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Daniel J Klionsky - One of the best experts on this subject based on the ideXlab platform.

  • dhh1 promotes autophagy related protein translation during nitrogen starvation
    PLOS Biology, 2019
    Co-Authors: Xu Liu, Zhiyuan Yao, Meiyan Jin, Sim Namkoong, Zhangyuan Yin, Jun Hee Lee, Daniel J Klionsky
    Abstract:

    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.

  • PP2C phosphatases promote autophagy by dephosphorylation of the Atg1 complex
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Gonen Memisoglu, Daniel J Klionsky, Vinay V. Eapen, Ying Yang, James E. Haber
    Abstract:

    Macroautophagy is orchestrated by the Atg1-Atg13 complex in budding yeast. Under nutrient-rich conditions, Atg13 is maintained in a hyperphosphorylated state by the TORC1 kinase. After nutrient starvation, Atg13 is dephosphorylated, triggering Atg1 kinase activity and macroautophagy induction. The phosphatases that dephosphorylate Atg13 remain uncharacterized. Here, we show that two redundant PP2C phosphatases, Ptc2 and Ptc3, regulate macroautophagy by dephosphorylating Atg13 and Atg1. In the absence of these phosphatases, starvation-induced macroautophagy and the cytoplasm-to-vacuole targeting pathway are inhibited, and the recruitment of the essential autophagy machinery to the phagophore assembly site is impaired. Expressing a genomic Atg13-8SA allele lacking key TORC1 phosphorylation sites partially bypasses the macroautophagy defect in ptc2Δ ptc3Δ strains. Moreover, Ptc2 and Ptc3 interact with the Atg1-Atg13 complex. Taken together, these results suggest that PP2C-type phosphatases promote macroautophagy by regulating the Atg1 complex.

  • Dhh1 promotes the translation of Atg1 and Atg13 under nitrogen-starvation conditions.
    2019
    Co-Authors: Xu Liu, Zhiyuan Yao, Meiyan Jin, Sim Namkoong, Zhangyuan Yin, Jun Hee Lee, Daniel J Klionsky
    Abstract:

    (A) WT (SEY6210) and dhh1Δ (XLY301) cells were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 6 h. Cell lysates were prepared, subjected to SDS-PAGE, and analyzed by western blot. For quantification of Atg1 protein level in cells after prolonged nitrogen starvation, the Atg1 protein level was first normalized to the loading control, Pgk1. The values of the dhh1Δ mutant samples were then normalized to that in WT cells. Average values ± s.d. of n = 3 independent experiments are shown as indicated. ****p < 0.0001. (B) Atg13–PA (ZYY202) and Atg13–PA dhh1Δ (ZYY203) cells were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 6 h. Cell lysates were prepared, subjected to SDS-PAGE, and analyzed by western blot. The quantification of Atg13 protein level was conducted as indicated in (A). The 5′-UTR and 3′-UTR of Atg13 in these strains were not changed. ****p < 0.0001. (C) Atg13–PA and Atg13–PA dhh1Δ cells were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 6 h. Total RNA for each sample was extracted, and the mRNA levels were quantified by RT-qPCR. The mRNA levels of the samples were first normalized to the level of the reference gene TAF10. Then, individual Atg1 and Atg13 mRNA levels were normalized to the mRNA level of the corresponding gene in WT cells. Error bars represent the SD of three independent experiments. (D) WT (XLY338) and Dhh1–AID (XLY340) cells were grown in YPD to mid-log phase and treated with either DMSO or 300 μM IAA for 30 min. They were then shifted to SD-N for 6 h in the presence of either DMSO or IAA. Cell lysates were prepared, subjected to SDS-PAGE, and analyzed by western blot. For quantification, the Atg1 protein levels were first normalized to the loading control, Pgk1. The values of the IAA-treated samples were then normalized to that in DMSO (IAA “-”)-treated samples. Average values ± s.d. of n = 3 independent experiments are shown as indicated. **p < 0.01. (See also S2 and S3 Figs; raw numerical values are shown in S1 Data). Atg, autophagy-related; DMSO, dimethyl sulfoxide; IAA, indoe-3-acetic acid; L.E., long exposure; NS, not significant; PA, protein A; Pgk1, 3-phosphoglycerate kinase 1; RT-qPCR, quantitative reverse transcription PCR; SD-N, synthetic minimal medium lacking nitrogen; S.E, short exposure; TAF10, TATA binding protein-associated factor 10; WT, wild type; YPD, yeast extract–peptone–dextrose.

  • Eap1 facilitates Atg1 and Atg13 translation during nitrogen starvation.
    2019
    Co-Authors: Xu Liu, Zhiyuan Yao, Meiyan Jin, Sim Namkoong, Zhangyuan Yin, Jun Hee Lee, Daniel J Klionsky
    Abstract:

    (A, B, C) Atg13–PA (ZYY202) and Atg13–PA eap1Δ (ZYY204) cells were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 6 h. Cell lysates were prepared, subjected to SDS-PAGE, and analyzed by western blot. Atg1 and Atg13–PA levels are shown in (A) and (B), respectively. The quantification of Atg1 and Atg13–PA protein levels was conducted as indicated in Fig 2A and was shown in (C). **p < 0.01. ***p < 0.001. (D) Atg13–PA (ZYY202) and Atg13–PA eap1Δ (ZYY204) cells were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 6 h. Total RNA for each sample was extracted, and the Atg1 and Atg13 mRNA levels were quantified by RT-qPCR. (E) WT (XLY338) and Eap1–AID (ZYY210) cells were grown in YPD to mid-log phase and treated with either DMSO or 300 μM IAA for 30 min. They were then shifted to SD-N for 6 h in the presence of either DMSO or IAA. Cell lysates were prepared, subjected to SDS-PAGE and analyzed by western blot. The quantification of Atg1 levels was conducted as indicated in Fig 2D. *p < 0.05. (F) Pgi1–GFP (XLY306) and Pgi1–GFP eap1Δ (XLY310) cells were grown in YPD to mid-log phase (-N, 0 d) and then shifted to SD-N for 1 or 3 d. Cell lysates were prepared, subjected to SDS-PAGE, and analyzed by western blot. The quantification of the ratio of free to total GFP was conducted as indicated in Fig 1C. *p < 0.05. **p < 0.01. (G) Schematic images of the domains of EIF4EBPs, including EIF4EBP1 (Homo sapiens), Caf20 (Saccharomyces cerevisiae), and Eap1 (S. cerevisiae). Although they share an EIF4E binding motif, Eap1 has a long C-terminal stretch that is absent in the other two proteins. (H) WT Eap1–GFP (ZYY215), Eap1Δ271–632–GFP (ZYY211), Eap1Δ441–632–GFP (ZYY212), and eap1Δ (ZYY204) cells were grown in YPD to mid-log phase (-N, 0 d) and then shifted to SD-N for 6 h. Cell lysates were prepared, subjected to SDS-PAGE, and analyzed by western blot. The quantification of Atg1 protein levels was conducted as indicated in Fig 2A. *p < 0.05. (See also S7 Fig; raw numerical values are shown in S1 Data). AID, auxin-inducible degron; Atg, autophagy-related; Caf20, cap associated factor 20; DMSO, dimethyl sulfoxide; Eap1, EIF4E–associated protein 1; EIF4E, eukaryotic translation initiation factor 4E; EIF4EBP, EIF4E binding protein; GFP, green fluorescent protein; IAA, indole-3-acetic acid; NS, not significant; PA, protein A; Pgi1, phosphoglucoisomerase 1; Pgk1, 3-phosphoglycerate kinase 1; RT-qPCR, quantitative reverse transcription PCR; SD-N, synthetic minimal medium lacking nitrogen; WT, wild type; YPD, yeast extract–peptone–dextrose.

  • The structured regions in the Atg1 and Atg13 ORFs are necessary for the translational regulation by Dhh1 after nitrogen starvation.
    2019
    Co-Authors: Xu Liu, Zhiyuan Yao, Meiyan Jin, Sim Namkoong, Zhangyuan Yin, Jun Hee Lee, Daniel J Klionsky
    Abstract:

    (A) The mutations (“mut”) made in the structured regions of Atg1 and Atg13 ORFs are shown as indicated; mutated nucleotides are presented in black boxes. (B) WT Atg1 (XLY316), WT Atg1 dhh1Δ (XLY317), mutant Atg1 (XLY318), and mutant Atg1 dhh1Δ (XLY319) cells were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 6 h. Cell lysates were prepared, subjected to SDS-PAGE, and analyzed by western blot. The quantification of Atg1 protein level was conducted as indicated in Fig 2A. NS, not significant. ****p < 0.0001. (C) WT Atg13–PA (ZYY202), WT Atg13–PA dhh1Δ (ZYY203), mutant Atg13–PA (ZYY205), and mutant Atg13–PA dhh1Δ (ZYY206) cells were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 6 h. Cell lysates were prepared, subjected to SDS-PAGE, and analyzed by western blot. The quantification of Atg13–PA protein level was conducted as indicated in Fig 2A. The 5′-UTR and 3′-UTR of Atg13 in these strains were not changed. ****p < 0.0001. (D) The WT strain with empty vector (XLY329), the dhh1Δ strain with either empty vector (XLY331), or vectors expressing WT Dhh1–PA (XLY333) or Dhh1D195A,E196A,S226A,T228A–PA (DEAA STAA; XLY342) were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 6 h. Cell lysates were prepared, subjected to SDS-PAGE, and analyzed by western blot. The quantification of the Atg1 protein level was conducted as indicated in Fig 2A. **p < 0.01. ***p < 0.001. (E) The strains in (D) were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 24 h. Cell lysates were prepared and subjected to SDS-PAGE. The processing of Pgi1–GFP was analyzed by western blot. The quantification of free GFP level was conducted as indicated in Fig 1D. **p < 0.01. ****p < 0.0001. (See also S5 Fig; raw numerical values are shown in S1 Data). Atg, autophagy-related; DEAA STAA, Dhh1D195A,E196A,S226A,T228A; GFP, green fluorescent protein; L.E., long exposure; NS, not significant; ORF, open reading frame; PA, protein A; Pgi1, phosphoglucoisomerase 1; Pgk1, 3-phosphoglycerate kinase 1; SD-N, synthetic minimal medium lacking nitrogen; S.E., short exposure; WT, wild type; YPD, yeast extract–peptone–dextrose.

Yoshinori Ohsumi - One of the best experts on this subject based on the ideXlab platform.

  • Atg13 horma domain recruits atg9 vesicles during autophagosome formation
    Proceedings of the National Academy of Sciences of the United States of America, 2015
    Co-Authors: Sho W Suzuki, Hayashi Yamamoto, Chika Kondokakuta, Yayoi Kimura, Hisashi Hirano, Yu Oikawa, Yoshinori Ohsumi
    Abstract:

    During autophagosome formation, autophagosome-related (Atg) proteins are recruited hierarchically to organize the preautophagosomal structure (PAS). Atg13, which plays a central role in the initial step of PAS formation, consists of two structural regions, the N-terminal HORMA (from Hop1, Rev7, and Mad2) domain and the C-terminal disordered region. The C-terminal disordered region of Atg13, which contains the binding sites for Atg1 and Atg17, is essential for the initiation step in which the Atg1 complex is formed to serve as a scaffold for the PAS. The N-terminal HORMA domain of Atg13 is also essential for autophagy, but its molecular function has not been established. In this study, we searched for interaction partners of the Atg13 HORMA domain and found that it binds Atg9, a multispanning membrane protein that exists on specific cytoplasmic vesicles (Atg9 vesicles). After the Atg1 complex is formed, Atg9 vesicles are recruited to the PAS and become part of the autophagosomal membrane. HORMA domain mutants, which are unable to interact with Atg9, impaired the PAS localization of Atg9 vesicles and exhibited severe defects in starvation-induced autophagy. Thus, Atg9 vesicles are recruited to the PAS via the interaction with the Atg13 HORMA domain. Based on these findings, we propose that the two distinct regions of Atg13 play crucial roles in distinct steps of autophagosome formation: In the first step, Atg13 forms a scaffold for the PAS via its C-terminal disordered region, and subsequently it recruits Atg9 vesicles via its N-terminal HORMA domain.

  • structural basis of starvation induced assembly of the autophagy initiation complex
    Nature Structural & Molecular Biology, 2014
    Co-Authors: Yuko Fujioka, Hayashi Yamamoto, Chika Kondokakuta, Rinji Akada, Sho Suzuki, Fuyuhiko Inagaki, Yayoi Kimura, Yoshinori Ohsumi, Hisashi Hirano, Nobuo N. Noda
    Abstract:

    Autophagy initiates with the assembly of a preautophagosomal structure (PAS), triggered by the yeast Atg1 complex. Ohsumi, Noda and colleagues present the crystal structures of Atg13–Atg1 and Atg13–Atg17, revealing how starvation-induced dephosphorylation of Atg13 triggers formationof the Atg1 complex and PAS assembly.

  • fine mapping of autophagy related proteins during autophagosome formation in saccharomyces cerevisiae
    Journal of Cell Science, 2013
    Co-Authors: Kuninori Suzuki, Hayashi Yamamoto, Chika Kondokakuta, Manami Akioka, Yoshinori Ohsumi
    Abstract:

    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.

  • Atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
    Nature Structural & Molecular Biology, 2013
    Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori Ohsumi
    Abstract:

    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.

  • Atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
    Nature Structural & Molecular Biology, 2013
    Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori Ohsumi
    Abstract:

    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.

  • the intrinsically disordered protein Atg13 mediates supramolecular assembly of autophagy initiation complexes
    Developmental Cell, 2016
    Co-Authors: Hayashi Yamamoto, Daisuke Noshiro, Chika Kondokakuta, Hironori Suzuki, Yuko Fujioka, Sho Suzuki, Toshio Ando, Yayoi Kimura, Hisashi Hirano, Nobuo N. Noda
    Abstract:

    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.

  • structure of the Atg101 Atg13 complex reveals essential roles of Atg101 in autophagy initiation
    Nature Structural & Molecular Biology, 2015
    Co-Authors: Hironori Suzuki, Takeshi Kaizuka, Noboru Mizushima, Nobuo N. Noda
    Abstract:

    Atg101 is an essential component of the autophagy-initiating ULK complex in higher eukaryotes, but it is absent from the functionally equivalent Atg1 complex in budding yeast. Here, we report the crystal structure of the fission yeast Atg101-Atg13 complex. Atg101 has a Hop1, Rev7 and Mad2 (HORMA) architecture similar to that of Atg13. Mad2 HORMA has two distinct conformations (O-Mad2 and C-Mad2), and, intriguingly, Atg101 resembles O-Mad2 rather than the C-Mad2-like Atg13. Atg13 HORMA from higher eukaryotes possesses an inherently unstable fold, which is stabilized by Atg101 via interactions analogous to those between O-Mad2 and C-Mad2. Mutational studies revealed that Atg101 is responsible for recruiting downstream factors to the autophagosome-formation site in mammals via a newly identified WF finger. These data define the molecular functions of Atg101, providing a basis for elucidating the molecular mechanisms of mammalian autophagy initiation by the ULK complex.

  • structural basis of starvation induced assembly of the autophagy initiation complex
    Nature Structural & Molecular Biology, 2014
    Co-Authors: Yuko Fujioka, Hayashi Yamamoto, Chika Kondokakuta, Rinji Akada, Sho Suzuki, Fuyuhiko Inagaki, Yayoi Kimura, Yoshinori Ohsumi, Hisashi Hirano, Nobuo N. Noda
    Abstract:

    Autophagy initiates with the assembly of a preautophagosomal structure (PAS), triggered by the yeast Atg1 complex. Ohsumi, Noda and colleagues present the crystal structures of Atg13–Atg1 and Atg13–Atg17, revealing how starvation-induced dephosphorylation of Atg13 triggers formationof the Atg1 complex and PAS assembly.

  • 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, 2014
    Co-Authors: Nobuo N. Noda, Fuyuhiko Inagaki
    Abstract:

    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.

  • Atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
    Nature Structural & Molecular Biology, 2013
    Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori Ohsumi
    Abstract:

    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.

  • structural basis of starvation induced assembly of the autophagy initiation complex
    Nature Structural & Molecular Biology, 2014
    Co-Authors: Yuko Fujioka, Hayashi Yamamoto, Chika Kondokakuta, Rinji Akada, Sho Suzuki, Fuyuhiko Inagaki, Yayoi Kimura, Yoshinori Ohsumi, Hisashi Hirano, Nobuo N. Noda
    Abstract:

    Autophagy initiates with the assembly of a preautophagosomal structure (PAS), triggered by the yeast Atg1 complex. Ohsumi, Noda and colleagues present the crystal structures of Atg13–Atg1 and Atg13–Atg17, revealing how starvation-induced dephosphorylation of Atg13 triggers formationof the Atg1 complex and PAS assembly.

  • 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, 2014
    Co-Authors: Nobuo N. Noda, Fuyuhiko Inagaki
    Abstract:

    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.

  • Atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
    Nature Structural & Molecular Biology, 2013
    Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori Ohsumi
    Abstract:

    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.

  • Atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
    Nature Structural & Molecular Biology, 2013
    Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori Ohsumi
    Abstract:

    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.

  • structure of the Atg12 atg5 conjugate reveals a platform for stimulating atg8 pe conjugation
    EMBO Reports, 2013
    Co-Authors: Nobuo N. Noda, Takao Hanada, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Abstract:

    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.

Hitoshi Nakatogawa - One of the best experts on this subject based on the ideXlab platform.

  • Atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
    Nature Structural & Molecular Biology, 2013
    Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori Ohsumi
    Abstract:

    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.

  • Atg12 atg5 conjugate enhances e2 activity of atg3 by rearranging its catalytic site
    Nature Structural & Molecular Biology, 2013
    Co-Authors: Machiko Sakohnakatogawa, Eri Asai, Kazuaki Matoba, Junko Ishii, Nobuo N. Noda, Fuyuhiko Inagaki, Hiromi Kirisako, Hitoshi Nakatogawa, Yoshinori Ohsumi
    Abstract:

    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.

  • noncanonical recognition and ubl loading of distinct e2s by autophagy essential atg7
    Nature Structural & Molecular Biology, 2012
    Co-Authors: Masaya Yamaguchi, Hayashi Yamamoto, Kazuaki Matoba, Yuko Fujioka, Rinji Akada, Yoshihiro Kobashigawa, Hitoshi Nakatogawa, Ryoko Sawada, Hisashi Hoshida, Yoshinori Ohsumi
    Abstract:

    The enzymes involved in autophagy-related UBL conjugation bear only passing resemblance to their counterparts in the better-known UBL conjugation pathways. New structural work provides insight into the mechanism by which the UBL proteins Atg8 and Atg12 are correctly charged by a single activating enzyme, Atg7, then transferred onto their cognate E2 proteins, Atg3 and Atg10, respectively.

  • 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, 2012
    Co-Authors: Hitoshi Nakatogawa, Hayashi Yamamoto, Chika Kondokakuta, Machiko Sakohnakatogawa, Nobuo N. Noda, Sho Suzuki, Hiromi Kirisako, Shiran Ohbayashi, Soichiro Kakuta, Yoshinori Ohsumi
    Abstract:

    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.

  • atg4 recycles inappropriately lipidated atg8 to promote autophagosome biogenesis
    Autophagy, 2012
    Co-Authors: Hitoshi Nakatogawa, Eri Asai, Junko Ishii, Yoshinori Ohsumi
    Abstract:

    Atg8 is a ubiquitin-like protein required for autophagy in the budding yeast Saccharomyces cerevisiae. A ubiquitin-like system mediates the conjugation of the C terminus of Atg8 to the lipid phosph...