1. Volume 38, Issue 1, Pages 86-99 (July 2016)
The Intrinsically Disordered Protein Atg13 Mediates Supramolecular Assembly of Autophagy Initiation Complexes 
Hayashi Yamamoto, Yuko Fujioka, Sho W. Suzuki, Daisuke Noshiro, Hironori Suzuki, Chika Kondo-Kakuta, Yayoi Kimura, Hisashi Hirano, Toshio Ando, Nobuo N. Noda, Yoshinori Ohsumi 
Developmental Cell 
Volume 38, Issue 1, Pages (July 2016)
DOI: /j.devcel
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Developmental Cell 2016 38, 86-99DOI: (10.1016/j.devcel.2016.06.015)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Identification of an Atg17-Binding Region of Atg13 Named Atg1317LR
(A) The Atg13-Atg17 interaction was analyzed by immunoprecipitation using GFP-Atg13 with a mutation in Atg1317BR. Yeast atg1Δ atg11Δ atg13Δ cells expressing GFP-Atg13 (wild-type [WT], S429D, or F430A) were treated with or without rapamycin for 1 hr, solubilized with 0.03% DDM, and subjected to immunoprecipitation with anti-GFP magnetic beads. The immunoprecipitates were analyzed by western blotting using antibodies against GFP, Atg17, and Pgk1.
(B) ITC results obtained by titration of the Atg1317BR-containing peptide into a solution of the Atg17-Atg31C complex. N, stoichiometry of binding.
(C) In vitro binding assay between GST-fused Atg13 peptide (GST-Atg13 pep.) and Atg17. The proteins bound to GST-accept beads were detected by Coomassie staining.
(D) Schematic representation of Atg17-binding regions of Atg13.
(E) ITC results obtained by titration of the Atg1317LR peptide into a solution of the Atg17-Atg31C complex.
(F) Successive HS-AFM images of Atg13-MBP captured at 10 fps (see also Movie S1). The arrowheads indicate the N-terminal Atg13HORMA that undergoes order-disorder transitions.
See also Table S1 and Movie S1.
Developmental Cell  , 86-99DOI: ( /j.devcel )
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4. Figure 2 Structural Basis of the Atg1317LR-Atg17 Interaction
(A) Crystal structure of the Atg17-Atg29-Atg31 dimeric complex co-crystallized with Atg1317LR and Atg1317BR peptides (PDB: 5JHF). The Atg1317LR and Atg1317BR peptides are shown as stick models in red. Two Atg17 protomers are shown as surface models in green (protomer A) or gray (protomer B). Atg29 and Atg31 are shown as ribbon models in yellow and orange, respectively.
(B) Stereo view of the Atg1317LR-Atg17 region. The amino acid numbers of LtAtg proteins are indicated with those corresponding to ScAtg proteins in parentheses.
(C) Schematic representation of Atg17-binding regions of Atg13 showing the specific serine residues phosphorylated under nutrient-rich conditions (Fujioka et al., 2014) and their conservation in fungal Atg13 homologs. 6S, six phosphorylated serine residues (Ser644, Ser646, Ser649, Ser652, Ser654, and Ser656). ∗p < 0.05 and ∗∗p < 0.01 by two-tailed Student's t test.
(D) In vitro binding assay between GST-Atg1317LR peptide and Atg17 detected by Coomassie staining. WT, wild-type.
(E) ITC results obtained by titration of the Atg1317LR peptide into a solution of the Atg17-Atg31C complex. The left panel (WT) is also used in Figure 1E.
See also Figure S1.
Developmental Cell  , 86-99DOI: ( /j.devcel )
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5. Figure 3
The Atg1317LR-Atg17 Interaction Is Enhanced by Dephosphorylation of Ser379 and Is Crucial for Autophagy In Vivo
(A) Western blots showing the phosphorylation state of Atg13-2×HA probed with anti-HA antibody or antibodies specific to phosphorylated Ser379 (pS379) as indicated. Yeast extracts were prepared from atg13Δ cells expressing Atg13-2×HA, grown in nutrient-rich medium (Nut.), starved in SD(–N) medium for 1 hr (–N), or treated with rapamycin for 1 hr (Rap.). p-Atg13-2×HA, the phosphorylated form of Atg13-2×HA.
(B) Atg1317LR is crucial for the Atg13-Atg17 interaction in vivo. The Atg13-Atg17 interaction was analyzed by immunoprecipitation using GFP-Atg13 with a mutation in Atg1317LR. Yeast lysates were prepared from atg1Δ atg11Δ atg13Δ cells expressing GFP-Atg13 (wild-type [WT], F375A, S379A, or S379D) starved for 1 hr, solubilized with 0.03% DDM, and subjected to immunoprecipitation as in Figure 1A. Immunoprecipitates were analyzed by western blotting using antibodies against GFP, Atg17, and Adh1. Atg17 co-precipitated with wild-type GFP-Atg13 under the starvation condition was set to 100%.
(C) The Atg1317LR-binding region of Atg17 is crucial for the Atg13-Atg17 interaction. Yeast lysates were prepared from atg1Δ atg11Δ atg13Δ atg17Δ cells expressing GFP-Atg13 and Atg17 (WT, I385A, P393A, or T395A) starved for 1 hr, solubilized with 0.03% DDM, and subjected to immunoprecipitation as in Figure 1A.
(D) Fluorescence microscopy of the PAS assembly of Atg17-GFP in Atg1317LR mutant cells. Atg17-GFP and the vacuolar marker protein Vph1-mCherry are shown in green and magenta, respectively. Cells with Atg17-GFP puncta were quantified.
(E) Atg1317LR is essential for autophagy. ALP assay showing autophagic activity of Atg1317LR mutant cells grown in nutrient-rich medium (white bars) or starved in SD(–N) medium for 4 hr (gray bars). Error bars denote SDs from three individual cell cultures. ∗∗p < 0.01 by two-tailed Student's t test.
(F) The Atg1317LR-binding region of Atg17 is required for autophagy. ALP assay showing autophagic activity of Atg17 mutant cells. The ALP assay was performed as in Figure 3E. Error bars denote SDs from three individual cell cultures. ∗∗p < 0.01 by two-tailed Student's t test.
(G) Atg1317LR is not required for the Cvt pathway. Yeast extracts were prepared from atg13Δ cells expressing Atg13 with a mutation in Atg1317LR and analyzed by western blotting using antibodies against Atg13, Ape1, and Pgk1. This experiment was performed in the presence of Atg11. prApe1 and mApe1 indicate the precursor form and the mature form of Ape1, respectively.
See also Figures S2 and S3.
Developmental Cell  , 86-99DOI: ( /j.devcel )
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6. Figure 4
Atg1317LR and Atg1317BR Interact with Atg17, but Not in an Intramolecular Manner
(A) In vitro binding assay between GST-fused Atg13 peptides (Atg1317BR, Atg1317BR2, and Atg1317LR peptides) and Atg17.
(B) Quantitation of Figure 4A. Error bars denote SDs from three individual experiments.
(C) ALP assay showing autophagic activity of Atg13 mutant cells (deletion of or mutation in Atg1317BR, Atg1317BR2, or Atg1317LR). 3SD and 6SD, three mutations (S646D S649D S652D) and six mutations (S644D S646D S649D S652D S654D S656D) in Atg1317BR2, respectively. The ALP assay was performed as in Figure 3E. ∗∗p < 0.01.
(D) Yeast lysates were prepared from atg1Δ atg11Δ atg13Δ cells expressing GFP-Atg13 (wild-type [WT], F430A, F375A, or F375A F430A), solubilized with 0.1% NP-40, and subjected to immunoprecipitation.
(E) Two possible models for the Atg13-Atg17 interaction.
(F) Fluorescence microscopy of the PAS assembly of Atg17-GFP in Atg13 mutant cells lacking linker residues between Atg1317LR and Atg1317BR. Atg17-GFP and the vacuolar marker protein Vph1-mCherry are shown in green and magenta, respectively. Cells with Atg17-GFP puncta were quantified. 17LR∗ and 17BR∗, the Atg1317LR and Atg1317BR regions observed in the determined crystal structure.
(G) ALP assay showing autophagic activity of the Atg13 mutant cells deleted of linker residues between Atg1317LR and Atg1317BR. The ALP assay was performed as in Figure 3E. ∗p < 0.05, ∗∗p < 0.01.
Developmental Cell  , 86-99DOI: ( /j.devcel )
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7. Figure 5 Atg13 Mediates the Multimeric Assembly of Atg1 Complexes
(A) In vitro tethering of Atg17-Atg29-Atg31 complexes by Atg13 fragments containing Atg1317LR and Atg1317BR. S and P, supernatant and precipitated fractions after centrifugation at 15,000 × g, respectively.
(B) Analytical ultracentrifugation showing in vitro multimeric self-assembly of the Atg1 complexes composed of Atg1ΔKD, Atg13-MBP, and the Atg17-Atg29-Atg31 complex.
(C) Gel-filtration analyses showing in vitro multimeric self-assembly of the Atg1 complexes composed of Atg1 (D211A), Atg13-MBP, and the Atg17-Atg29-Atg31 complex (left panels). Quantitation of the results of gel-filtration analyses (right panels).
See also Figures S4 and S5.
Developmental Cell  , 86-99DOI: ( /j.devcel )
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8. Figure 6
Supramolecular Atg1 Complex Assembly Is Responsible for Autophosphorylation of Atg1 and Recruitment of Atg9 Vesicles
(A) Fluorescence microscopy of the PAS assembly of Atg17-GFP in Atg13 mutant cells (truncation or mutation in Atg1317BR, Atg1317BR2, or Atg1317LR), analyzed as in Figure 4F. Atg17-GFP and Atg13-mCherry are shown in green and magenta, respectively. Cells with Atg17-GFP and Atg13-mCherry double-positive puncta were quantified.
(B) Fluorescence microscopy of the PAS assembly of the Atg9 vesicles in Atg13 mutant cells, analyzed as in Figure 4F. Cells with Atg9-GFP and Atg13-mCherry double-positive puncta were quantified.
(C) Atg1317BR and Atg1317LR are required for autophosphorylation of Atg1 and phosphorylation of Atg9 by Atg1. Yeast extracts were prepared from atg11Δ atg13Δ cells expressing Atg13 with a mutation in Atg1317BR or Atg1317LR and analyzed by Phos-tag-containing SDS-PAGE (lower panels) followed by western blotting using antibodies against Atg13, Atg1, and Atg9. p-Atg1 and p-Atg9, the phosphorylated form of Atg1 and Atg9, respectively.
(D) Summary of the Atg13-Atg17 interactions involved in supramolecular self-assembly of the Atg1 complexes. Atg13 is shown as a blue string representing the IDR with featured regions: from N terminus to C terminus, Atg13HORMA (blue surface model), Atg1317LR (red box), Atg1317BR (blue box), and Atg13MIM (blue box). Ser379 in Atg1317LR and Ser428 and Ser429 in Atg1317BR are dephosphorylated in response to starvation, thereby interacting with Atg17 in an intermolecular manner.
(E) Atg13 links the Atg17-Atg29-Atg31 complexes with each other via two distinct regions, Atg1317LR and Atg1317BR, to mediate self-assembly of the Atg1 complexes. The supramolecular self-assembly of the Atg1 complexes will lead to autophosphorylation of Atg1, recruitment of the Atg9 vesicles, and phosphorylation of Atg9 by the autoactivated Atg1.
Developmental Cell  , 86-99DOI: ( /j.devcel )
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