The Experts below are selected from a list of 12654 Experts worldwide ranked by ideXlab platform
Daniel J Klionsky - One of the best experts on this subject based on the ideXlab platform.
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Psp2, a novel regulator of Autophagy that promotes Autophagy-Related Protein translation
Cell Research, 2019Co-Authors: Aileen Ariosa, Haina Huang, Katrin Karbstein, Daniel J KlionskyAbstract:MacroAutophagy/Autophagy defines an evolutionarily conserved catabolic process that targets cytoplasmic components for lysosomal degradation. The process of Autophagy from initiation to closure is tightly executed and controlled by the concerted action of Autophagy-Related (Atg) Proteins. Although substantial progress has been made in characterizing transcriptional and post-translational regulation of ATG /Atg genes/Proteins, little is known about the translational control of Autophagy. Here we report that Psp2, an RGG motif Protein, positively regulates Autophagy through promoting the translation of Atg1 and Atg13, two Proteins that are crucial in the initiation of Autophagy. During nitrogen starvation conditions, Psp2 interacts with the 5′ UTR of ATG1 and ATG13 transcripts in an RGG motif-dependent manner and with eIF4E and eIF4G2, components of the translation initiation machinery, to regulate the translation of these transcripts. Deletion of the PSP2 gene leads to a decrease in the synthesis of Atg1 and Atg13, which correlates with reduced Autophagy activity and cell survival. Furthermore, deactivation of the methyltransferase Hmt1 constitutes a molecular switch that regulates Psp2 arginine methylation status as well as its mRNA binding activity in response to starvation. These results reveal a novel mechanism by which Atg Proteins become upregulated to fulfill the increased demands of Autophagy activity as part of translational reprogramming during stress conditions, and help explain how ATG genes bypass the general block in Protein translation that occurs during starvation.
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dhh1 promotes Autophagy Related Protein translation during nitrogen starvation
PLOS Biology, 2019Co-Authors: Xu Liu, Zhiyuan Yao, Meiyan Jin, Sim Namkoong, Zhangyuan Yin, Jun Hee Lee, Daniel J KlionskyAbstract:MacroAutophagy (hereafter Autophagy) is a well-conserved cellular process through which cytoplasmic components are delivered to the vacuole/lysosome for degradation and recycling. Studies have revealed the molecular mechanism of transcriptional regulation of Autophagy-Related (ATG) genes upon nutrient deprivation. However, little is known about their translational regulation. Here, we found that Dhh1, a DExD/H-box RNA helicase, is required for efficient translation of Atg1 and Atg13, two Proteins essential for Autophagy induction. Dhh1 directly associates with ATG1 and ATG13 mRNAs under nitrogen-starvation conditions. The structured regions shortly after the start codons of the two ATG mRNAs are necessary for their translational regulation by Dhh1. Both the RNA-binding ability and helicase activity of Dhh1 are indispensable to promote Atg1 translation and Autophagy. Moreover, eukaryotic translation initiation factor 4E (EIF4E)-associated Protein 1 (Eap1), a target of rapamycin (TOR)-regulated EIF4E binding Protein, physically interacts with Dhh1 after nitrogen starvation and facilitates the translation of Atg1 and Atg13. These results suggest a model for how some ATG genes bypass the general translational suppression that occurs during nitrogen starvation to maintain a proper level of Autophagy.
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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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quantitative analysis of Autophagy Related Protein stoichiometry by fluorescence microscopy
Journal of Cell Biology, 2008Co-Authors: Jiefei Geng, Misuzu Baba, Usha Nair, Daniel J KlionskyAbstract:In yeast, ∼31 Autophagy-Related (Atg) Proteins have been identified. Most of them reside at the phagophore assembly site (PAS), although the function of the PAS mostly remains unclear. One reason for the latter is the lack of stoichiometric information regarding the Atg Proteins at this site. We report the application of fluorescence microscopy to study the amount of Atg Proteins at the PAS. We find that an increase in the amount of Atg11 at the PAS enhances the recruitment of Atg8 and Atg9 to this site and facilitates the formation of more cytoplasm-to-vacuole targeting vesicles. In response to Autophagy induction, the amount of most Atg Proteins remains unchanged at the PAS, whereas we see an enhanced recruitment of Atg8 and 9 at this site. During Autophagy, the amount of Atg8 at the PAS showed a periodic change, indicating the formation of autophagosomes. Application of this method and further analysis will provide more insight into the functions of Atg Proteins.
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physiological functions of atg6 beclin 1 a unique Autophagy Related Protein
Cell Research, 2007Co-Authors: Yang Cao, Daniel J KlionskyAbstract:The most striking morphological feature of eukaryotic cells is the presence of various membrane-enclosed compartments. These compartments, including organelles and transient transport intermediates, are not static. Rather, dynamic exchange of Proteins and membrane is needed to maintain cellular homeostasis. One of the most dramatic examples of membrane mobilization is seen during the process of macroAutophagy. MacroAutophagy is the primary cellular pathway for degradation of long-lived Proteins and organelles. In response to environmental cues, such as starvation or other types of stress, the cell produces a unique membrane structure, the phagophore. The phagophore sequesters cytoplasm as it forms a double-membrane cytosolic vesicle, an autophagosome. Upon completion, the autophagosome fuses with a lysosome or a vacuole in yeast, which delivers hydrolases that break down the inner autophagosome membrane along with its cargo, and the resulting macromolecules are released back into the cytosol for reuse. Autophagy is therefore a recycling process, allowing cells to survive periods of nutrient limitation; however, it has a wider physiological role, participating in development and aging, and also in protection against pathogen invasion, cancer and certain neurodegenerative diseases. In many cases, the role of Autophagy is identified through studies of an Autophagy-Related Protein, Atg6/Beclin 1. This Protein is part of a lipid kinase complex, and recent studies suggest that it plays a central role in coordinating the cytoprotective function of Autophagy and in opposing the cellular death process of apoptosis. Here, we summarize our current knowledge of Atg6/Beclin 1 in different model organisms and its unique function in the cell.
Fuyuhiko Inagaki - One of the best experts on this subject based on the ideXlab platform.
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structure of the novel c terminal domain of vacuolar Protein sorting 30 Autophagy Related Protein 6 and its specific role in Autophagy
Journal of Biological Chemistry, 2012Co-Authors: Nobuo N. Noda, Yoshinori Ohsumi, Takafumi Kobayashi, Wakana Adachi, Yuko Fujioka, Fuyuhiko InagakiAbstract:Vacuolar Protein sorting 30 (Vps30)/Autophagy-Related Protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in Autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar Protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for Autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, Autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar Protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this Protein family in various biological processes.
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STRUCTURE OF THE NOVEL C-TERMINAL DOMAIN OF VACUOLAR Protein SORTING 30/Autophagy-Related Protein 6 AND ITS SPECIFIC ROLE IN Autophagy
Journal of Biological Chemistry, 2012Co-Authors: Nobuo N. Noda, Yoshinori Ohsumi, Takafumi Kobayashi, Wakana Adachi, Yuko Fujioka, Fuyuhiko InagakiAbstract:Vacuolar Protein sorting 30 (Vps30)/Autophagy-Related Protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in Autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar Protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for Autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, Autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar Protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this Protein family in various biological processes.
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Autophagy-Related Protein 32 acts as autophagic degron and directly initiates mitophagy.
Journal of Biological Chemistry, 2012Co-Authors: Noriko Kondo-okamoto, Nobuo N. Noda, Sho Suzuki, Hitoshi Nakatogawa, Ikuko Takahashi, Miou Matsunami, Ayako Hashimoto, Fuyuhiko Inagaki, Yoshinori Ohsumi, Koji OkamotoAbstract:Autophagy-Related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, Autophagy-Related Protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its N- and C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like Protein localized to the autophagosome, and Atg11, a scaffold Protein required for selective Autophagy-Related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole Protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted Autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle Autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy.
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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
Journal of Biological Chemistry, 2010Co-Authors: Masaya Yamaguchi, Nobuo N. Noda, Hitoshi Nakatogawa, Yoshinori Ohsumi, Hiroyuki Kumeta, Fuyuhiko InagakiAbstract: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.
Nobuo N. Noda - One of the best experts on this subject based on the ideXlab platform.
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the Autophagy Related Protein kinase atg1 interacts with the ubiquitin like Protein atg8 via the atg8 family interacting motif to facilitate autophagosome formation
Journal of Biological Chemistry, 2012Co-Authors: Hitoshi Nakatogawa, Nobuo N. Noda, Sho Suzuki, Shiran Ohbayashi, Machiko Sakohnakatogawa, Soichiro Kakuta, Hiromi Kirisako, Chika Kondokakuta, Hayashi Yamamoto, Yoshinori OhsumiAbstract:In Autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires Autophagy-Related (Atg) Proteins. Atg8 is a ubiquitin-like Protein that localizes to the isolation membrane; a subpopulation of this Protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via Autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of Autophagy, receptor Proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in Autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.
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structure of the novel c terminal domain of vacuolar Protein sorting 30 Autophagy Related Protein 6 and its specific role in Autophagy
Journal of Biological Chemistry, 2012Co-Authors: Nobuo N. Noda, Yoshinori Ohsumi, Takafumi Kobayashi, Wakana Adachi, Yuko Fujioka, Fuyuhiko InagakiAbstract:Vacuolar Protein sorting 30 (Vps30)/Autophagy-Related Protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in Autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar Protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for Autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, Autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar Protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this Protein family in various biological processes.
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STRUCTURE OF THE NOVEL C-TERMINAL DOMAIN OF VACUOLAR Protein SORTING 30/Autophagy-Related Protein 6 AND ITS SPECIFIC ROLE IN Autophagy
Journal of Biological Chemistry, 2012Co-Authors: Nobuo N. Noda, Yoshinori Ohsumi, Takafumi Kobayashi, Wakana Adachi, Yuko Fujioka, Fuyuhiko InagakiAbstract:Vacuolar Protein sorting 30 (Vps30)/Autophagy-Related Protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in Autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar Protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for Autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, Autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar Protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this Protein family in various biological processes.
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Autophagy-Related Protein 32 acts as autophagic degron and directly initiates mitophagy.
Journal of Biological Chemistry, 2012Co-Authors: Noriko Kondo-okamoto, Nobuo N. Noda, Sho Suzuki, Hitoshi Nakatogawa, Ikuko Takahashi, Miou Matsunami, Ayako Hashimoto, Fuyuhiko Inagaki, Yoshinori Ohsumi, Koji OkamotoAbstract:Autophagy-Related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, Autophagy-Related Protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its N- and C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like Protein localized to the autophagosome, and Atg11, a scaffold Protein required for selective Autophagy-Related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole Protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted Autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle Autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy.
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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
Journal of Biological Chemistry, 2010Co-Authors: Masaya Yamaguchi, Nobuo N. Noda, Hitoshi Nakatogawa, Yoshinori Ohsumi, Hiroyuki Kumeta, Fuyuhiko InagakiAbstract: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.
Yoshinori Ohsumi - One of the best experts on this subject based on the ideXlab platform.
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the Autophagy Related Protein kinase atg1 interacts with the ubiquitin like Protein atg8 via the atg8 family interacting motif to facilitate autophagosome formation
Journal of Biological Chemistry, 2012Co-Authors: Hitoshi Nakatogawa, Nobuo N. Noda, Sho Suzuki, Shiran Ohbayashi, Machiko Sakohnakatogawa, Soichiro Kakuta, Hiromi Kirisako, Chika Kondokakuta, Hayashi Yamamoto, Yoshinori OhsumiAbstract:In Autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires Autophagy-Related (Atg) Proteins. Atg8 is a ubiquitin-like Protein that localizes to the isolation membrane; a subpopulation of this Protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via Autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of Autophagy, receptor Proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in Autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.
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structure of the novel c terminal domain of vacuolar Protein sorting 30 Autophagy Related Protein 6 and its specific role in Autophagy
Journal of Biological Chemistry, 2012Co-Authors: Nobuo N. Noda, Yoshinori Ohsumi, Takafumi Kobayashi, Wakana Adachi, Yuko Fujioka, Fuyuhiko InagakiAbstract:Vacuolar Protein sorting 30 (Vps30)/Autophagy-Related Protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in Autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar Protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for Autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, Autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar Protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this Protein family in various biological processes.
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STRUCTURE OF THE NOVEL C-TERMINAL DOMAIN OF VACUOLAR Protein SORTING 30/Autophagy-Related Protein 6 AND ITS SPECIFIC ROLE IN Autophagy
Journal of Biological Chemistry, 2012Co-Authors: Nobuo N. Noda, Yoshinori Ohsumi, Takafumi Kobayashi, Wakana Adachi, Yuko Fujioka, Fuyuhiko InagakiAbstract:Vacuolar Protein sorting 30 (Vps30)/Autophagy-Related Protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in Autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar Protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for Autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, Autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar Protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this Protein family in various biological processes.
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Autophagy-Related Protein 32 acts as autophagic degron and directly initiates mitophagy.
Journal of Biological Chemistry, 2012Co-Authors: Noriko Kondo-okamoto, Nobuo N. Noda, Sho Suzuki, Hitoshi Nakatogawa, Ikuko Takahashi, Miou Matsunami, Ayako Hashimoto, Fuyuhiko Inagaki, Yoshinori Ohsumi, Koji OkamotoAbstract:Autophagy-Related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, Autophagy-Related Protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its N- and C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like Protein localized to the autophagosome, and Atg11, a scaffold Protein required for selective Autophagy-Related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole Protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted Autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle Autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy.
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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
Journal of Biological Chemistry, 2010Co-Authors: Masaya Yamaguchi, Nobuo N. Noda, Hitoshi Nakatogawa, Yoshinori Ohsumi, Hiroyuki Kumeta, Fuyuhiko InagakiAbstract: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.
Sébastien Besteiro - One of the best experts on this subject based on the ideXlab platform.
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toxoplasma gondii Autophagy Related Protein atg9 is crucial for the survival of parasites in their host
Cellular Microbiology, 2017Co-Authors: Hoa Mai Nguyen, Laurence Berry, Hiba El Hajj, Rana El Hajj, Nadim Tawil, Maryse Lebrun, Yann Bordat, Sébastien BesteiroAbstract:Autophagy is a conserved, life-promoting, catabolic process involved in the recycling of nonessential cellular components in response to stress. The parasite Toxoplasma gondii is an early-diverging eukaryote in which part of the Autophagy machinery is not exclusively involved in a catabolic process but instead has been repurposed for an original function in organelle inheritance during cell division. This function, depending essentially on Protein TgATG8 and its membrane conjugation system, is crucial for parasite survival and prevented an in depth study of Autophagy in the mutants generated so far in Toxoplasma. Thus, in order to decipher the primary function of canonical Autophagy in the parasites, we generated a cell line deficient for TgATG9, a Protein thought to be involved in the early steps of the Autophagy process. Although the Protein proved to be dispensable for the development of these obligate intracellular parasites in vitro, the absence of TgATG9 led to a reduced ability to sustain prolonged extracellular stress. Importantly, depletion of the Protein significantly reduced parasites survival in macrophages and markedly attenuated their virulence in mice. Altogether, this shows TgATG9 is important for the fate of Toxoplasma in immune cells and contributes to the overall virulence of the parasite, possibly through an involvement in a canonical Autophagy pathway.
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Repurposing of conserved Autophagy-Related Protein ATG8 in a divergent eukaryote
Communicative and Integrative Biology, 2016Co-Authors: Maude Lévêque, Hoa Mai Nguyen, Sébastien BesteiroAbstract:Toxoplasma gondii and other apicomplexan parasites contain a peculiar non-photosynthetic plastid called the apicoplast, which is essential for their survival. The localization of Autophagy-Related Protein ATG8 to the apicoplast in several apicomplexan species and life stages has recently been described, and we have shown this Protein is essential for proper inheritance of this complex plastid into daughter cells during cell division. Although the mechanism behind ATG8 association to the apicoplast in T. gondii is Related to the canonical conjugation system leading to autophagosome formation, its singular role seems independent from the initial catabolic purpose of Autophagy. Here we also discuss further the functional evolution and innovative adaptations of the Autophagy machinery to maintain this organelle during parasite division.
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Autophagy-Related Protein ATG8 Has a Noncanonical Function for Apicoplast Inheritance in Toxoplasma gondii
mBio, 2015Co-Authors: Maude F. Lévêque, Hoa Mai Nguyen, Laurence Berry, Michael J. Cipriano, Boris Striepen, Sébastien BesteiroAbstract:Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of un- wanted Proteins and organelles through the lysosomal pathway. This mechanism involves the formation of a double-membrane structure called the autophagosome that sequesters cellular components to be degraded. To orchestrate this process, yeasts and animals rely on a conserved set of Autophagy-Related Proteins (ATGs). Key among these factors is ATG8, a cytoplasmic Protein that is recruited to nascent autophagosomal membranes upon the induction of Autophagy. Toxoplasma gondii is a potentially harmful human pathogen in which only a subset of ATGs appears to be present. Although this eukaryotic parasite seems able to generate autophagosomes upon stresses such as nutrient starvation, the full functionality and biological relevance of a canonical Autophagy pathway are as yet unclear. Intriguingly, in T. gondii, ATG8 localizes to the apicoplast under normal intracellular growth conditions. The apicoplast is a nonphotosynthetic plastid enclosed by four membranes resulting from a secondary endo- symbiosis. Using superresolution microscopy and biochemical techniques, we show that TgATG8 localizes to the outermost membrane of this organelle. We investigated the unusual function of TgATG8 at the apicoplast by generating a conditional knockdown mutant. Depletion of TgATG8 led to rapid loss of the organelle and subsequent intracellular replication defects, in- dicating that the Protein is essential for maintaining apicoplast homeostasis and thus for survival of the tachyzoite stage. More precisely, loss of TgATG8 led to abnormal segregation of the apicoplast into the progeny because of a loss of physical interactions of the organelle with the centrosomes. IMPORTANCE By definition, Autophagy is a catabolic process that leads to the digestion and recycling of eukaryotic cellular components. The molecular machinery of Autophagy was identified mainly in model organisms such as yeasts but remains poorly characterized in phylogenetically distant apicomplexan parasites. We have uncovered an unusual function for Autophagy-Related Protein ATG8 in Toxoplasma gondii: TgATG8 is crucial for normal replication of the parasite inside its host cell. Seemingly unRelated to the catabolic Autophagy process, TgATG8 associates with the outer membrane of the non- photosynthetic plastid harbored by the parasite called the apicoplast, and there it plays an important role in the centrosome-driven inheritance of the organelle during cell division. This not only reveals an unexpected function for an Autophagy-Related Protein but also sheds new light on the division process of an organelle that is vital to a group of impor- tant human and animal pathogens.
