 E3 complex, but the mechanism is not fully understood. We recently found that ATG12 of the E3 binds to a short sequence in the flexible region (FR) of ATG3 with high affinity, and that this interaction is critical for E2–E3 complex formation. These findings, together with detailed structural analyses of this interaction, define the properties of ATG12 and provide new insights of how LC3 transfer begins with ATG3 recruitment by ATG12.     

Structural insights into E2–E3 interaction for LC3 lipidation

The members of the LC3/Atg8 family of proteins are covalently attached to phagophore and autophagosomal membranes. At the last step of the LC3 lipidation cascade, LC3 is transferred from the E2 enzyme ATG3 to phosphatidylethanolamine (PE). This transfer is stimulated by the ATG12–ATG5-ATG16L1 E3 complex, but the mechanism is not fully understood. We recently found that ATG12 of the E3 binds to a short sequence in the flexible region (FR) of ATG3 with high affinity, and that this interaction is critical for E2–E3 complex formation. These findings, together with detailed structural analyses of this interaction, define the properties of ATG12 and provide new insights of how LC3 transfer begins with ATG3 recruitment by ATG12.     

Solution structure of microtubule-associated protein light chain 3 and identification of its functional subdomains

Microtubule-associated protein (MAP) light chain 3 (LC3) is a human homologue of yeast Apg8/Aut7/Cvt5 (Atg8), which is essential for autophagy. MAP-LC3 is cleaved by a cysteine protease to produce LC3-I, which is located in cytosolic fraction. LC3-I, in turn, is converted to LC3-II through the actions of E1- and E2-like enzymes. LC3-II is covalently attached to phosphatidylethanolamine on its C terminus, and it binds tightly to autophagosome membranes. We determined the solution structure of LC3-I and found that it is divided into N- and C-terminal subdomains. Additional analysis using a photochemically induced dynamic nuclear polarization technique also showed that the N-terminal subdomain of LC3-I makes contact with the surface of the C-terminal subdomain and that LC3-I adopts a single compact conformation in solution. Moreover, the addition of dodecylphosphocholine into the LC3-I solution induced chemical shift perturbations primarily in the C-terminal subdomain, which implies that the two subdomains have different sensitivities to dodecylphosphocholine micelles. On the other hand, deletion of the N-terminal subdomain abolished binding of tubulin and microtubules. Thus, we showed that two subdomains of the LC3-I structure have distinct functions, suggesting that MAP-LC3 can act as an adaptor protein between microtubules and autophagosomes.     

ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation

Expanded polyglutamine 72 repeat (polyQ72) aggregates induce endoplasmic reticulum (ER) stress-mediated cell death with caspase-12 activation and vesicular formation (autophagy). We examined this relationship and the molecular mechanism of autophagy formation. Rapamycin, a stimulator of autophagy, inhibited the polyQ72-induced cell death with caspase-12 activation. PolyQ72, but not polyQ11, stimulated Atg5-Atg12-Atg16 complex-dependent microtubule-associated protein 1 (MAP1) light chain 3 (LC3) conversion from LC3-I to -II, which plays a key role in autophagy. The eucaryotic translation initiation factor 2 alpha (eIF2alpha) A/A mutation, a knock-in to replace a phosphorylatable Ser51 with Ala51, and dominant-negative PERK inhibited polyQ72-induced LC3 conversion. PolyQ72 as well as ER stress stimulators upregulated Atg12 mRNA and proteins via eIF2alpha phosphorylation. Furthermore, Atg5 deficiency as well as the eIF2alpha A/A mutation increased the number of cells showing polyQ72 aggregates and polyQ72-induced caspase-12 activation. Thus, autophagy formation is a cellular defense mechanism against polyQ72-induced ER-stress-mediated cell death by degrading polyQ72 aggregates, with PERK/eIF2alpha phosphorylation being involved in polyQ72-induced LC3 conversion.     

Visualization of Atg3 during Autophagosome Formation in Saccharomyces cerevisiae

Macroautophagy (autophagy) is a highly conserved cellular recycling process involved in degradation of eukaryotic cellular components. During autophagy, macromolecules and organelles are sequestered into the double-membrane autophagosome and degraded in the vacuole/lysosome. Autophagy-related 8 (Atg8), a core Atg protein essential for autophagosome formation, is a marker of several autophagic structures: the pre-autophagosomal structure (PAS), isolation membrane (IM), and autophagosome. Atg8 is conjugated to phosphatidylethanolamine (PE) through a ubiquitin-like conjugation system to yield Atg8-PE; this reaction is called Atg8 lipidation. Although the mechanisms of Atg8 lipidation have been well studied in vitro, the cellular locale of Atg8 lipidation remains enigmatic. Atg3 is an E2-like enzyme that catalyzes the conjugation reaction between Atg8 and PE. Therefore, we hypothesized that the localization of Atg3 would provide insights about the site of the lipidation reaction. To explore this idea, we constructed functional GFP-tagged Atg3 (Atg3-GFP) by inserting the GFP portion immediately after the handle region of Atg3. During autophagy, Atg3-GFP transiently formed a single dot per cell on the vacuolar membrane. This Atg3-GFP dot colocalized with 2× mCherry-tagged Atg8, demonstrating that Atg3 is localized to autophagic structures. Furthermore, we found that Atg3-GFP is localized to the IM by fine-localization analysis. The localization of Atg3 suggests that Atg3 plays an important role in autophagosome formation at the IM.