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Structure of the Atg101–Atg13 complex reveals essential roles of Atg101 in autophagy initiation

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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.

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Figure 1: Structure of the SpAtg101–SpAtg13HORMA complex.
Figure 2: Stereo view of the SpAtg101-SpAtg13HORMA interactions observed in the crystal.
Figure 3: The Atg13-binding surface, but not the WF finger, of Atg101 is essential for the interaction with Atg13.
Figure 4: Both the Atg13-binding surface and the WF finger are essential for autophagy.
Figure 5: The Atg13-binding surface is required for the recruitment of Atg101 to the autophagosome-formation site.
Figure 6: The Atg13-binding surface and WF finger of Atg101 are important for the recruitment of downstream Atg proteins.
Figure 7: Proposed model of the molecular functions of Atg101 in the ULK complex in comparison with those of Atg29–31 in the Atg1 complex.

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Acknowledgements

We thank T. Kitamura (University of Tokyo) and S. Yamaoka (Tokyo Medical and Dental University) for retroviral vectors and Plat-E cells and S. Sugano (University of Tokyo) for the pEF321-T plasmid. Synchrotron radiation experiments were performed at beamline BL41XU at SPring-8, Japan. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas 25111005 (to N.M.) and 25111004 (to N.N.N.) and by funding from the Platform for Drug Discovery, Informatics and Structural Life Science (to N.N.N.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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  1. Hironori Suzuki and Takeshi Kaizuka: These authors contributed equally to this work.

Authors and Affiliations

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

    Hironori Suzuki & Nobuo N Noda

  2. Graduate School of Medicine, University of Tokyo, Tokyo, Japan

    Takeshi Kaizuka & Noboru Mizushima

  3. Core Research for Evolutionary Science and Technology, Japan Science and Technology Agency, Tokyo, Japan

    Nobuo N Noda

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  1. Hironori Suzuki
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  2. Takeshi Kaizuka
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Contributions

H.S. and N.N.N. performed structural studies; H.S. performed in vitro experiments; T.K. performed experiments in mammalian cells; H.S., T.K., N.M. and N.N.N. analyzed data; and H.S. and N.N.N. wrote the manuscript. All authors discussed the results and commented on the manuscript. N.N.N. and N.M. supervised the work.

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Correspondence to Noboru Mizushima or Nobuo N Noda.

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Integrated supplementary information

Supplementary Figure 1 Crystallographic study of the Atg101–Atg13 complex.

(a) Oligomerization states of the SpAtg101, SpAtg13HORMA, and SpAtg101-SpAtg13HORMA complex studied by gel filtration chromatography. Absorbance at 280 nm of SpAtg101 alone, SpAtg13HORMA alone, and SpAtg101-SpAtg13HORMA mixture are shown with blue, green, and red lines, respectively. The apparent molecular mass of the proteins estimated from the elution volumes was indicated in parentheses. (b) Oligomerization states of the SpAtg101, SpAtg13HORMA, and SpAtg101-SpAtg13HORMA complex studied by SAXS. Top panel shows experimental scattering patterns and middle four panels show the concentration dependence of I(0)/Concentration, where I(0) is the forward scattering intensity derived using the Guinier law. (c) Sequence alignment of Atg101 homologs and human Mad2. Secondary structure elements that are colored as in Fig. 1a are shown below the sequence. The residues des are shown below the sequence. The residues interacting with SpAtg13HORMA are surrounded with red (Atg13 binding surface) or blue (WF finger) squares. Species are abbreviated as follows: S. pombe (Sp), Dorosophila melanogaster (Dm), Mus musculus (Mm), and Homo sapiens (Hs). As for Mad2, sequence of human Mad2 structurally aligned with SpAtg101 was shown, in which unaligned residues were shaded. (d) Structure and topology of LtAtg13HORMA (PDB ID 4J2G). The C-terminal segments including the safety belt and the cap are purple and yellow, respectively. (e) Close-up stereo view of the interaction of β1 with α-helices in SpAtg101. β1 is colored orange while the other regions are colored light blue. The side-chains of the residues involved in the interactions are shown by a stick model. (f) Close-up stereo view of the interaction of β1 with α-helices in O-Mad2 (PDB ID 2V64). β1 is colored orange while the other regions are colored light blue. The side-chains of the residues involved in the interactions are shown by a stick model. (g) Close-up stereo view of the interaction between the cap and the safety belt in LtAtg13HORMA (PDB ID 4J2G). Coloring is as in d. The side-chains of the residues involved in the interactions are shown by a stick model. (h) Sequence alignment of the HORMA domain of Atg13 homologs and human Mad2. Secondary structure elements that are colored as in Fig. 1a are shown below the sequence. The residues described in g are marked by dots on the secondary structure elements. Species are abbreviated as in c and as follows: S. cerevisiae (Sc), L. thermotolerans (Lt), Kluyveromyces marxianus (Km), Kluyveromyces lactis (Kl), Candida glabrata (Cg).

Supplementary Figure 2 Interactions between SpAtg101 and SpAtg13HORMA observed in the crystal.

(a) Close-up stereo view of the intermolecular β-sheet between SpAtg101 and SpAtg13HORMA. The main-chain of the residues forming the intermolecular β-sheet is shown with a stick model. Coloring is as in Fig. 1a. (b) Artificial lattice contact between SpAtg101 and SpAtg13HORMA. Coloring is as in Fig. 1a except for the neighboring SpAtg13HORMA, which is colored sea green. (c) Close-up stereo view of the lattice contact interaction between the WF finger of SpAtg101 and a neighboring SpAtg13HORMA molecule in the crystal. Coloring is as in (b). (d) In vitro pulldown assay between GST-fused SpAtg101 and SpAtg13HORMA. Uncropped images of SDS-PAGE gels are shown in Supplementary Data Set 1.

Supplementary Figure 3 Mutational analyses of the Atg101-Atg13 interaction.

(a) Gel filtration profiles of SpAtg101 mutants mixed with wild-type SpAtg13HORMA or SpAtg13HORMA mutants mixed with wild-type SpAtg101. Black line denotes the absorption at 220nm. SDS-PAGE analysis of the fraction No. 1 and 2 stained with Oriole (Bio-rad) was also shown. (b) CD spectra of wild-type and representative mutants of SpAtg101 (top panel) and SpAtg13HORMA (bottom panel). (c) Mutation detected in Atg101 KO MEFs. The sequences of the wild-type Atg101 allele and mutated alleles identified in Atg101 KO MEFs. The position of the PAM sequence used for CRISPR-mediated gene targeting is shown with a red line. (d) Close-up stereo view of the interaction between SpAtg101 and β8’ of SpAtg13HORMA. The side-chain of the residues involved in the interaction is shown with a stick model. Coloring is as in Fig. 1a. (e) Close-up stereo view of the interaction between O-Mad2 and β8’ of C-Mad2. The side-chain of the residues involved in the interaction is shown with a stick model. Coloring is as in Fig. 1b. (f) The cytosolic fractions of wild-type or Atg101 KO MEFs were separated by size exclusion chromatography. Each fraction was analyzed by immunoblotting with indicated antibodies. Positions of the molecular mass standards (in kDa) are shown. V, void fraction. (g) Cytosolic fractions of Atg101 KO MEFs stably expressing Atg101 W110A P111A F112A separated by gel filtration chromatography as in Fig. 3e. Uncropped images of SDS-PAGE gels and immunoblotting are shown in Supplementary Data Set 1.

Supplementary Figure 4 Low expression of wild-type Atg101 can restore Atg13 stability and autophagy.

Western blotting analyses for detecting LC3 turnover, p62 accumulation, and expression level of Atg13. Samples are lysates of Atg101 KO MEFs stably expressing wild type HA-HsAtg101 with a high expression level (used in the main Figure) or low expression levels (clones 3, 6, and 10), and Atg101 KO MEFs stably expressing the L30R/H31R mutant of HA-HsAtg101 cultured in regular or starvation medium in the presence or absence of 100 nM bafilomycin A1 for 2 h. Uncropped images of immunoblotting are shown in Supplementary Data Set 1.

Supplementary Figure 5 Colocalization between Atg13 and FIP200 in Atg101 mutant–expressing cells.

Colocalization analysis of GFP-Atg13 and FIP200 by immunostaining using anti-GFP and anti-FIP200 antibodies. Samples are Atg101 KO MEFs stably expressing GFP-Atg13 and indicated HA-Atg101 mutants cultured in regular medium or starvation medium for 1 h. Scale bars, white: 20 µm, yellow (insets): 1 µm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 6298 kb)

Supplementary Data Set 1

Uncropped images for western blots and SDS-PAGE gels (PDF 3375 kb)

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Suzuki, H., Kaizuka, T., Mizushima, N. et al. Structure of the Atg101–Atg13 complex reveals essential roles of Atg101 in autophagy initiation. Nat Struct Mol Biol 22, 572–580 (2015). https://doi.org/10.1038/nsmb.3036

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