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Structural basis of starvation-induced assembly of the autophagy initiation complex

Nature Structural & Molecular Biology volume 21, pages 513–521 (2014)Cite this article

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Abstract

Assembly of the preautophagosomal structure (PAS) is essential for autophagy initiation in yeast. Starvation-induced dephosphorylation of Atg13 is required for the formation of the Atg1–Atg13–Atg17–Atg29–Atg31 complex (Atg1 complex), a prerequisite for PAS assembly. However, molecular details underlying these events have not been established. Here we studied the interactions of yeast Atg13 with Atg1 and Atg17 by X-ray crystallography. Atg13 binds tandem microtubule interacting and transport domains in Atg1, using an elongated helix-loop-helix region. Atg13 also binds Atg17, using a short region, thereby bridging Atg1 and Atg17 and leading to Atg1-complex formation. Dephosphorylation of specific serines in Atg13 enhanced its interaction with not only Atg1 but also Atg17. These observations update the autophagy-initiation model as follows: upon starvation, dephosphorylated Atg13 binds both Atg1 and Atg17, and this promotes PAS assembly and autophagy progression.

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Figure 1: Structural basis of the Atg1tMIT-Atg13MIM interaction.
Figure 2: Molecular characterization of the Atg1tMIT-Atg13MIM interaction both in vitro and in vivo.
Figure 3: Structural basis of the Atg1317BR-Atg17 interaction.
Figure 4: Phosphorylation at Atg13MIM(C) negatively regulates Atg1 binding.
Figure 5: Dephosphorylation of Ser429 in Atg13 is required for Atg17 binding, PAS assembly and autophagy.

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Acknowledgements

The synchrotron radiation experiments were performed at beamlines BL41XU at SPring8 and NW12A at Photon Factory, Japan. This work was supported in part by Japan Society for the Promotion of Sciences KAKENHI, grant nos. 24113725 and 25111004 (to N.N.N.), 2440279 (to Y.F.), 30114416 (to Y.O.) and 24770182 (to H.Y.), and by the Targeted Proteins Research Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y.O. and F.I.).

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Author notes

  1. Yuko Fujioka, Sho W Suzuki and Hayashi Yamamoto: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Microbial Chemistry (BIKAKEN), Tokyo, Japan

    Yuko Fujioka & Nobuo N Noda

  2. Frontier Research Center, Tokyo Institute of Technology, Yokohama, Japan

    Sho W Suzuki, Hayashi Yamamoto, Chika Kondo-Kakuta & Yoshinori Ohsumi

  3. Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan

    Sho W Suzuki

  4. Graduate School of Medical Life Science and Advanced Medical Research Center, Yokohama City University, Yokohama, Japan

    Yayoi Kimura & Hisashi Hirano

  5. Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Ube, Japan

    Rinji Akada

  6. Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan

    Fuyuhiko Inagaki

  7. Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan

    Fuyuhiko Inagaki & Nobuo N Noda

Authors
  1. Yuko Fujioka
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Contributions

Y.F. and N.N.N. performed structural studies; Y.F. performed biochemical studies; S.W.S., H.Y. and C.K.-K. performed yeast experiments; R.A. cloned the Atg homolog from K. marxianus; Y.K. and H.H. performed LC-MS/MS analysis; and Y.F., S.W.S., H.Y., F.I., Y.O. and N.N.N. analyzed data and wrote the manuscript. All authors discussed the results and commented on the manuscript. N.N.N. and Y.O. supervised the work.

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Correspondence to Yoshinori Ohsumi or Nobuo N Noda.

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Integrated supplementary information

Supplementary Figure 1 Crystallographic study of the Atg1tMIT–Atg13MIM complex.

(a) Prediction of intrinsically disordered regions in S. cerevisiae Atg13 using the DISOPRED server1. The residues predicted to be disordered are colored red. (b) SDS-PAGE of in vitro pulldown assay between Atg13–GST and Atg1tMIT. As for Atg1tMIT bands, they were also detected by Western blotting using anti-Atg1 antibody. (c) CD spectra of Atg13MIM–GB1, GB1 alone, ubiquitin-associated domain (UBA) of p62 (p62UBA)–GB1 as a model of helix-rich domain2, and the flexible region (FR) of Arabidopsis thaliana (At) Atg3 (AtAtg3FR)–GB1 as a model of intrinsically disordered protein3,4. For CD spectroscopy, GB1, a small globular domain consisting of one α-helix and one four-stranded β-sheet, was used as a stabilization tag5. (d) Stereo view of the 2Fo-Fc electron density map of the Atg1tMIT–Atg13MIM complex countered at 1.5 σ level. The final model was shown with a stick model, in which carbon, nitrogen, oxygen and sulfur atoms were colored yellow, blue, red and green, respectively. This figure was prepared using COOT6. (e) Stereo view of the Cα-traces of the Atg1tMIT–Atg13MIM complex. (f) Superimposition of the Atg1–Atg13 complex (copy 2; colored gray) on that of copy 1 (colored red and blue). (g) Ribbon representation of the structure of Vta1tMIT–Vps60MIM complex (PDB ID 2LUH). MIT1 and MIT2 of Vta1 are colored blue and cyan, respectively, and Vps60 is colored salmon pink. Amino- and carboxy termini are denoted by N and C, respectively. Uncropped gel image for b is shown in Supplementary Fig. 6k.

Supplementary Figure 2 The Atg1tMIT-Atg13MIM interaction is evolutionarily conserved.

(a,b) Amino-acid sequence alignment of Atg13 (a) and Atg1 (b) homologs using the ConSurf server7. Purple and cyan indicate high and low sequence conservation, respectively. The position of α-helices is indicated above the sequence. Surface representation of Atg1tMIT is inserted in b, whose coloring is the same as the sequence alignment. Atg13MIM bound to Atg1tMIT is shown by a ribbon model. (c) Sequence alignment between the Atg1/ULK1 binding regions of ScAtg13 and HsAtg13. Hydrophobic residues important for Atg1 binding are indicated with asterisks. The position of α-helices is indicated below the sequence. (d) SDS-PAGE of GST–HsAtg13 (458–517) co-purified with MmULK1 (834–1051). Uncropped gel image for d is shown in Supplementary Fig. 6l.

Supplementary Figure 3 Functional analysis of the Atg1tMIT-Atg13MIM interaction.

(a) CD spectra of Atg13MIM–GB1 (black) and Atg13MIM(F468A L472A)–GB1 (red). (b) SDS-PAGE of in vitro pulldown assay between Atg13MIM–GST and Atg1tMIT. (c) Western blots showing in vivo Atg1–Atg13 interaction analyzed by coimmunoprecipitation in the absence of Atg11 and Atg17. Samples are yeast total lysates (Input) prepared from ATG1–GFP atg11Δ atg17Δcells grown in nutrient-rich medium (Rapamycin, –) or treated with rapamycin for 1 h (Rapamycin, +) and immunoprecipitates isolated with anti-GFP magnetic beads (20xEluate). Asterisks indicate nonspecific bands. Atg13–P indicates phosphorylated forms of Atg13. (d) Western blots of yeast total lysates. Samples are yeast total lysates prepared from cells grown in nutrient-rich medium (Rapamycin, –) or treated with rapamycin for 1 h (Rapamycin, +). To detect slow-migrating phosphorylated forms of Atg1, SDS-PAGE was performed for prolonged period (~120 min) by using 10% polyacrylamide gel (10% gel). prApe1 and mApe1 indicate precursor and mature forms of Ape1, respectively. Uncropped gel images for b, c and d are shown in Supplementary Fig. 6m–o.

Supplementary Figure 4 Molecular characterization of the Atg1317BR-Atg17 interaction.

(a) SDS-PAGE of in vitro pulldown assay between GST–Atg13 and Atg17. (b) Stereo view of the electron density map of Atg1317BR bound to Atg17. The simulated annealing Fo-Fc difference Fourier map was calculated by omitting Atg1317BR, and is shown with black meshes countered at 3.0σ. Structural model of Atg13 and Atg17 is shown similarly with Fig. 3c, right. (c) Stereo view of the Fo-Fc difference Fourier map (countered at 3.0σ) at the Atg1317BR binding region of another Atg17 protomer. (d) CD spectra of Atg17(Wild-type), Atg17(D247A) and Atg17(D247A E250A) bound to Atg31(174–196). (e) SDS-PAGE of in vitro pulldown assay between Atg31(174–196)-bound Atg17 and GST–fused Atg1317BR. (f) ITC result obtained by titrating Atg1317BR into a solution of Atg17(D247A E250A). Uncropped gel images for a and e are shown in Supplementary Fig. 6p,q.

Supplementary Figure 5 Regulation of the interactions of Atg13 with Atg1 and Atg17.

(a) Western blots showing in vivo Atg1–Atg13 interaction analyzed by coimmunoprecipitation in different buffer conditions. Samples are yeast total lysates (Input) prepared from ATG1–GFP cells grown in nutrient-rich medium (Rapamycin, –) or treated with rapamycin for 1 h (Rapamycin, +) and immunoprecipitates isolated with anti-GFP magnetic beads (20xEluate) prepared from buffer conditions 1 and 2. Buffer condition 1 is the same as that used in other coimmunoprecipitation experiments performed in this manuscript. Buffer condition 2 is the same as that used in the recent paper by Kraft et al. Asterisks indicate nonspecific bands. Atg13–P indicates phosphorylated forms of Atg13. (b) Western blots using anti-HA antibody showing in vivo Atg1–Atg13 interaction analyzed by coimmunoprecipitation. Samples are yeast total lysates (Input) prepared from ATG1–GFP cells grown in nutrient-rich medium (Rapamycin, –) or treated with rapamycin for 1 h (Rapamycin, +) and immunoprecipitates isolated with anti-GFP magnetic beads (20xEluate). Atg13–2xHA–P indicates phosphorylated forms of Atg13–2xHA. λPPase indicates treatment of the immunoprecipitates with lambda protein phosphatase. (c) Western blots showing the effects of S429D mutation in Atg13 on the Atg1–Atg13 interaction analyzed by coimmunoprecipitation. Coimmunoprecipitation experiments and western blots were performed as in b. (d) Western blots showing the phosphorylation state of Atg13–2xHA probed with anti-HA antibody and phospho-specific anti-S428/9–P antibodies. Samples are immunoprecipitates of anti-HA immunoprecipitation prepared from cells grown in nutrient-rich medium (Rapamycin, –) or treated with rapamycin for 1 h (Rapamycin, +). (e) Immunoprecipitation and western blots performed as in d. λPPase indicates treatment of the immunoprecipitates with lambda protein phosphatase. Uncropped gel images are shown in Supplementary Fig. 6r–u.

Supplementary Figure 6 Uncropped images for western blots and SDS-PAGE gels.

Broken box marks the borders of the final cropped image.

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Fujioka, Y., Suzuki, S., Yamamoto, H. et al. Structural basis of starvation-induced assembly of the autophagy initiation complex. Nat Struct Mol Biol 21, 513–521 (2014). https://doi.org/10.1038/nsmb.2822

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