GATE-16 and GABARAP are authentic modifiers mediated by Apg7 and Apg3

GATE-16, GABARAP, and LC3 are three mammalian counterparts of yeast Apg8p/Aut7p. Here, we show that GATE-16 and GABARAP are authentic modifiers, as is the case of LC3 modification. The C-terminal Phe(117) of proGATE-16 and the C-terminal Leu(117) of proGABARAP are post-translationally cleaved to expose an essential Gly(116) within GATE-16 and GABARAP, with the products designated GATE-16-I and GABARAP-I, respectively. The Gly(116) within GATE-16 and GABARAP are essential for further formation of the intermediates between them and Apg7p(C572S) and Apg3p(C264S). When Apg7p and Apg3p are expressed, GATE-16-I and GABARAP-I are modified to a secondary ubiquitin-like modified form, GATE-16-II and GABARAP-II, respectively. GATE-16-I and GABARAP-I, but not LC3-I, localize to membrane compartments before their modification. These results indicate that GATE-16 and GABARAP are authentic modifiers, but that they have different biochemical characteristics from those of LC3.     

Atg8L/Apg8L is the fourth mammalian modifier of mammalian Atg8 conjugation mediated by human Atg4B, Atg7 and Atg3

Murine Atg8L/Apg8L has significant homology with the other known mammalian Atg8 homologs, LC3, GABARAP and GATE-16. However, it is unclear whether murine Atg8L modification is mediated by human Atg4B, Atg7 and Atg3. Expression of Atg8L in HEK293 cells led to cleavage of its C-terminus. In vitro, the C-terminus of Atg8L was cleaved by human Atg4B, but not human Atg4A or Atg4C. Atg8L-I formed an E1-substrate intermediate with Atg7(C572S), and an E2-substrate intermediate with Atg3(C264S). A modified form of Atg8L was detected in the pelletable fraction in the presence of lysosomal protease inhibitors under nutrient-rich conditions. Cyan fluorescent protein (CFP)-Atg8L colocalized with yellow fluorescent protein (YFP)-LC3 in HeLa cells in the presence of the inhibitors. However, little accumulation of the modified form of Atg8L was observed under conditions of starvation. These results indicate that Atg8L is the fourth modifier of mammalian Atg8 conjugation.     

Human autophagins,a family of cysteine proteinases potentially implicated in cell degradation by autophagy

We have cloned four human cDNAs encoding putative cysteine proteinases that have been tentatively called autophagins. These proteins are similar to Apg4/Aut2, a yeast enzyme involved in the activation of Apg8/Aut7 during the process of autophagy. The identified proteins ranging in length from 393 to 474 amino acids also contain several structural features characteristic of cysteine proteinases including a conserved cysteine residue that is essential for the catalytic properties of these enzymes. Northern blot analysis demonstrated that autophagins are broadly distributed in human tissues, being especially abundant in skeletal muscle. Functional and morphological analysis in autophagy-defective yeast strains lacking Apg4/Aut2 revealed that human autophagins-1 and -3 were able to complement the deficiency in the yeast protease, restoring the phenotypic and biochemical characteristics of autophagic cells. Enzymatic studies performed with autophagin-3, the most widely expressed human autophagin, revealed that the recombinant protein hydrolyzed the synthetic substrate Mca-Thr-Phe-Gly-Met-Dpa-NH(2) whose sequence derives from that present around the Apg4 cleavage site in yeast Apg8/Aut7. This proteolytic activity was diminished by N-ethylmaleimide, an inhibitor of cysteine proteases including yeast Apg4/Aut2. These results provide additional evidence that the autophagic process widely studied in yeast can also be fully reconstituted in human tissues and open the possibility to explore the relevance of the autophagin-based proteolytic system in the induction, regulation, and execution of autophagy.     

Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hApg5p

Autophagy is a process of bulk degradation of cytoplasmic components by the lysosome/vacuole and has a significant relationship to several neurodegenerative disorders and myopathies in mammals. One of APG gene products essential for autophagy in yeast, Apg3p, is a protein-conjugating enzyme for Apg8p lipidation (Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., Noda, T., and Ohsumi, Y. (2000) Nature 408, 488-492). In this study, the cloning of a human Apg3p homologue (hApg3p) as an E2 enzyme essential for human Apg8p homologues (i.e. GATE-16, GABARAP, and MAP-LC3) is shown, and its unique characteristics are described. The predicted amino acid sequence of the isolated clone shows 34.1% identity and 48.1% similarity to yeast Apg3p. Site-directed mutagenesis revealed that Cys(264) of hApg3p is an authentic active-site cysteine residue essential for the formation of hApg3p small middle dothApg8p homologue intermediates. Overexpression of hApg7p enhances the formation of a stable E2-substrate complex between hApg3p(C264S) and each of the hApg8p homologues, and MAP-LC3 is preferred as the substrate over the other two Apg8p homologues. These results indicate that hApg3p is an E2-like enzyme essential for three human Apg8p homologues. Co-immunoprecipitation of hApg7p with hApg3p indicates that hApg3p forms an E1.E2 complex with hApg7p as in the case of yeast Apg3p and Apg7p. Furthermore, hApg3p coimmunoprecipitates with hApg12p, and the overexpression of hApg3p facilitates the formation of the GFPhApg12p.thApg5p conjugate, suggesting that hApg3p cross-talks with the hApg12p conjugation system.     

The carboxyl terminal 17 amino acids within Apg7 are essential for Apg8 lipidation,but not for Apg12 conjugation

In the yeast, Saccharomyces cerevisiae, two ubiquitin-like modifications, Apg12 conjugation with Apg5 and Apg8 lipidation with phosphatidylethanolamine, are essential for autophagy and the cytoplasm-to-vacuole transport of aminopeptidase I (Cvt pathway). As a unique E1-like enzyme, Apg7 activates two modifiers (Apg12 and Apg8) in an ATP-dependent manner and, for this activity, the carboxyl terminal 40 amino acids are essential. For a better understanding of the function of the carboxyl terminus of Apg7, we performed a sequential deletion of the region. A mutant expressing Apg7DeltaC17 protein, which lacks the carboxyl 17 amino acids of Apg7, showed defects in both the Cvt pathway and autophagy. Apg8 lipidation is inhibited in the mutant, while Apg12 conjugation occurs normally. A mutant expressing Apg7DeltaC13 protein showed a defect in the Cvt pathway, but not autophagy, suggesting that the activity of Apg7 for Apg8 lipidation is more essential for the Cvt pathway than for autophagy. Mutant Apg7DeltaC17 protein is still able to interact with Apg8, Apg12 and Apg3, and forms a homodimer, indicating that the deletion of the carboxyl terminal 17 amino acids has little effect on these interactions and Apg7 dimerization. These results suggest that the carboxyl terminal 17 amino acids of Apg7 play a specific role in Apg8 lipidation indispensable for the Cvt pathway and autophagy.     

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.     

Apg16p is required for the function of the Apg12p-Apg5p conjugate in the yeast autophagy pathway.

Autophagy is an intracellular bulk degradation system that is ubiquitous for eukaryotic cells. In this process, cytoplasmic components are enclosed in autophagosomes and delivered to lysosomes/vacuoles. We recently found that a protein conjugation system, in which Apg12p is covalently attached to Apg5p, is indispensable for autophagy in yeast. Here, we describe a novel coiled-coil protein, Apg16p, essential for autophagy. Apg16p interacts with Apg12p-conjugated Apg5p and less preferentially with unconjugated Apg5p. Moreover, the coiled-coil domain of Apg16p mediates self-multimerization that leads to cross-linking of Apg5p molecules and formation of a stable protein complex. Apg16p is not essential for the Apg12p-Apg5p conjugation reaction. These results suggest that the Apg12p-Apg5p conjugate requires Apg16p to accomplish its role in the autophagy pathway, and Apg16p is a key molecule as a linker to form the Apg12p-Apg5p-Apg16p multimer.     

Mammalian Apg12p,but not the Apg12p.Apg5p conjugate,facilitates LC3 processing

A dynamic membrane rearrangement occurs in cells during autophagy to form autophagosomes. In this dynamic process, two ubiquitin-like modifications, Apg12p-conjugation and LC3-modification, are essential for the formation of autophagosomes. Apg7p and Apg10p catalyze the conjugation of Apg12p to Apg5p. The same Apg7p and Apg3p catalyze the processing of LC3 to a membrane-bound form, LC3-II. In this paper, we investigated whether Apg12p has an influence on the second LC3-modification system. A cross-linking experiment revealed that Apg3p interacts with the endogenous Apg12p.Apg5p conjugate. However, Apg3p itself interacts with free Apg12p more preferentially than the Apg12p.Apg5p conjugate, when free Apg12p exists. When Apg12p was overexpressed, LC3 processing was significantly enhanced in the presence of Apg7p. In contrast, when the Apg12p.Apg5p conjugate itself was accumulated by the overexpression of Apg12p and Apg5p, LC3 processing was dominantly inhibited, even in the presence of Apg7p. These results indicate that both Apg12p and the Apg12p.Apg5p conjugate are regulatory factors for LC3 processing.     

Formation of the approximately 350-kDa Apg12-Apg5.Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast

Autophagy, responsible for the delivery of cytoplasmic components to the lysosome/vacuole for degradation, is the major degradative pathway in eukaryotic cells. This process requires a ubiquitin-like protein conjugation system, in which Apg12 is covalently bound to Apg5. In the yeast Saccharomyces cerevisiae, the Apg12-Apg5 conjugate further interacts with a small coiled-coil protein, Apg16. The Apg12-Apg5 and Apg16 are localized in the cytosol and pre-autophagosomal structures and play an essential role in autophagosome formation. Here we show that the Apg12-Apg5 conjugate and Apg16 form a approximately 350-kDa complex in the cytosol. Because Apg16 was suggested to form a homo-oligomer, we generated an in vivo system that allowed us to control the oligomerization state of Apg16. With this system, we demonstrated that formation of the approximately 350-kDa complex and autophagic activity depended on the oligomerization state of Apg16. These results suggest that the Apg12-Apg5 conjugate and Apg16 form a multimeric complex mediated by the Apg16 homo-oligomer, and formation of the approximately 350-kDa complex is required for autophagy in yeast.     

The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway

Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles. Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature. We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself. First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG. Subsequently, Apg8FG forms a conjugate with an unidentified molecule "X" and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme. Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4. The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes. A reaction similar to ubiquitination is probably involved in the second modification. The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway.     

The human homolog of Saccharomyces cerevisiae Apg7p is a Protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3

Autophagy is a process that involves the bulk degradation of cytoplasmic components by the lysosomal/vacuolar system. In the yeast, Saccharomyces cerevisiae, an autophagosome is formed in the cytosol. The outer membrane of the autophagosome is fused with the vacuole, releasing the inner membrane structure, an autophagic body, into the vacuole. The autophagic body is subsequently degraded by vacuolar hydrolases. Taking advantage of yeast genetics, apg (autophagy-defective) mutants were isolated that are defective in terms of formation of autophagic bodies under nutrient starvation conditions. One of the APG gene products, Apg12p, is covalently attached to Apg5p via the C-terminal Gly of Apg12p as in the case of ubiquitylation, and this conjugation is essential for autophagy. Apg7p is a novel E1 enzyme essential for the Apg12p-conjugation system. In mammalian cells, the human Apg12p homolog (hApg12p) also conjugates with the human Apg5p homolog. In this study, the unique characteristics of hApg7p are shown. A two-hybrid experiment indicated that hApg12p interacts with hApg7p. Site-directed mutagenesis revealed that Cys(572) of hApg7p is an authentic active site cysteine residue essential for the formation of the hApg7p.hApg12p intermediate. Overexpression of hApg7p enhances the formation of the hApg5p.hApg12p conjugate, indicating that hApg7p is an E1-like enzyme essential for the hApg12p conjugation system. Cross-linking experiments and glycerol-gradient centrifugation analysis showed that the mammalian Apg7p homolog forms a homodimer as in yeast Apg7p. Each of three human Apg8p counterparts, i.e. the Golgi-associated ATPase enhancer of 16 kDa, GABA(A) receptor-associated protein, and microtubule-associated protein light chain 3, coimmunoprecipitates with hApg7p and conjugates with mutant hApg7p(C572S) to form a stable intermediate via an ester bond. These results indicate that hApg7p is an authentic protein-activating enzyme for hApg12p and the three Apg8p homologs.     

Cloning and analysis of human Apg16L.

Autophagy is an intracellular bulk degradation system, which delivers cytoplasmic components to the lysosome/vacuole. In yeast and mammalian cells, the Apg12-Apg5 conjugate, together with Apg16, form a multimeric complex, which plays an essential role in autopihageosome formation. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA encoding a putative protein with 607 amino acid residues, which shows 90% identity and 93% similarity to mouse Apg16L. This protein, designated human Apg16L, contains a coiled-coil domain and a motif with seven WD repeats, which are also shared by mouse Apg16L. Database searching revealed that Apg16L is mapped to chromosome 2q37.1 and there exist at least four splice variants.     

Molecular mechanism of autophagy in yeast, Saccharomyces cerevisiae

Bulk degradation of cytosol and organelles is important for cellular homeostasis under nutrient limitation, cell differentiation and development. This process occurs in a lytic compartment, and autophagy is the major route to the lysosome and/or vacuole. We found that yeast, Saccharomyces cerevisiae, induces autophagy under various starvation conditions. The whole process is essentially the same as macroautophagy in higher eukaryotic cells. However, little is known about the mechanism of autophagy at a molecular level. To elucidate the molecules involved, a genetic approach was carried out and a total of 16 autophagy-defective mutants (apg) were isolated. So far, 14 APG genes have been cloned. Among them we recently found a unique protein conjugation system essential for autophagy. The C-terminal glycine residue of a novel modifier protein Apg12p, a 186-amino-acid protein, is conjugated to a lysine residue of Apg5p, a 294-amino-acid protein, via an isopeptide bond. We also found that apg7 and apg10 mutants were unable to form an Apg12p-Apg5p conjugate. The conjugation reaction is mediated via Apg7p, E1-like activating enzyme and Apg10p, indicating that it is a ubiquitination-like system. These APG genes have mammalian homologues, suggesting that the Apg12 system is conserved from yeast to human. Further molecular and cell biological analyses of APG gene products will give us crucial clues to uncover the mechanism and regulation of autophagy.     

Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy

In the yeast Saccharomyces cerevisiae, the Apg12p-Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc-tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p-Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p-Apg5p conjugation.     

Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells

In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein-tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.     

Role of the Apg12 conjugation system in mammalian autophagy

The Apg12 system is one of the ubiquitin-like protein conjugation systems conserved in eukaryotes. It was first discovered in yeast during systematic analyses of the apg mutants defective in autophagy, which is the intracellular bulk degradation system. Covalent attachment of Apg12-Apg5 is essential for autophagy. Enzymes catalyzing this conjugation reaction were also identified based on the apg mutant analyses. These are Apg7 and Apg10, corresponding to E1 and E2 enzymes, respectively. Studies using mammalian cells further revealed the function of the Apg12 system. The Apg12-Apg5 conjugate localizes to elongating autophagic isolation membranes. Apg12 conjugation of Apg5 is required for elongation of the isolation membrane to form a complete spherical autophagosome. Discovery of the Apg12 system has facilitated our understanding of the molecular mechanism of autophagosome formation.     

Apg2p functions in autophagosome formation on the perivacuolar structure

Autophagy is a degradative process in which cytoplasmic components are non-selectively sequestered by double-membrane structures, termed autophagosomes, and transported to the vacuole. We have identified and characterized a novel protein Apg2p essential for autophagy in yeast. Biochemical and fluorescence microscopic analyses indicate that Apg2p functions at the step of autophagosome formation. Apg2p localizes to some membranous structure distinct from any known organelle. Using fluorescent protein-tagged Apg2p, we showed that Apg2p localizes to a dot structure close to the vacuole, where Apg8p also exists, but not on autophagosomes unlike Apg8p. This punctate localization of Apg2p depends on the function of Apg1p kinase, phosphatidylinositol 3-kinase complex and Apg9p. Apg2p(G83E), encoded by an apg2-2 allele, shows a severely reduced activity of autophagy and a dispersed localization in the cytoplasm. Overexpression of the mutant Apg2p lessens the defect in autophagy. These results suggest that the dot structure is physiologically important. Apg2p and Apg8p are independently recruited to the structure but coordinately function there to form the autophagosome.     

Tor-mediated induction of autophagy via an Apg1 protein kinase complex

Autophagy is a membrane trafficking to vacuole/lysosome induced by nutrient starvation. In Saccharomyces cerevisiae, Tor protein, a phosphatidylinositol kinase-related kinase, is involved in the repression of autophagy induction by a largely unknown mechanism. Here, we show that the protein kinase activity of Apg1 is enhanced by starvation or rapamycin treatment. In addition, we have also found that Apg13, which binds to and activates Apg1, is hyperphosphorylated in a Tor-dependent manner, reducing its affinity to Apg1. This Apg1-Apg13 association is required for autophagy, but not for the cytoplasm-to-vacuole targeting (Cvt) pathway, another vesicular transport mechanism in which factors essential for autophagy (Apg proteins) are also employed under vegetative growth conditions. Finally, other Apg1-associating proteins, such as Apg17 and Cvt9, are shown to function specifically in autophagy or the Cvt pathway, respectively, suggesting that the Apg1 complex plays an important role in switching between two distinct vesicular transport systems in a nutrient-dependent manner.     

LC3 conjugation system in mammalian autophagy

Autophagy is the bulk degradation of proteins and organelles, a process essential for cellular maintenance, cell viability, differentiation and development in mammals. Autophagy has significant associations with neurodegenerative diseases, cardiomyopathies, cancer, programmed cell death, and bacterial and viral infections. During autophagy, a cup-shaped structure, the preautophagosome, engulfs cytosolic components, including organelles, and closes, forming an autophagosome, which subsequently fuses with a lysosome, leading to the proteolytic degradation of internal components of the autophagosome by lysosomal lytic enzymes. During the formation of mammalian autophagosomes, two ubiquitylation-like modifications are required, Atg12-conjugation and LC3-modification. LC3 is an autophagosomal ortholog of yeast Atg8. A lipidated form of LC3, LC3-II, has been shown to be an autophagosomal marker in mammals, and has been used to study autophagy in neurodegenerative and neuromuscular diseases, tumorigenesis, and bacterial and viral infections. The other Atg8 homologues, GABARAP and GATE-16, are also modified by the same mechanism. In non-starved rats, the tissue distribution of LC3-II differs from those of the lipidated forms of GABARAP and GATE-16, GABARAP-II and GATE-16-II, suggesting that there is a functional divergence among these three modified proteins. Delipidation of LC3-II and GABARAP-II is mediated by hAtg4B. We review the molecular mechanism of LC3-modification, the crosstalk between LC3-modification and mammalian Atg12-conjugation, and the cycle of LC3-lipidation and delipidation mediated by hAtg4B, as well as recent findings concerning the other two Atg8 homologues, GABARAP and GATE-16. We also highlight recent findings regarding the pathobiology of LC3-modification, including its role in microbial infection, cancer and neuromuscular diseases.     

The mouse APG10 homologue,an E2-like enzyme for Apg12p conjugation,facilitates MAP-LC3 modification

Autophagy is a process for the bulk degradation of cytosolic compartments by lysosomes/vacuoles. The formation of autophagosomes involves a dynamic rearrangement of the membrane for which two ubiquitin-like modifications (the conjugation of Apg12p and the modification of a soluble form of MAP-LC3 to a membrane-bound form) are essential. In yeast, Apg10p is an E2-like enzyme essential for Apg12p conjugation. The isolated mouse APG10 gene product interacts with mammalian Apg12p dependent on mammalian Apg7p (E1-like enzyme), and facilitates Apg12p conjugation. The interaction of Apg10p with Apg12p is dependent on the carboxyl-terminal glycine of Apg12p. Mutational analysis of the predicted active site cysteine (Cys161) within mouse Apg10p shows that mutant Apg10pC161S, which can form a stable intermediate with Apg12p, inhibits Apg12p conjugation even in the presence of Apg7p, while overexpression of Apg7p facilitates formation of an Apg12p-Apg5p conjugate. Furthermore, the coexpression of Apg10p with Apg7p facilitates the modification of a soluble form of MAP-LC3 to a membrane-bound form, a second modification essential for autophagy. Mouse Apg10p interacts with MAP-LC3 in HEK293 cells, while no mutant Apg10pC161S forms any intermediate with MAP-LC3. Direct interaction between Apg10p and MAP-LC3 is also demonstrated by yeast two-hybrid analysis. The inability of mutant Apg10pC161S to form any intermediate with MAP-LC3 has ruled out the possibility that MAP-LC3 interacts with Apg10p as a substrate.