The Experts below are selected from a list of 42690 Experts worldwide ranked by ideXlab platform
Yoshinori Ohsumi - One of the best experts on this subject based on the ideXlab platform.
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Atg7 Activates an Autophagy-Essential Ubiquitin-like Protein ATG8 through Multi-Step Recognition.
Journal of Molecular Biology, 2018Co-Authors: Masaya Yamaguchi, Hironori Suzuki, Yuko Fujioka, Fuyuhiko Inagaki, Yoshinori Ohsumi, Kenji Satoo, Nobuo N. NodaAbstract:ATG8 is a unique ubiquitin-like protein that is covalently conjugated with a phosphatidylethanolamine through reactions similar to ubiquitination and plays essential roles in autophagy. Atg7 is the E1 enzyme for ATG8, and it activates the C-terminal Gly116 of ATG8 using ATP. Here, we report the crystal structure of ATG8 bound to the C-terminal domain of Atg7 in an unprecedented mode. ATG8 neither contacts with the central β-sheet nor binds to the catalytic site of Atg7, both of which were observed in previously reported Atg7-ATG8 structures. Instead, ATG8 binds to the C-terminal α-helix and crossover loop, thereby changing the autoinhibited conformation of the crossover loop observed in the free Atg7 structure into a short helix and a disordered loop. Mutational analyses suggested that this interaction mode is important for the activation reaction. We propose that Atg7 recognizes ATG8 through multiple steps, which would be necessary to induce a conformational change in Atg7 that is optimal for the activation reaction.
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localization of atg3 to autophagy related membranes and its enhancement by the ATG8 family interacting motif to promote expansion of the membranes
FEBS Letters, 2015Co-Authors: Machiko Sakohnakatogawa, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori OhsumiAbstract:The E2 enzyme Atg3 conjugates the ubiquitin-like protein ATG8 to phosphatidylethanolamine (PE) to drive autophagosome formation in Saccharomyces cerevisiae. In this study, we show that Atg3 localizes to the pre-autophagosomal structure (PAS) and the isolation membrane (IM), providing crucial evidence that ATG8-PE conjugates are produced on these structures. We also find that mutations in the ATG8-family interacting motif (AIM) of Atg3 significantly impairs the PAS/IM localization of Atg3, resulting in inefficient IM expansion. It is suggested that the AIM-mediated PAS/IM localization of Atg3 facilitates membrane expansion in these structures probably by ensuring active production of ATG8-PE on the membranes.
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fine mapping of autophagy related proteins during autophagosome formation in saccharomyces cerevisiae
Journal of Cell Science, 2013Co-Authors: Kuninori Suzuki, Hayashi Yamamoto, Chika Kondokakuta, Manami Akioka, Yoshinori OhsumiAbstract:Autophagy is a bulk degradation system mediated by biogenesis of autophagosomes under starvation conditions. In Saccharomyces cerevisiae, a membrane sac called the isolation membrane (IM) is generated from the pre-autophagosomal structure (PAS); ultimately, the IM expands to become a mature autophagosome. Eighteen autophagy-related (Atg) proteins are engaged in autophagosome formation at the PAS. However, the cup-shaped IM was visualized just as a dot by fluorescence microscopy, posing a challenge to further understanding the detailed functions of Atg proteins during IM expansion. In this study, we visualized expanding IMs as cup-shaped structures using fluorescence microscopy by enlarging a selective cargo of autophagosomes, and finely mapped the localizations of Atg proteins. The PAS scaffold proteins (Atg13 and Atg17) and phosphatidylinositol 3-kinase complex I were localized to a position at the junction between the IM and the vacuolar membrane, termed the vacuole-IM contact site (VICS). By contrast, Atg1, ATG8 and the Atg16-Atg12-Atg5 complex were present at both the VICS and the cup-shaped IM. We designate this localization the 'IM' pattern. The Atg2-Atg18 complex and Atg9 localized to the edge of the IM, appearing as two or three dots, in close proximity to the endoplasmic reticulum exit sites. Thus, we designate these dots as the 'IM edge' pattern. These data suggest that Atg proteins play individual roles at spatially distinct locations during IM expansion. These findings will facilitate detailed investigations of the function of each Atg protein during autophagosome formation.
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atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
Nature Structural & Molecular Biology, 2013Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori OhsumiAbstract:In the yeast autophagy system, the Atg12–Atg5 conjugate acts as an E3 to promote the E2 activity of Atg3, which conjugates ATG8 to phosphatidylethanolamine. Now structural and biochemical analyses reveal that Atg12–Atg5 induces a rearrangement in the catalytic center of Atg3, which employs a threonine residue in addition to the active cysteine to catalyze the conjugation reaction.
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atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
Nature Structural & Molecular Biology, 2013Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori OhsumiAbstract:In the yeast autophagy system, the Atg12–Atg5 conjugate acts as an E3 to promote the E2 activity of Atg3, which conjugates ATG8 to phosphatidylethanolamine. Now structural and biochemical analyses reveal that Atg12–Atg5 induces a rearrangement in the catalytic center of Atg3, which employs a threonine residue in addition to the active cysteine to catalyze the conjugation reaction.
Fuyuhiko Inagaki - One of the best experts on this subject based on the ideXlab platform.
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Atg7 Activates an Autophagy-Essential Ubiquitin-like Protein ATG8 through Multi-Step Recognition.
Journal of Molecular Biology, 2018Co-Authors: Masaya Yamaguchi, Hironori Suzuki, Yuko Fujioka, Fuyuhiko Inagaki, Yoshinori Ohsumi, Kenji Satoo, Nobuo N. NodaAbstract:ATG8 is a unique ubiquitin-like protein that is covalently conjugated with a phosphatidylethanolamine through reactions similar to ubiquitination and plays essential roles in autophagy. Atg7 is the E1 enzyme for ATG8, and it activates the C-terminal Gly116 of ATG8 using ATP. Here, we report the crystal structure of ATG8 bound to the C-terminal domain of Atg7 in an unprecedented mode. ATG8 neither contacts with the central β-sheet nor binds to the catalytic site of Atg7, both of which were observed in previously reported Atg7-ATG8 structures. Instead, ATG8 binds to the C-terminal α-helix and crossover loop, thereby changing the autoinhibited conformation of the crossover loop observed in the free Atg7 structure into a short helix and a disordered loop. Mutational analyses suggested that this interaction mode is important for the activation reaction. We propose that Atg7 recognizes ATG8 through multiple steps, which would be necessary to induce a conformational change in Atg7 that is optimal for the activation reaction.
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architecture of the atg12 atg5 atg16 complex and its molecular role in autophagy
Autophagy: Cancer Other Pathologies Inflammation Immunity Infection and Aging#R##N#Volume 3 - Mitophagy, 2014Co-Authors: Nobuo N. Noda, Fuyuhiko InagakiAbstract:Atg5 is covalently modified with Atg12 via reactions that are similar to ubiquitination, and it noncovalently interacts with Atg16. Formation of the Atg12–Atg5–Atg16 complex is essential for its E3-like function: facilitation of ATG8 transfer from Atg3 to phosphatidylethanolamine at autophagic membranes. Structural studies on the Atg12–Atg5–Atg16 complex revealed that the unique architecture of this protein complex is totally distinct from the other E3 enzymes. The Atg12–Atg5–Atg16 complex interacts directly with Atg3 via Atg12, and enhances the conjugase activity of Atg3 by rearranging its catalytic center, while it is targeted to the membranes via Atg5 and Atg16, and promotes the transfer of ATG8 from Atg3 to the membranes.
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atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
Nature Structural & Molecular Biology, 2013Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori OhsumiAbstract:In the yeast autophagy system, the Atg12–Atg5 conjugate acts as an E3 to promote the E2 activity of Atg3, which conjugates ATG8 to phosphatidylethanolamine. Now structural and biochemical analyses reveal that Atg12–Atg5 induces a rearrangement in the catalytic center of Atg3, which employs a threonine residue in addition to the active cysteine to catalyze the conjugation reaction.
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atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
Nature Structural & Molecular Biology, 2013Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori OhsumiAbstract:In the yeast autophagy system, the Atg12–Atg5 conjugate acts as an E3 to promote the E2 activity of Atg3, which conjugates ATG8 to phosphatidylethanolamine. Now structural and biochemical analyses reveal that Atg12–Atg5 induces a rearrangement in the catalytic center of Atg3, which employs a threonine residue in addition to the active cysteine to catalyze the conjugation reaction.
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structure of the atg12 atg5 conjugate reveals a platform for stimulating ATG8 pe conjugation
EMBO Reports, 2013Co-Authors: Nobuo N. Noda, Takao Hanada, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko InagakiAbstract:Atg12 is conjugated to Atg5 through enzymatic reactions similar to ubiquitination. The Atg12–Atg5 conjugate functions as an E3-like enzyme to promote lipidation of ATG8, whereas lipidated ATG8 has essential roles in both autophagosome formation and selective cargo recognition during autophagy. However, the molecular role of Atg12 modification in these processes has remained elusive. Here, we report the crystal structure of the Atg12–Atg5 conjugate. In addition to the isopeptide linkage, Atg12 forms hydrophobic and hydrophilic interactions with Atg5, thereby fixing its position on Atg5. Structural comparison with unmodified Atg5 and mutational analyses showed that Atg12 modification neither induces a conformational change in Atg5 nor creates a functionally important architecture. Rather, Atg12 functions as a binding module for Atg3, the E2 enzyme for ATG8, thus endowing Atg5 with the ability to interact with Atg3 to facilitate ATG8 lipidation.
Daniel J Klionsky - One of the best experts on this subject based on the ideXlab platform.
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multiple structural rearrangements mediated by high plasticity regions in atg3 are key for efficient conjugation of ATG8 to pe during autophagy
Autophagy, 2021Co-Authors: Hana Popelka, Daniel J KlionskyAbstract:The Atg3 protein is highly homologous from yeast to human. Atg3 functions as an E2-like enzyme promoting conjugation of ATG8-family proteins to phosphatidylethanolamine (PE), a lipid molecule embedded in the growing phagophore membrane during stress-induced autophagy. Over the last decade, Atg3 became one of the most explored autophagy proteins, resulting in observations that provided specific insights into the structural mechanisms of its function. In this article, we describe a recent study by Ye et al. that reveals, using the human ATG3, how the membrane binding capability of the enzyme is tightly linked to its conjugation activity. We summarize the current knowledge on important mechanisms that involve protein-protein or protein-membrane interactions of Atg3 and that ultimately lead to efficient ATG8-PE conjugation.Abbreviations: AH: amphipathic helix; FR: flexible region; HR: handle region; NMR: nuclear magnetic resonance.
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noncanonical e2 recruitment by the autophagy e1 revealed by atg7 atg3 and atg7 atg10 structures
Nature Structural & Molecular Biology, 2012Co-Authors: Stephen E. Kaiser, Asad M. Taherbhoy, Alan Deng, Igor Kurinov, Jennifer L Olszewski, David M Duda, Timothy D Fenn, Kai Mao, Daniel J KlionskyAbstract:An early step in the autophagy process is the conjugation of the ubiquitin-like proteins (UBLs) ATG8 and Atg12 to their targets. Structural and functional experiments reveal how the autophagy E1 Atg7 uses a trans mechanism to catalyze the charging of the autophagy UBLs onto their respective carrier E2 proteins, Atg3 and Atg10.
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look people atg is an abbreviation for autophagy related that s it
Autophagy, 2012Co-Authors: Daniel J KlionskyAbstract:Prior to the adoption of the unified nomenclature for naming autophagy-related genes and proteins there were at least ten different names being used in fungal systems. Accordingly, in 2003 the majority of the researchers (at that time) working in fungal autophagy decided it would be advantageous to agree on a single name so that it was no longer necessary to search through the literature (or hope that the authors of the paper you were reading would inform you) to determine that APG1 was the same gene as AUT3, CVT10, GSA10, PAZ1 or PDD7—this gene now has a standard name of ATG1.1 This nomenclature has been adopted in most other eukaryotic systems, further simplifying the naming of these genes and proteins. As noted in the nomenclature paper, “ATG” and “Atg” stand for “autophagy-related” gene or protein, respectively. That is, “ATG” means “autophagy-related,” and that is it. It does not mean “autophagy-related gene” or “autophagy-related protein.” The abbreviation derives from just the first word, autophagy, as in AuTophaGy-related. It does not make sense for “ATG” to represent “autophagy-related gene;” otherwise, when people refer to an “ATG gene” this would translate into “autophagy-related gene gene,” which sounds rather absurd. Similarly, “Atg” does not represent “autophagy-related protein” when referring to a protein, for obvious reasons; otherwise, the “Atg1 protein” would be spelled out as “autophagy-related protein 1 protein,” which seems a little redundant. So, “ATG” and “Atg” are simply abbreviations for “autophagy-related.” If you want to say “autophagy-related gene” or “autophagy-related protein,” you can use “ATG gene” or “Atg protein.” Note that I am not going to cite incorrect examples of the use of these abbreviations because there are far too many. Also, I am using the capitalization that applies to yeast in these examples. If I was referring to humans the abbreviations would be “ATG” and “ATG” for the gene and protein, respectively, or “Atg” and “ATG” for the mouse system.2 That said, while we are on the subject of names, “Cvt” is an abbreviation for “cytoplasm to vacuole targeting” (or “cytoplasm-to-vacuole targeting,” with dashes).3 “Cvt” does not stand for “cytoplasm-to-vacuole,”4,5 which ignores the letter “t.” It also does not stand for “cytosol-to-vacuole-targeting,”6 “cytoplasm to vacuole (cvt) trafficking,”7 or “cytoplasm-to-vacuole transport.”8 In a similar vein, the abbreviation “TAKA” when used to refer to the TAKA assay is an abbreviation for “transport of Atg9 after knocking out ATG1.”9 “TAKA” does not stand for “take Atg1 kinase away,”4 or any other permutations you might be able to come up with. A final note about nomenclature concerns the Atg12 conjugation complex. Both Atg12 and ATG8 are unusual in that they become covalently attached to another molecule. Noncovalent interactions are typically indicated with a standard dash “-” as in “Atg1-Atg13.” To denote the covalent attachment we use an en dash “–” as in “ATG8–PE” as opposed to “ATG8-PE”. Now, going back to the Atg12 complex, Atg16 binds Atg5 directly, not Atg12. Thus, it makes sense to write this as “Atg5-Atg16” using a standard dash. One could write “Atg16-Atg5,” but in general we list the lower number first unless we are trying to indicate something specific about the interactions (as with “Atg17-Atg31-Atg29” because Atg29 appears to interact with Atg31 directly, and not with Atg17). So, where is “Atg12” added to this interaction? If we agree on the order “Atg5-Atg16,” there is only once choice, and that is “Atg12–Atg5-Atg16” because Atg12 is covalently attached to Atg5 (note the use of the en dash between these two proteins) and not Atg16. Therefore, please use the correct designations of “Atg12–Atg5” and “Atg12–Atg5-Atg16” and not “Atg5-Atg12,”10-17 “Atg5/Atg12,”18 “ATG5/ATG12,”19 “Atg5-Atg12/Atg16,”20 “Atg5-Atg12/Atg16L1,”21 “ATG16/ATG5/ATG12”19 or “Atg5-Atg12-Atg16”15 (I am citing some arbitrary examples where the incorrect nomenclature was used, but I could list many more). Thus, if you want to use these abbreviations correctly, consider the definitions as explained here. Alternatively, take a look at “A comprehensive glossary of autophagy-related molecules and processes”22 (the second edition).
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a role for ATG8 pe deconjugation in autophagosome biogenesis
Autophagy, 2012Co-Authors: Usha Nair, Fulvio Reggiori, Wei Lien Yen, Muriel Mari, Yang Cao, Zhiping Xie, Misuzu Baba, Daniel J KlionskyAbstract:Formation of the autophagosome is likely the most complex step of macroautophagy, and indeed it is the morphological and functional hallmark of this process; accordingly, it is critical to understand the corresponding molecular mechanism. ATG8 is the only known autophagy-related (Atg) protein required for autophagosome formation that remains associated with the completed sequestering vesicle. Approximately one-fourth of all of the characterized Atg proteins that participate in autophagosome biogenesis affect ATG8, regulating its conjugation to phosphatidylethanolamine (PE), localization to the phagophore assembly site and/or subsequent deconjugation. An unanswered question in the field regards the physiological role of the deconjugation of ATG8-PE. Using an ATG8 mutant that bypasses the initial Atg4-dependent processing, we demonstrate that ATG8 deconjugation is an important step required to facilitate multiple events during macroautophagy. The inability to deconjugate ATG8-PE results in the mislocalization of this protein to the vacuolar membrane. We also show that the deconjugation of ATG8-PE is required for efficient autophagosome biogenesis, the assembly of Atg9-containing tubulovesicular clusters into phagophores/autophagosomes, and for the disassembly of PAS-associated Atg components.
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for the last time it is gfp ATG8 not ATG8 gfp and the same goes for lc3
Autophagy, 2011Co-Authors: Daniel J KlionskyAbstract:GFP-ATG8/LC3 is a very useful marker to follow macroautophagy (hereafter autophagy). In contrast, ATG8/LC3-GFP has relatively limited uses. Unfortunately, many authors do not seem to appreciate the difference between these two constructs. I hope to put an end to that now. The title of this missive pretty much makes my point, but I will elaborate briefly. ATG8/LC3 is of course a key autophagy-related protein (for the purposes of this article I am going to ignore the other LC3 family members, but the same holds true for them). Although its function is not clear, we do know that it is a ubiquitin-like protein that undergoes conjugation to phosphatidylethanolamine (PE). Importantly, ATG8/LC3 remains associated with the completed autophagosome, and for this reason it is the key autophagy-related marker protein; when fused to GFP or similar fluorophores it can be used to follow the phagophore or autophagosome in vivo. Let’s quickly review what is known about the conjugation process. In most organisms ATG8/LC3 is initially synthesized with a C-terminal amino acid(s) that follow the critical glycine residue. The additional amino acid (or amino acids) is removed by the Atg4 cysteine protease, exposing the glycine residue. Subsequent activation and conjugation involve Atg7 and Atg3, respectively (Fig. 1). Figure 1. Schematic comparison of the fate of C-terminal versus N-terminal GFP fused to ATG8. (A) If GFP is fused to the C terminus of ATG8 (or LC3), it is removed when Atg4 hydrolyzes the bond between the penultimate glycine residue and arginine. The ... Now, let’s return to that first step, Atg4-dependent cleavage of the C terminus. You see, that is my point. Atg4 cleaves the C terminus of ATG8/LC3 after the glycine residue. To put it another way, anything after that glycine will be removed from ATG8/LC3 early on in the process of autophagy. Got it? Removed. Gone. No longer connected to ATG8/LC3. In yeast, a C-terminal fusion of GFP to ATG8 (written as ATG8-GFP) has been used to monitor Atg4 activity (Fig. 2A). Otherwise, the fusion is always at the N terminus. With an N-terminal fusion, GFP-ATG8/LC3 can be used as a marker of the phagophore or the autophagosome as noted above. A tandem mCherry-GFP-LC3 (or similar construct) can be used to monitor flux, or at least the conversion of autophagosomes to autolysosomes. Similarly, GFP-ATG8/LC3 can be followed by western blot, and the appearance of free GFP is indicative of autophagic flux (Fig. 2B). Figure 2. ATG8-GFP cannot be used to monitor autophagic flux. (A) Wild-type (WT), atg4Δ or atg1Δ cells expressing ATG8-GFP were grown to mid-log phase and protein extracts were examined by western blot with antisera to ATG8 or GFP. The ... The point is, if you want to use a fluorescently tagged ATG8/LC3 to monitor anything other than Atg4 activity, you have to use an N-terminal fusion (written as GFP-ATG8/LC3). Because of these two potential uses for ATG8/LC3, it is important to specify the construct correctly. Furthermore, the incorrect written usage of ATG8/LC3-GFP indicates a fundamental lack of understanding of the conjugation process (or simply carelessness). Nonetheless, I still see authors incorrectly referring to ATG8/LC3-GFP. For example, looking at abstracts from a few papers published in 2009 and 2010, I found the following (if I extend my search beyond the abstracts I could provide many more examples, including papers published in 2011): “...L. monocytogenes was encapsulated by LC3-GFP...” “...NRK-52E cell lines stably transfected with LC3-GFP...” “...large vesicles that intensely stained for a transfected LC3-GFP construct...” “...LC3-GFP fusion protein was accumulated as granular dots in autophagosomes.” “An autophagosome marker protein ATG8-GFP...” I highly doubt the technical validity of these statements. For example, if Atg4 does not remove GFP from the C terminus, there is no way the fusion protein is going to get conjugated to PE. Similarly, I find it unlikely that the authors used ATG8/LC3-GFP, because they wanted to monitor autophagosome formation, not Atg4 activity. I could give you more examples, but I think you get the point. So, unless you are talking about an assay for Atg4, please use the correct designation, GFP-ATG8/LC3.
Richard David Vierstra - One of the best experts on this subject based on the ideXlab platform.
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the atg1 atg13 protein kinase complex is both a regulator and a target of autophagic recycling in arabidopsis
The Plant Cell, 2011Co-Authors: Anongpat Suttangkakul, Taijoon Chung, Richard David VierstraAbstract:Autophagy is an intracellular recycling route in eukaryotes whereby organelles and cytoplasm are sequestered in vesicles, which are subsequently delivered to the vacuole for breakdown. The process is induced by various nutrient-responsive signaling cascades converging on the Autophagy-Related1 (ATG1)/ATG13 kinase complex. Here, we describe the ATG1/13 complex in Arabidopsis thaliana and show that it is both a regulator and a target of autophagy. Plants missing ATG13 are hypersensitive to nutrient limitations and senesce prematurely similar to mutants lacking other components of the ATG system. Synthesis of the ATG12-ATG5 and ATG8-phosphatidylethanolamine adducts, which are essential for autophagy, still occurs in ATG13-deficient plants, but the biogenesis of ATG8-decorated autophagic bodies does not, indicating that the complex regulates downstream events required for autophagosome enclosure and/or vacuolar delivery. Surprisingly, levels of the ATG1a and ATG13a phosphoproteins drop dramatically during nutrient starvation and rise again upon nutrient addition. This turnover is abrogated by inhibition of the ATG system, indicating that the ATG1/13 complex becomes a target of autophagy. Consistent with this mechanism, ATG1a is delivered to the vacuole with ATG8-decorated autophagic bodies. Given its responsiveness to nutrient demands, the turnover of the ATG1/13 kinase likely provides a dynamic mechanism to tightly connect autophagy to a plant’s nutritional status.
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ATG8 lipidation and ATG8 mediated autophagy in arabidopsis require atg12 expressed from the differentially controlled atg12a and atg12b loci
Plant Journal, 2010Co-Authors: Taijoon Chung, Allison R Phillips, Richard David VierstraAbstract:Summary Autophagic recycling of intracellular plant constituents is maintained at a basal level under normal growth conditions but can be induced in response to nutritional demand, biotic stress, and senescence. One route requires the ubiquitin-fold proteins Autophagy-related (ATG)-8 and ATG12, which become attached to the lipid phosphatidylethanolamine (PE) and the ATG5 protein, respectively, during formation of the engulfing vesicle and delivery of its cargo to the vacuole for breakdown. Here, we genetically analyzed the conjugation machinery required for ATG8/12 modification in Arabidopsis thaliana with a focus on the two loci encoding ATG12. Whereas single atg12a and atg12b mutants lack phenotypic consequences, atg12a atg12b double mutants senesce prematurely, are hypersensitive to nitrogen and fixed carbon starvation, and fail to accumulate autophagic bodies in the vacuole. By combining mutants eliminating ATG12a/b, ATG5, or the ATG10 E2 required for their condensation with a method that unequivocally detects the ATG8-PE adduct, we also show that ATG8 lipidation requires the ATG12–ATG5 conjugate. Unlike ATG8, ATG12 does not associate with autophagic bodies, implying that its role(s) during autophagy is restricted to events before the vacuolar deposition of vesicles. The expression patterns of the ATG12a and ATG12b genes and the effects of single atg12a and atg12b mutants on forming the ATG12–ATG5 conjugate reveal that the ATG12b locus is more important during basal autophagy while the ATG12a locus is more important during induced autophagy. Taken together, we conclude that the formation of the ATG12–ATG5 adduct is essential for ATG8-mediated autophagy in plants by promoting ATG8 lipidation.
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ATG8 lipidation and ATG8 mediated autophagy in arabidopsis require atg12 expressed from the differentially controlled atg12a and atg12b loci
Plant Journal, 2010Co-Authors: Taijoon Chung, Allison R Phillips, Richard David VierstraAbstract:Summary Autophagic recycling of intracellular plant constituents is maintained at a basal level under normal growth conditions but can be induced in response to nutritional demand, biotic stress, and senescence. One route requires the ubiquitin-fold proteins Autophagy-related (ATG)-8 and ATG12, which become attached to the lipid phosphatidylethanolamine (PE) and the ATG5 protein, respectively, during formation of the engulfing vesicle and delivery of its cargo to the vacuole for breakdown. Here, we genetically analyzed the conjugation machinery required for ATG8/12 modification in Arabidopsis thaliana with a focus on the two loci encoding ATG12. Whereas single atg12a and atg12b mutants lack phenotypic consequences, atg12a atg12b double mutants senesce prematurely, are hypersensitive to nitrogen and fixed carbon starvation, and fail to accumulate autophagic bodies in the vacuole. By combining mutants eliminating ATG12a/b, ATG5, or the ATG10 E2 required for their condensation with a method that unequivocally detects the ATG8-PE adduct, we also show that ATG8 lipidation requires the ATG12–ATG5 conjugate. Unlike ATG8, ATG12 does not associate with autophagic bodies, implying that its role(s) during autophagy is restricted to events before the vacuolar deposition of vesicles. The expression patterns of the ATG12a and ATG12b genes and the effects of single atg12a and atg12b mutants on forming the ATG12–ATG5 conjugate reveal that the ATG12b locus is more important during basal autophagy while the ATG12a locus is more important during induced autophagy. Taken together, we conclude that the formation of the ATG12–ATG5 adduct is essential for ATG8-mediated autophagy in plants by promoting ATG8 lipidation.
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the atg12 conjugating enzyme atg10 is essential for autophagic vesicle formation in arabidopsis thaliana
Genetics, 2008Co-Authors: Allison R Phillips, Anongpat Suttangkakul, Richard David VierstraAbstract:Autophagy is an important intracellular recycling system in eukaryotes that utilizes small vesicles to traffic cytosolic proteins and organelles to the vacuole for breakdown. Vesicle formation requires the conjugation of the two ubiquitin-fold polypeptides ATG8 and ATG12 to phosphatidylethanolamine and the ATG5 protein, respectively. Using Arabidopsis thaliana mutants affecting the ATG5 target or the ATG7 E1 required to initiate ligation of both ATG8 and ATG12, we previously showed that the ATG8/12 conjugation pathways together are important when plants encounter nutrient stress and during senescence. To characterize the ATG12 conjugation pathway specifically, we characterized a null mutant eliminating the E2-conjugating enzyme ATG10 that, similar to plants missing ATG5 or ATG7, cannot form the ATG12-ATG5 conjugate. atg10-1 plants are hypersensitive to nitrogen and carbon starvation and initiate senescence and programmed cell death (PCD) more quickly than wild type, as indicated by elevated levels of senescence- and PCD-related mRNAs and proteins during carbon starvation. As detected with a GFP-ATG8a reporter, atg10-1 and atg5-1 mutant plants fail to accumulate autophagic bodies inside the vacuole. These results indicate that ATG10 is essential for ATG12 conjugation and that the ATG12-ATG5 conjugate is necessary to form autophagic vesicles and for the timely progression of senescence and PCD in plants.
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the atg12 conjugating enzyme atg10 is essential for autophagic vesicle formation in arabidopsis thaliana
Genetics, 2008Co-Authors: Allison R Phillips, Anongpat Suttangkakul, Richard David VierstraAbstract:Autophagy is an important intracellular recycling system in eukaryotes that utilizes small vesicles to traffic cytosolic proteins and organelles to the vacuole for breakdown. Vesicle formation requires the conjugation of the two ubiquitin-fold polypeptides ATG8 and ATG12 to phosphatidylethanolamine and the ATG5 protein, respectively. Using Arabidopsis thaliana mutants affecting the ATG5 target or the ATG7 E1 required to initiate ligation of both ATG8 and ATG12, we previously showed that the ATG8/12 conjugation pathways together are important when plants encounter nutrient stress and during senescence. To characterize the ATG12 conjugation pathway specifically, we characterized a null mutant eliminating the E2-conjugating enzyme ATG10 that, similar to plants missing ATG5 or ATG7, cannot form the ATG12-ATG5 conjugate. atg10-1 plants are hypersensitive to nitrogen and carbon starvation and initiate senescence and programmed cell death (PCD) more quickly than wild type, as indicated by elevated levels of senescence- and PCD-related mRNAs and proteins during carbon starvation. As detected with a GFP-ATG8a reporter, atg10-1 and atg5-1 mutant plants fail to accumulate autophagic bodies inside the vacuole. These results indicate that ATG10 is essential for ATG12 conjugation and that the ATG12-ATG5 conjugate is necessary to form autophagic vesicles and for the timely progression of senescence and PCD in plants.
Nobuo N. Noda - One of the best experts on this subject based on the ideXlab platform.
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Atg7 Activates an Autophagy-Essential Ubiquitin-like Protein ATG8 through Multi-Step Recognition.
Journal of Molecular Biology, 2018Co-Authors: Masaya Yamaguchi, Hironori Suzuki, Yuko Fujioka, Fuyuhiko Inagaki, Yoshinori Ohsumi, Kenji Satoo, Nobuo N. NodaAbstract:ATG8 is a unique ubiquitin-like protein that is covalently conjugated with a phosphatidylethanolamine through reactions similar to ubiquitination and plays essential roles in autophagy. Atg7 is the E1 enzyme for ATG8, and it activates the C-terminal Gly116 of ATG8 using ATP. Here, we report the crystal structure of ATG8 bound to the C-terminal domain of Atg7 in an unprecedented mode. ATG8 neither contacts with the central β-sheet nor binds to the catalytic site of Atg7, both of which were observed in previously reported Atg7-ATG8 structures. Instead, ATG8 binds to the C-terminal α-helix and crossover loop, thereby changing the autoinhibited conformation of the crossover loop observed in the free Atg7 structure into a short helix and a disordered loop. Mutational analyses suggested that this interaction mode is important for the activation reaction. We propose that Atg7 recognizes ATG8 through multiple steps, which would be necessary to induce a conformational change in Atg7 that is optimal for the activation reaction.
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architecture of the atg12 atg5 atg16 complex and its molecular role in autophagy
Autophagy: Cancer Other Pathologies Inflammation Immunity Infection and Aging#R##N#Volume 3 - Mitophagy, 2014Co-Authors: Nobuo N. Noda, Fuyuhiko InagakiAbstract:Atg5 is covalently modified with Atg12 via reactions that are similar to ubiquitination, and it noncovalently interacts with Atg16. Formation of the Atg12–Atg5–Atg16 complex is essential for its E3-like function: facilitation of ATG8 transfer from Atg3 to phosphatidylethanolamine at autophagic membranes. Structural studies on the Atg12–Atg5–Atg16 complex revealed that the unique architecture of this protein complex is totally distinct from the other E3 enzymes. The Atg12–Atg5–Atg16 complex interacts directly with Atg3 via Atg12, and enhances the conjugase activity of Atg3 by rearranging its catalytic center, while it is targeted to the membranes via Atg5 and Atg16, and promotes the transfer of ATG8 from Atg3 to the membranes.
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atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
Nature Structural & Molecular Biology, 2013Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori OhsumiAbstract:In the yeast autophagy system, the Atg12–Atg5 conjugate acts as an E3 to promote the E2 activity of Atg3, which conjugates ATG8 to phosphatidylethanolamine. Now structural and biochemical analyses reveal that Atg12–Atg5 induces a rearrangement in the catalytic center of Atg3, which employs a threonine residue in addition to the active cysteine to catalyze the conjugation reaction.
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atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
Nature Structural & Molecular Biology, 2013Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori OhsumiAbstract:In the yeast autophagy system, the Atg12–Atg5 conjugate acts as an E3 to promote the E2 activity of Atg3, which conjugates ATG8 to phosphatidylethanolamine. Now structural and biochemical analyses reveal that Atg12–Atg5 induces a rearrangement in the catalytic center of Atg3, which employs a threonine residue in addition to the active cysteine to catalyze the conjugation reaction.
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structure of the atg12 atg5 conjugate reveals a platform for stimulating ATG8 pe conjugation
EMBO Reports, 2013Co-Authors: Nobuo N. Noda, Takao Hanada, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko InagakiAbstract:Atg12 is conjugated to Atg5 through enzymatic reactions similar to ubiquitination. The Atg12–Atg5 conjugate functions as an E3-like enzyme to promote lipidation of ATG8, whereas lipidated ATG8 has essential roles in both autophagosome formation and selective cargo recognition during autophagy. However, the molecular role of Atg12 modification in these processes has remained elusive. Here, we report the crystal structure of the Atg12–Atg5 conjugate. In addition to the isopeptide linkage, Atg12 forms hydrophobic and hydrophilic interactions with Atg5, thereby fixing its position on Atg5. Structural comparison with unmodified Atg5 and mutational analyses showed that Atg12 modification neither induces a conformational change in Atg5 nor creates a functionally important architecture. Rather, Atg12 functions as a binding module for Atg3, the E2 enzyme for ATG8, thus endowing Atg5 with the ability to interact with Atg3 to facilitate ATG8 lipidation.