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Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion

Nature Structural & Molecular Biology volume 27, pages 1185–1193 (2020)Cite this article

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An Author Correction to this article was published on 12 November 2020

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Abstract

The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9’s ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.

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Fig. 1: Atg9 is a lipid scramblase.
Fig. 2: Architecture of Atg9.
Fig. 3: The LP and VP are important for autophagy.
Fig. 4: The LP and VP are important for IM expansion.
Fig. 5: The lipid scramblase activity of Atg9 is essential for autophagy.

Data availability

All relevant data are available upon request to the authors. The cryo-EM density maps have been deposited into the Electron Microscopy Data Bank with accession numbers EMD-30535 (hexamer) and EMD-30545 (trimer). Atomic coordinates are deposited into the wwPDB under accession number PDB 7D0I (hexamer). Uncropped gel and blot images are available in Supplementary Fig. 2. Source data are provided with this paper.

Change history

  • 12 November 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank H. Yamamoto at the University of Tokyo and Y. Ohashi and R.L. Williams at the MRC Laboratory of Molecular Biology for providing plasmids and strains for yeast experiments; Y. Jin for useful advice regarding QF-FRL analyses; T. Ando and Y. Ishii for assistance with protein preparation; Y. Nakada-Nakura, K. Liu and T. Uemura for technical assistance in the generation of antibodies; and A. I. May for proofreading. This work was supported in part by JSPS KAKENHI grant nos. 25111004, 18H03989, 19H05707 (to N.N.N.), 15K21608, 18K06097 (to K.M.), 19K16071 (to T.K.), 19K07265 (to T.T.), (19K06532 to T.M.), 19K16344 (to D.N), 26119006, 19H05645 (to Y.S.), 16H06375, 19H05708 (to Y.O.), 15H05902, 18H04023 (to T.F.), 17H01430, 19H05708 (to H.N.), JST CREST grant no. JPMJCR13M7 (to N.N.N. and H.N.), JPMJCR14M1 (to M.K.), grants from the Takeda Science Foundation (to N.N.N.) and from the Naito Foundation (to N.N.N.), and by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under grant no. JP19am0101001 (support no. 0053) and 19am0101079 (support no. 0739), and from Research on Development of New Drugs from the AMED.

Author information

Author notes

  1. These authors contributed equally: Tetsuya Kotani, Akihisa Tsutsumi, Takuma Tsuji.

Authors and Affiliations

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

    Kazuaki Matoba, Daisuke Noshiro & Nobuo N. Noda

  2. School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan

    Tetsuya Kotani & Hitoshi Nakatogawa

  3. Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Akihisa Tsutsumi & Masahide Kikkawa

  4. Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo, Japan

    Takuma Tsuji & Toyoshi Fujimoto

  5. RIKEN Cluster for Pioneering Research, Wako, Japan

    Takaharu Mori & Yuji Sugita

  6. RIKEN Center for Computational Science, Kobe, Japan

    Yuji Sugita

  7. RIKEN Center for Biosystems Dynamics Research, Kobe, Japan

    Yuji Sugita

  8. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Norimichi Nomura & So Iwata

  9. RIKEN SPring-8 Center, Hyogo, Japan

    So Iwata

  10. Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan

    Yoshinori Ohsumi

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  1. Kazuaki Matoba
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Contributions

K.M. and N.N.N. conceived the project. K.M. purified recombinant proteins and prepared proteoliposomes. K.M., A.T. and M.K. performed cryo-EM experiments. K.M., T.K., Y.O. and H.N. performed yeast experiments. T.T. and T.F. performed quick-freezing and freeze-fracture replica labeling method experiments. D.N. performed HS-AFM observation. T.M. and Y.S. performed MD simulation. N.N. and S.I. prepared antibodies against Atg9. All authors analyzed the data. K.M. and N.N.N. wrote the manuscript with input from all other authors. N.N.N. supervised the work.

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Correspondence to Nobuo N. Noda.

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Extended data

Extended Data Fig. 1 In vitro analysis of lipid scramblase activity of Atg9.

a, b, Dithionite assay. Assay was performed using proteoliposomes (green) in the existence of ATP/Mg (red) or calcium ion (cyan) (a) and proteoliposomes containing NBD-C6-PC, NBD-C6-PE, or NBD-C6-PS (b). FI(t) and FI(0) indicate the fluorescence intensity at each time and 0, respectively. c, SDS-PAGE of the purified PI3K complex I stained by Coomassie Brilliant Blue. d, Left, results of the floatation assay of liposomes with either 5% PI3P (PC/PI/PS/PE/PI3P) or 18% PI (PC/PI/PS/PE) detected by p40phox PX-EYFP. Fluorescence intensity (FI) of p40phox PX-EYFP was detected at Fraction 1 only when PI3P liposomes were used, indicating that p40phox PX-EYFP specifically recognizes PI3P liposomes but not PI liposomes. Right, results of the floatation assay of 18% PI liposomes treated with PI3K and various concentrations of ATP. As a positive control, 5% PI3P liposomes were also analyzed. The graph shows the fluorescence intensity at Fraction 1. Fluorescence intensity was proportional to the ATP concentration, indicating that catalytic conversion of PI to PI3P proceeded in liposomes.

Extended Data Fig. 2 Cryo-EM analysis of Atg9.

a, SEC profiles of SpAtg9-GFP fusion (black and red) or SpAtg9 alone (blue). b, Summary of cryo-EM analysis of SpAtg9 hexamer. A representative micrograph of SpAtg9 (upper left), local resolution map (lower left), workflow of cryo-EM (upper middle), Fourier shell correlation curves (lower middle), and 2D class average images (upper right), where selected particle class was surrounded by a red line. Lower right, representative density map of some regions with a structural model. Each amino-acid sequence is also provided. c, Summary of cryo-EM analysis of SpAtg9 trimer. Workflow of cryo-EM (left), Fourier shell correlation curves (middle), and 2D class average images (right). d, Superimposed cryo-EM map of hexamer (resolution 3.0 Å, colored yellow) and trimer (resolution 4.7 Å, colored grey). Protomer boundaries are shown with blue, red, and black lines. e, comparison of the cryo-EM map between plant Atg9 (EMD-9681) (top) and yeast trimer (resolution 4.7 Å) (bottom). f, DISOPRED result of SpAtg9 (upper) and schematic drawing of the domain organization of Atg9 homologs (bottom), where structured and disordered regions predicted by DISOPRED are shown with a square and a black line, respectively. Arrow line (cyan) indicates the region whose model was built by cryo-EM experiment.

Extended Data Fig. 3 Detailed interactions observed in the cryo-EM structure.

a, Close−up view of H3 and H6. Prolines responsible for kinking helices are indicated. b-e, Close−up view of the interactions observed in ARCH. f, H4 and H5 bound to adjacent protomers within a trimer. One protomer is shown in a ribbon model whereas the other two protomers are shown in a surface model. g, Schematic drawing of the trimer structure. h, Close-up view of the interactions observed between H1, H3, and H4. i, Close-up view of the trimer-trimer interface observed in the hexamer structure (left) and proposed transmembrane regions (TM) within the hexamer (right).

Extended Data Fig. 4 Characterization of Atg9 trimer.

a, Thickness of lipid bilayer membrane on mica measured by HS-AFM. b, 2D class average images of Atg9-nanodiscs recognized by Fab. Scale bar, 50 Å. c, Density map of LMNG at the LP. Two LMNG molecules are colored differently. d, Comparison of the VP radius between cryo-EM and MD models. e, Comparison of different classes of trimer Atg9 structure. Left and right show density map at 5.3 and 6.2 Å resolution, on which structural model of SpAtg9 is superimposed by real-space refinement. The distance is between Cα atoms of Leu539 located at VP. f, Comparison of the SEC profiles between WT and Hexamer mutant (I641D, V643A, V654A, and F655A) of SpAtg9 using a Superose 6 increase 10/300GL column.

Extended Data Fig. 5 Mutational analysis of Atg9.

a, Circular dichroism (CD) spectra of WT and mutant forms of SpAtg9. b, SEC profiles of WT and mutant forms of SpAtg9. Blue bar indicates the fraction used for in vitro analysis. c, d, Fluorescence images of colocalization of Atg17 puncta with Atg9-GFP mutants (c) and with Atg2-mNeongreen in cells expressing Atg9 mutants (d).

Extended Data Fig. 6 Random mutational analysis of Atg9.

a, Ape1 maturation assay of atg9Δ cells expressing Atg9 with random mutations. b, Left table shows the random mutation sites in ScAtg9 that partially or severely impaired Ape1 maturation and their equivalent residues in SpAtg9. Right figure shows the location of residues at LP and VP that were shown to be important for autophagy by random mutagenesis experiments.

Extended Data Fig. 7 Structural comparison between human ATG9A and yeast Atg9.

a, Human ATG9A (PDB 6WQZ)29 and SpAtg9 are shown with a cyan and orange surface model, respectively. LMNG molecules bound to LP are shown with a stick model. b, Superimposed cartoon model of trimer (left) and protomer (right). Coloring is as in a.

Supplementary information

Supplementary Information

Supplementary Figures 1 and 2, Supplementary Notes 1 and 2 and Supplementary Tables 1 and 2.

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Supplementary Video 1

Visualization of the cryo-EM map of the SpAtg9 hexamer with the final atomic model.

Supplementary Video 2

HS-AFM observation of the cytoplasmic side of Atg9 embedded in the lipid bilayer membrane. Z-scale, 0–6.5 nm.

Supplementary Video 3

HS-AFM observation of the luminal side of Atg9 embedded in the lipid bilayer membrane. Z-scale, 0–1.9 nm.

Supplementary Video 4

Density map of LMNG bound to Atg9, related to Fig. 3a and Extended Data Fig. 4c.

Supplementary Video 5

Mutation sites in VP, related to Fig. 3b.

Supplementary Video 6

Vacuolar protease−deficient cells expressing Atg9 mutants were treated with rapamycin for 24 h before autophagic body accumulation in these cells was observed by phase contrast microscopy. Time-lapse images were taken at 32.65 ms per frame.

Supplementary Video 7

Observation of Atg9 vesicles in yeast cells. The numbers correspond to mutant numbers (mutant number 72 corresponds to the Wall mutant).

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Matoba, K., Kotani, T., Tsutsumi, A. et al. Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion. Nat Struct Mol Biol 27, 1185–1193 (2020). https://doi.org/10.1038/s41594-020-00518-w

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