The Experts below are selected from a list of 3810 Experts worldwide ranked by ideXlab platform
Fuyuhiko Inagaki - One of the best experts on this subject based on the ideXlab platform.
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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, 2020Co-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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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, Hiromi Kirisako, Eri Asai, Hitoshi Nakatogawa, Nobuo N Noda, Fuyuhiko Inagaki, Kazuaki Matoba, Junko Ishii, 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 Sakoh-nakatogawa, Hiromi Kirisako, Eri Asai, Hitoshi Nakatogawa, Nobuo N Noda, Fuyuhiko Inagaki, Kazuaki Matoba, Junko Ishii, 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, Yoshinori Ohsumi, Yuko Fujioka, Takao Hanada, 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.
Yoshinori Ohsumi - One of the best experts on this subject based on the ideXlab platform.
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Two distinct mechanisms target the autophagy-related E3 complex to the pre-autophagosomal structure.
eLife, 2019Co-Authors: Kumi Harada, Hiromi Kirisako, Yoshinori Ohsumi, Machiko Sakoh-nakatogawa, Hayashi Yamamoto, Yayoi Kimura, Hisashi Hirano, Tetsuya Kotani, Yu Oikawa, Hitoshi NakatogawaAbstract:In autophagy, Atg proteins organize the pre-autophagosomal structure (PAS) to initiate autophagosome formation. Previous studies in yeast revealed that the autophagy-related E3 complex ATG12-Atg5-Atg16 is recruited to the PAS via Atg16 interaction with Atg21, which binds phosphatidylinositol 3-phosphate (PI3P) produced at the PAS, to stimulate conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine. Here, we discover a novel mechanism for the PAS targeting of ATG12-Atg5-Atg16, which is mediated by the interaction of ATG12 with the Atg1 kinase complex that serves as a scaffold for PAS organization. While autophagy is partially defective without one of these mechanisms, cells lacking both completely lose the PAS localization of ATG12-Atg5-Atg16 and show no autophagic activity. As with the PI3P-dependent mechanism, ATG12-Atg5-Atg16 recruited via the ATG12-dependent mechanism stimulates Atg8 lipidation, but also has the specific function of facilitating PAS scaffold assembly. Thus, this study significantly advances our understanding of the nucleation step in autophagosome formation.
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membrane morphology is actively transformed by covalent binding of the protein atg8 to pe lipids
PLOS ONE, 2014Co-Authors: Roland L Knorr, Yoshinori Ohsumi, Hitoshi Nakatogawa, Reinhard Lipowsky, Tobias Baumgart, Rumiana DimovaAbstract:Autophagy is a cellular degradation pathway involving the shape transformation of lipid bilayers. During the onset of autophagy, the water-soluble protein Atg8 binds covalently to phosphatdylethanolamines (PEs) in the membrane in an ubiquitin-like reaction coupled to ATP hydrolysis. We reconstituted the Atg8 conjugation system in giant and nm-sized vesicles with a minimal set of enzymes and observed that formation of Atg8-PE on giant vesicles can cause substantial tubulation of membranes even in the absence of ATG12-Atg5-Atg16. Our findings show that ubiquitin-like processes can actively change properties of lipid membranes and that membrane crowding by proteins can be dynamically regulated in cells. Furthermore we provide evidence for curvature sorting of Atg8-PE. Curvature generation and sorting are directly linked to organelle shapes and, thus, to biological function. Our results suggest that a positive feedback exists between the ubiquitin-like reaction and the membrane curvature, which is important for dynamic shape changes of cell membranes, such as those involved in the formation of autophagosomes.
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hrr25 triggers selective autophagy related pathways by phosphorylating receptor proteins
Journal of Cell Biology, 2014Co-Authors: Chikara Tanaka, Keisuke Mochida, Hiromi Kirisako, Michiko Koizumi, Eri Asai, Machiko Sakohnakatogawa, Yoshinori Ohsumi, Hitoshi NakatogawaAbstract:In selective autophagy, degradation targets are specifically recognized, sequestered by the autophagosome, and transported into the lysosome or vacuole. Previous studies delineated the molecular basis by which the autophagy machinery recognizes those targets, but the regulation of this process is still poorly understood. In this paper, we find that the highly conserved multifunctional kinase Hrr25 regulates two distinct selective autophagy–related pathways in Saccharomyces cerevisiae. Hrr25 is responsible for the phosphorylation of two receptor proteins: Atg19, which recognizes the assembly of vacuolar enzymes in the cytoplasm-to-vacuole targeting pathway, and Atg36, which recognizes superfluous peroxisomes in pexophagy. Hrr25-mediated phosphorylation enhances the interactions of these receptors with the common adaptor Atg11, which recruits the core autophagy-related proteins that mediate the formation of the autophagosomal membrane. Thus, this study introduces regulation of selective autophagy as a new role of Hrr25 and, together with other recent studies, reveals that different selective autophagy–related pathways are regulated by a uniform mechanism: phosphoregulation of the receptor–adaptor interaction.
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Hrr25 triggers selective autophagy–related pathways by phosphorylating receptor proteins
Journal of Cell Biology, 2014Co-Authors: Chikara Tanaka, Keisuke Mochida, Hiromi Kirisako, Michiko Koizumi, Eri Asai, Yoshinori Ohsumi, Machiko Sakoh-nakatogawa, Hitoshi NakatogawaAbstract:In selective autophagy, degradation targets are specifically recognized, sequestered by the autophagosome, and transported into the lysosome or vacuole. Previous studies delineated the molecular basis by which the autophagy machinery recognizes those targets, but the regulation of this process is still poorly understood. In this paper, we find that the highly conserved multifunctional kinase Hrr25 regulates two distinct selective autophagy–related pathways in Saccharomyces cerevisiae. Hrr25 is responsible for the phosphorylation of two receptor proteins: Atg19, which recognizes the assembly of vacuolar enzymes in the cytoplasm-to-vacuole targeting pathway, and Atg36, which recognizes superfluous peroxisomes in pexophagy. Hrr25-mediated phosphorylation enhances the interactions of these receptors with the common adaptor Atg11, which recruits the core autophagy-related proteins that mediate the formation of the autophagosomal membrane. Thus, this study introduces regulation of selective autophagy as a new role of Hrr25 and, together with other recent studies, reveals that different selective autophagy–related pathways are regulated by a uniform mechanism: phosphoregulation of the receptor–adaptor interaction.
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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, Hiromi Kirisako, Eri Asai, Hitoshi Nakatogawa, Nobuo N Noda, Fuyuhiko Inagaki, Kazuaki Matoba, Junko Ishii, 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.
Nobuo N Noda - One of the best experts on this subject based on the ideXlab platform.
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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, 2020Co-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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Evolution from covalent conjugation to non-covalent interaction in the ubiquitin-like ATG12 system
Nature Structural & Molecular Biology, 2019Co-Authors: Yu Pang, Hayashi Yamamoto, Nobuo N Noda, Hirokazu Sakamoto, Joe Kimanthi Mutungi, Mayurbhai Himatbhai Sahani, Yoshitaka Kurikawa, Kiyoshi Kita, Yasuyoshi SakaiAbstract:Ubiquitin or ubiquitin-like proteins can be covalently conjugated to multiple proteins that do not necessarily have binding interfaces. Here, we show that an evolutionary transition from covalent conjugation to non-covalent interaction has occurred in the ubiquitin-like autophagy-related 12 (ATG12) conjugation system. ATG12 is covalently conjugated to its sole substrate, ATG5, by a ubiquitylation-like mechanism. However, the apicomplexan parasites Plasmodium and Toxoplasma and some yeast species such as Komagataella phaffii (previously Pichia pastoris) lack the E2-like enzyme ATG10 and the most carboxy (C)-terminal glycine of ATG12, both of which are required for covalent linkage. Instead, ATG12 in these organisms forms a non-covalent complex with ATG5. This non-covalent ATG12–ATG5 complex retains the ability to facilitate ATG8–phosphatidylethanolamine conjugation. These results suggest that ubiquitin-like covalent conjugation can evolve to a simpler non-covalent interaction, most probably when the system has a limited number of targets.
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the intrinsically disordered protein atg13 mediates supramolecular assembly of autophagy initiation complexes
Developmental Cell, 2016Co-Authors: Hayashi Yamamoto, Yuko Fujioka, Daisuke Noshiro, Hironori Suzuki, Chika Kondokakuta, Yayoi Kimura, Hisashi Hirano, Toshio Ando, Sho Suzuki, Nobuo N NodaAbstract:Autophagosome formation in yeast entails starvation-induced assembly of the pre-autophagosomal structure (PAS), in which multiple Atg1 complexes (composed of Atg1, Atg13, and the Atg17-Atg29-Atg31 subcomplex) are initially engaged. However, the molecular mechanisms underlying the multimeric assembly of these complexes remain unclear. Using structural and biological techniques, we herein demonstrate that Atg13 has a large intrinsically disordered region (IDR) and interacts with two distinct Atg17 molecules using two binding regions in the IDR. We further reveal that these two binding regions are essential not only for Atg1 complex assembly in vitro, but also for PAS organization in vivo. These findings underscore the structural and functional significance of the IDR of Atg13 in autophagy initiation: Atg13 provides intercomplex linkages between Atg17-Atg29-Atg31 complexes, thereby leading to supramolecular self-assembly of Atg1 complexes, in turn accelerating the initial events of autophagy, including autophosphorylation of Atg1, recruitment of Atg9 vesicles, and phosphorylation of Atg9 by Atg1.
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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, Hiromi Kirisako, Eri Asai, Hitoshi Nakatogawa, Nobuo N Noda, Fuyuhiko Inagaki, Kazuaki Matoba, Junko Ishii, 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.
Jayanta Debnath - One of the best experts on this subject based on the ideXlab platform.
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ATG12 atg3 coordinates basal autophagy endolysosomal trafficking and exosome release
Molecular and Cellular Oncology, 2018Co-Authors: Lyndsay Murrow, Jayanta DebnathAbstract:ABSTRACTWe recently identified an interaction between ATG12–Atg3, a complex between two core autophagy regulators, and the Endosomal Sorting Complexes Required for Transport (ESCRT)-associated protein Pdcd6ip (programmed cell death 6 interacting protein, commonly known as Alix), which coordinately regulates basal autophagy, late endosome-to-lysosome trafficking, and exosome release. Because these processes all serve fundamental roles in cancer progression and therapy, ATG12-Atg3 may be an attractive anti-cancer target.
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ATG12–Atg3 coordinates basal autophagy, endolysosomal trafficking and exosome release
Molecular and Cellular Oncology, 2018Co-Authors: Lyndsay Murrow, Jayanta DebnathAbstract:ABSTRACTWe recently identified an interaction between ATG12–Atg3, a complex between two core autophagy regulators, and the Endosomal Sorting Complexes Required for Transport (ESCRT)-associated protein Pdcd6ip (programmed cell death 6 interacting protein, commonly known as Alix), which coordinately regulates basal autophagy, late endosome-to-lysosome trafficking, and exosome release. Because these processes all serve fundamental roles in cancer progression and therapy, ATG12-Atg3 may be an attractive anti-cancer target.
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ATG12 atg3 connects basal autophagy and late endosome function
Autophagy, 2015Co-Authors: Lyndsay Murrow, Jayanta DebnathAbstract:In addition to supporting cell survival in response to starvation or stress, autophagy promotes basal protein and organelle turnover. Compared to our understanding of stress-induced autophagy, little is known about how basal autophagy is regulated and how its activity is coordinated with other cellular processes. We recently identified a novel interaction between the ATG12–ATG3 conjugate and the ESCRT-associated protein PDCD6IP/Alix that promotes basal autophagy and endolysosomal trafficking. Moreover, ATG12–ATG3 is required for diverse PDCD6IP-mediated functions including late endosome distribution, exosome secretion, and viral budding. Our results highlight the importance of late endosomes for basal autophagic flux and reveal distinct roles for the core autophagy proteins ATG12 and ATG3 in controlling late endosome function.
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ATG12–ATG3 connects basal autophagy and late endosome function
Autophagy, 2015Co-Authors: Lyndsay Murrow, Jayanta DebnathAbstract:In addition to supporting cell survival in response to starvation or stress, autophagy promotes basal protein and organelle turnover. Compared to our understanding of stress-induced autophagy, little is known about how basal autophagy is regulated and how its activity is coordinated with other cellular processes. We recently identified a novel interaction between the ATG12–ATG3 conjugate and the ESCRT-associated protein PDCD6IP/Alix that promotes basal autophagy and endolysosomal trafficking. Moreover, ATG12–ATG3 is required for diverse PDCD6IP-mediated functions including late endosome distribution, exosome secretion, and viral budding. Our results highlight the importance of late endosomes for basal autophagic flux and reveal distinct roles for the core autophagy proteins ATG12 and ATG3 in controlling late endosome function.
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ATG12 atg3 interacts with alix to promote basal autophagic flux and late endosome function
Nature Cell Biology, 2015Co-Authors: Lyndsay Murrow, Ritu Malhotra, Jayanta DebnathAbstract:The ubiquitin-like molecule ATG12 is required for the early steps of autophagy. Recently, we identified ATG3, the E2-like enzyme required for LC3 lipidation during autophagy, as an ATG12 conjugation target. Here, we demonstrate that cells lacking ATG12-ATG3 have impaired basal autophagic flux, accumulation of perinuclear late endosomes, and impaired endolysosomal trafficking. Furthermore, we identify an interaction between ATG12-ATG3 and the ESCRT-associated protein Alix (also known as PDCD6IP) and demonstrate that ATG12-ATG3 controls multiple Alix-dependent processes including late endosome distribution, exosome biogenesis and viral budding. Similar to ATG12-ATG3, Alix is functionally required for efficient basal, but not starvation-induced, autophagy. Overall, these results identify a link between the core autophagy and ESCRT machineries and uncover a role for ATG12-ATG3 in late endosome function that is distinct from the canonical role of either ATG in autophagosome formation.
Yuko Fujioka - One of the best experts on this subject based on the ideXlab platform.
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the intrinsically disordered protein atg13 mediates supramolecular assembly of autophagy initiation complexes
Developmental Cell, 2016Co-Authors: Hayashi Yamamoto, Yuko Fujioka, Daisuke Noshiro, Hironori Suzuki, Chika Kondokakuta, Yayoi Kimura, Hisashi Hirano, Toshio Ando, Sho Suzuki, Nobuo N NodaAbstract:Autophagosome formation in yeast entails starvation-induced assembly of the pre-autophagosomal structure (PAS), in which multiple Atg1 complexes (composed of Atg1, Atg13, and the Atg17-Atg29-Atg31 subcomplex) are initially engaged. However, the molecular mechanisms underlying the multimeric assembly of these complexes remain unclear. Using structural and biological techniques, we herein demonstrate that Atg13 has a large intrinsically disordered region (IDR) and interacts with two distinct Atg17 molecules using two binding regions in the IDR. We further reveal that these two binding regions are essential not only for Atg1 complex assembly in vitro, but also for PAS organization in vivo. These findings underscore the structural and functional significance of the IDR of Atg13 in autophagy initiation: Atg13 provides intercomplex linkages between Atg17-Atg29-Atg31 complexes, thereby leading to supramolecular self-assembly of Atg1 complexes, in turn accelerating the initial events of autophagy, including autophosphorylation of Atg1, recruitment of Atg9 vesicles, and phosphorylation of Atg9 by Atg1.
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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, Yoshinori Ohsumi, Yuko Fujioka, Takao Hanada, 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.
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Structure of the ATG12–Atg5 conjugate reveals a platform for stimulating Atg8–PE conjugation
EMBO Reports, 2012Co-Authors: Nobuo N Noda, Yoshinori Ohsumi, Yuko Fujioka, Takao Hanada, 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.
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crystallization of the ATG12 atg5 conjugate bound to atg16 by the free interface diffusion method
Journal of Synchrotron Radiation, 2008Co-Authors: Nobuo N Noda, Yoshinori Ohsumi, Yuko Fujioka, Fuyuhiko InagakiAbstract:Autophagy mediates the bulk degradation of cytoplasmic components in lysosomes/vacuoles. Five autophagy-related (Atg) proteins are involved in a ubiquitin-like protein conjugation system. ATG12 is conjugated to its sole target, Atg5, by two enzymes, Atg7 and Atg10. The ATG12–Atg5 conjugates form a multimeric complex with Atg16. Formation of the ATG12–Atg5–Atg16 ternary complex is crucial for the functions of these proteins on autophagy. Here, the expression, purification and crystallization of the ATG12–Atg5 conjugate bound to the N-terminal region of Atg16 (Atg16N) are reported. The ATG12–Atg5 conjugates were formed by co-expressing Atg5, Atg7, Atg10 and ATG12 in Eschericia coli. The ATG12–Atg5–Atg16N ternary complex was formed by mixing purified ATG12–Atg5 conjugates and Atg16N, and was further purified by gel-filtration chromatography. Crystallization screening was performed by the free-interface diffusion method. Using obtained microcrystals as seeds, large crystals for diffraction data collection were obtained by the sitting-drop vapour-diffusion method. The crystal contained one ternary complex per asymmetric unit, and diffracted to 2.6 A resolution.
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Crystallization of the ATG12–Atg5 conjugate bound to Atg16 by the free-interface diffusion method
Journal of Synchrotron Radiation, 2008Co-Authors: Nobuo N Noda, Yoshinori Ohsumi, Yuko Fujioka, Fuyuhiko InagakiAbstract:Autophagy mediates the bulk degradation of cytoplasmic components in lysosomes/vacuoles. Five autophagy-related (Atg) proteins are involved in a ubiquitin-like protein conjugation system. ATG12 is conjugated to its sole target, Atg5, by two enzymes, Atg7 and Atg10. The ATG12–Atg5 conjugates form a multimeric complex with Atg16. Formation of the ATG12–Atg5–Atg16 ternary complex is crucial for the functions of these proteins on autophagy. Here, the expression, purification and crystallization of the ATG12–Atg5 conjugate bound to the N-terminal region of Atg16 (Atg16N) are reported. The ATG12–Atg5 conjugates were formed by co-expressing Atg5, Atg7, Atg10 and ATG12 in Eschericia coli. The ATG12–Atg5–Atg16N ternary complex was formed by mixing purified ATG12–Atg5 conjugates and Atg16N, and was further purified by gel-filtration chromatography. Crystallization screening was performed by the free-interface diffusion method. Using obtained microcrystals as seeds, large crystals for diffraction data collection were obtained by the sitting-drop vapour-diffusion method. The crystal contained one ternary complex per asymmetric unit, and diffracted to 2.6 A resolution.
