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Structure, lipid scrambling activity and role in autophagosome formation of ATG9A

Nature Structural & Molecular Biology volume 27, pages 1194–1201 (2020)Cite this article

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Abstract

De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly smaller autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.

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Fig. 1: The structure of ATG9A.
Fig. 2: Dissection of the ATG9A architecture.
Fig. 3: Conformational stability of ATG9A.
Fig. 4: Functional studies of ATG9A.
Fig. 5: Models.

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Data availability

Cryo-EM density maps and atomic coordinates for human ATG9A have been deposited in the Electron Microscopy Data Bank and wwPDB, respectively, under accession codes EMD-22375 and PDB 7JLO (amphipol); EMD-22376 and PDB 7JLP (nanodisc); and EMD-22377 and PDB 7JLQ (LMNG). Source data are provided with this paper.

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Acknowledgements

We thank K. Ohashi, S. Chowdhury and G. C. Lander for their contributions to preliminary studies of this project; S. Chowdhury and G. C. Lander for also providing cryo-EM training; M. Shirakawa, K. Igarashi, Y. Ishida, C. Saito, and I. Koyama-Honda for technical assistance with cellular studies; B. Anderson for technical assistance with cryo-EM operation; J-C. Ducom and the Scripps HPC Facility for computational support for cryo-EM data processing and MD simulations; and I. J. Macrae for critical reading of the manuscript. The MultiBac expression system, cDNA of BRIL, and pMSP2N2 (Addgene plasmid no. 29520), pMXs-puro, Atg9a KO MEFs, pCG-gag-pol and pCG-VSV-G retrovirus plasmids were gifts from I. Berger, R. Stevens, S. Sligar, T. Kitamura, T. Saitoh and T. Yasui, respectively. This work was supported by grants from the National Institute of Health (GM092740 to T.O., GM069832 to S.F. and GM127390 to N.V.G.), the Welch Foundation (I-1505 to N.V.G.), a Grant-in-Aid for Exploratory Research for Advanced Technology (ERATO) (JPMJER1702 to N.M.) from the Japan Science and Technology Agency (JST). Computational analyses of EM data were performed using shared instrumentation at The Scripps Research Institute funded by NIH S10OD021634.

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Authors and Affiliations

  1. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA

    Shintaro Maeda, Christina M. Garza, Chinatsu Otomo, Stefano Forli & Takanori Otomo

  2. Department of Biochemistry and Molecular Biology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Hayashi Yamamoto, Satoru Takahashi & Noboru Mizushima

  3. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA

    Lisa N. Kinch & Nick V. Grishin

  4. Department of Neurosurgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan

    Satoru Takahashi

  5. Department of Biophysics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA

    Nick V. Grishin

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  1. Shintaro Maeda
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Contributions

S.M. purified proteins, collected and processed cryo-EM data and performed lipid scrambling assays; H.Y. performed autophagy assays, fluorescence and 3D CLEM imaging, and analyses. L.N.K. performed bioinformatics analyses under supervision of N.V.G. C.M.G performed MD simulations under supervision of S.F. S.T. collected and analyzed 3D CLEM data. C.O. generated plasmids and expressed proteins. N.M. supervised cellular studies. T.O. conceived the project, assisted structural and biochemical analyses and wrote the initial draft of the manuscript. All authors discussed the results and edited or commented on the manuscript.

Corresponding author

Correspondence to Takanori Otomo.

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The authors declare no competing interests.

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Peer review information Anke Sparmann and Inês Chen were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Conformational variability of ATG9A.

a, Superposition of ATG9A structures in amphipols (gray) and nanodiscs (colored). b, Conservation mapping of ATG9A. c, The structure of ATG9A in LMNG micelles. d,e, Superpositions of ATG9A structures in nanodiscs (colored) and LMNG (gray and violet): overall superposition (d) and focused on a protomer (e). f,g, Top views of the nanodisc (f) and LMNG (g) structures visualizing the dilation of the central pore. h, CHAP analysis of the LMNG structure.

Extended Data Fig. 2 Similarities between the N- and C-terminal halves of ATG9A.

a,b, The bundles of α-helices flanking the TMH1–2 (a) and TMH3–4 (b) hairpins. c, Superposition of the bundles. d, Structural overlay of the N- and C-terminal halves generated by superposing TMH1–2 and TMH3–4. e, FATCAT flexible structural alignment of the N- and C-terminal halves. f, Sequence alignment between the N- and C-terminal halves of ATG9A.

Extended Data Fig. 3 Structural alignments of ATG9A with ABCexps.

a,b, Structures of YbtQ (a, PDB 6P6J) and MRP1 (b, PDB 5UJA) bound to their substrates. TMH1–3 are colored violet. c, A cutaway view of the central pore of ATG9A. d,e, Superpositions of ATG9A with YbtQ (d) and MRP1 (e). ATG9A’s TMH3–4 can be superimposed with TMH1–2 from the identified ABCexp structures using as a guide for the helix register the conserved Asp (D398 in ATG9 with D53 in MsbA, D56 in YbtQ, D355 in MRP1). In all cases, ABCexps’ substrates locate to the central pore of ATG9A.

Extended Data Fig. 4 MD simulation of the nanodisc structure.

a, The inter-residue distances measured are indicated on the nanodisc structure. b–d, Analyses of the simulation. The top panels are structure snapshots at t = 894 ns. The middle panels show time-dependent changes of the three pore openings between three selected pairs of residues at the A-C (b), B-A (c), and C-B (d) chain interfaces. The bottom panels show the trajectory of individual lipids that penetrated through the interface during the simulation. Each trace shows the Z position of the phosphate head of a lipid of interest, normalized by the average z coordinate of the luminal (0) and cytosolic (1) leaflets. The simulation shows lipid movements induced by the dynamic behavior of the protein. The luminal sides of the A-C and B-A interfaces (blue and orange lines in b and c) opened early (t = ~100–200 ns), followed by insertions of the headgroups of the lipids #59 and #42 from the luminal leaflet through the A-C and B-A interfaces, respectively (b, c, cyan lines, t = ~300–450 ns). As these headgroups moved toward the cytosolic side, the lipids #5 and #61 in the luminal leaflet inserted their headgroups into the pore through the A-C and B-A interfaces, respectively (b, c). Subsequently, the cytosolic side of the A-C and B-A interfaces opened (b, c, green lines), and at the A-C interface, the lipid #258 in the cytosolic leaflet entered the pore. Conversely, the C-B interface remained closed longer with respect to the A-C and B-A interfaces, but it eventually started to open after t = ~700 ns, allowing lipid #2 in the luminal leaflet to insert its headgroup into the luminal side of the pore (d). All the lipids mentioned here became laterally orientated following the placement of their headgroups into the pore. Source data for plots are available online.

Source data

Extended Data Fig. 5 Dithionite permeation assays.

a,b, Top panels show the schemes of the experiments with no protein (a) and ATG9A (b). 26 µM NBD-phosphoethanolamine (PEA), which was synthesized by conjugating NBD-chloride and PEA and purified by agarose gel electrophoresis, was included in the (proteo)liposome reconstitution mixtures. After reconstitutions, NBD-PEA was removed by dialysis. Fluorescence was recorded as described for the dithionite lipid scramblase assays. 30 mM dithionite was added to the reaction mixtures at t = 100 s. The initial drops of fluorescence indicate that the residual amounts of NBD-PEA at the outside of the (proteo)liposomes were quenched. Between the two experiments, the addition of dithionite quenched the same amount of fluorescence and left the same amount of fluorescence, indicating that the same amount of NBD-PEA was included in the (proteo)liposomes. Fluorescence remained almost unchanged after reaching plateaus quickly. At 600 s, 0.1 % TritonX-100 was added to the reaction mixtures to dissolve the membranes for full quenching. The same amount of fluorescence was lost between the two experiments, confirming that the same amounts of NBD-PEA were included in liposomes and proteoliposomes. Thus, the ATG9A proteoliposomes were impermeable to dithionite. The experiments were repeated three times. All results are shown in different colors. Source data are available online.

Source data

Extended Data Fig. 6 BSA back extraction lipid scrambling assays.

a, Schematic for the BSA back extraction-based lipid scrambling assay. NBD-lipids are preferentially extracted by fatty acid-free BSA. Upon extraction, the fluorescence of BSA-bound NBD-lipids is reduced by ~ 50%. b, The results with reconstituted liposomes and ATG9A-containing proteoliposomes. 1.5 mg/ml BSA was added to each reaction at t = 100 s. BSA extracts NBD-lipids only from the outer leaflet of the membranes. Thus, in the absence of scrambling, ~50% of NBD-lipids on the outer leaflet lose their fluorescence by ~50%, reducing the total fluorescence by ~25%. In the presence of ATG9A, an additional ~15% of fluorescence was lost upon BSA addition, which indicates that NBD-lipids that were originally in the inner leaflet were exposed to BSA upon flipping. Source data are available online.

Source data

Extended Data Fig. 7 GFP-LC3-RFP autophagic flux assays with ATG9A mutants.

a, Locations of the mutations on the ATG9A nanodisc structure. Hydrophilic and hydrophobic amino acids were replaced with hydrophobic and hydrophilic ones, respectively. b,c, Flow cytometry autophagy flux assays. GFP/RFP fluorescence ratios of Torin 1-treated ATG9A KO HeLa cells stably expressing GFP-LC3-mRFP and ATG9A-FLAG mutants are plotted. Data shown in (b) and (c) are the results of the first and second rounds of mutant screenings, respectively. Data of cell populations (n = 4800 for b and n = 2500 for c) are presented as box plots. The box limit, the horizontal line, and the whisker show the first and third quartiles, the median, and the minimum and maximum values, respectively. No outliers are shown. The assay was repeated for the mutants shown in Fig. 4, and they were reproducible. The data shown in blue and green are of mutations in the lateral cavity and central pore, respectively. d,e, Western blotting of ATG9A KO HeLa cells stably expressing ATG9A-FLAG mutants. Source data for the box plots are available online.

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Extended Data Fig. 8 Autophagosome formation in ATG9A mutant-expressing cells.

a,b, ATG9A KO HeLa cells (a) and Atg9a KO MEFs (b) stably expressing GFP-LC3-mRFP and ATG9A-FLAG mutants were observed by fluorescence microscopy. GFP fluorescence was monitored. In (a) additional mutants M26 (T412W) and M32 (M8 + M26) are shown. ATG9A KO HeLa ells and WT ATG9A-FLAG-expressing cells are the same image as shown in Fig. 4. Images were acquired after 3 h starvation. M26 yielded typical GFP-LC3 puncta while M32 generated smaller autophagosomes. The results are similar to those of M28 (T419W) and M33 (M8 + M28), which were selected for followed-up 3D CLEM and biochemical studies, as shown in Fig. 4. (b) MEFs were starved for 1 h, and GFP fluorescence was monitored. In Atg9a KO MEFs, GFP-LC3 forms larger puncta than typical autophagosomes. Expression of WT ATG9A-FLAG, as well as M26 and M28, generated ring-shaped autophagosomes, while M8-expressing, M32-expressing and M33-expressing cells yielded markedly smaller GFP-puncta. These observations are consistent with the results with HeLa cells, leading to the conclusion that the observed effects of the mutations in ATG9A are cell type-independent.

Supplementary information

Supplementary Information

Supplementary Note 1, Supplementary Figures 1–11 and Supplementary Tables 1 and 2.

Reporting Summary

Supplementary Data 1

HHpred hits with various ATG9 sequences and sequence alignments.

Supplementary Video 1

Movie of the MD simulation in a top cutaway view and three side views.

Supplementary Video 2

3D reconstruction of a cup-shaped phagophore observed in M33-expressing cells. A cup-shaped phagophore, a closed autophagosome and the ER are colored green, light green and red, respectively.

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Source Data Extended Data Fig. 7

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Maeda, S., Yamamoto, H., Kinch, L.N. et al. Structure, lipid scrambling activity and role in autophagosome formation of ATG9A. Nat Struct Mol Biol 27, 1194–1201 (2020). https://doi.org/10.1038/s41594-020-00520-2

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