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Membrane perturbation by lipidated Atg8 underlies autophagosome biogenesis

Nature Structural & Molecular Biology volume 28, pages 583–593 (2021)Cite this article

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Abstract

Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.

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Fig. 1: Membrane deformation of GUVs by Atg8 lipidation.
Fig. 2: NMR observation of Atg8–PE reconstituted onto lipid bilayer nanodiscs.
Fig. 3: Identification of Atg8 residues essential for membrane deformation in vitro.
Fig. 4: Role of Atg8 aromatic residues in maintaining vacuolar membrane morphology.
Fig. 5: Role of Atg8 aromatic residues on autophagy.
Fig. 6: Aromatic residues in mammalian Atg8 are important for efficient degradation of p62 gels.
Fig. 7: Role of membrane perturbation by Atg8.

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Data availability

Microscopic images are available upon request. NMR spectra are deposited online at https://doi.org/10.6084/m9.figshare.14727903. Source data are provided with this paper.

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Acknowledgements

We thank T. Ando for assistance with nanodisc preparation; T. Kotani and C. Kakuta (Tokyo Institute of Technology) for preparing yeast strains; M. Lazarou (Monash University) for providing HeLa cells with disruption of six mammalian ATG8s; and N. Mizushima, I. Koyama-Honda and Y. Sakai (The University of Tokyo) for discussion. This work was supported in part by JSPS KAKENHI grant nos. 18H03989 and 19H05707 (to N.N.N.), 20K06552 (to T.F.), 19H05712 (to T.K), 19H05708 (to H.N. and Y.O.), 16H06375 (to Y.O.), 17H06097 (to I.S.), 20K06549 (to S.K.), 19H05706 and 21H04771 (to M.K.); JSPS A3 foresight program (to M.K.); CREST, Japan Science and Technology Agency Grant number JPMJCR13M7 (to N.N.N. and H.N.); JPMJCR20E3 (to N.N.N.); AMED-CREST, Japan Agency for Medical Research and Development Grant no. 21gm1410004s0102 (to M.K. and H.N.); and grants from the Takeda Science Foundation (to N.N.N. and M.K.), Mochida Memorial Foundation for Medical and Pharmaceutical Research (to N.N.N.) and the Tokyo Biochemical Research foundation (to N.N.N. and J.M.A.).

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Author notes

  1. These authors contributed equally: Tatsuro Maruyama, Jahangir Md. Alam.

Authors and Affiliations

  1. Institute of Microbial Chemistry, Tokyo, Japan

    Tatsuro Maruyama, Jahangir Md. Alam, Yuki Ishii & Nobuo N. Noda

  2. Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan

    Tomoyuki Fukuda & Tomotake Kanki

  3. Juntendo University Graduate School of Medicine, Tokyo, Japan

    Shun Kageyama & Masaaki Komatsu

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

    Hiromi Kirisako & Hitoshi Nakatogawa

  5. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Ichio Shimada

  6. RIKEN Center for Biosystems Dynamics Research, Yokohama, Japan

    Ichio Shimada

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

    Yoshinori Ohsumi

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Contributions

T.M., J.M.A. and N.N.N. designed the research. T.M. and Y.I. performed sample preparation. T.M. and I.S. performed NMR experiments. T.M. and J.M.A. performed GUV experiments. H.K., H.N. and Y.O. performed budding yeast experiments. T.F. and T.K. performed fission yeast experiments. S.K. and M.K. performed mammalian experiments. T.M., J.M.A., T.F., S.K., M.K., T.K., H.N. and N.N.N. analyzed data. T.M. and N.N.N. wrote the manuscript. All authors discussed the results and commented on the manuscript. N.N.N. supervised the work.

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Correspondence to Nobuo N. Noda.

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The authors declare no competing interests.

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Peer review information Nature Structural & Molecular Biology thanks Liang Ge and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Inês Chen and Anke Sparmann 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 Membrane deformation upon Atg8 lipidation.

a, Membrane deformation of GUV upon lipidation of mCherry–Atg8. GUVs are produced from lipid films containing POPC:POPE:PI=30:60:10 (mol%). Experiments were repeated independently 10 times with similar results. Scale bar, 10 μm. b, Effect of chemical anchoring of mCherry-Atg8G116C on GUVs incorporating POPC:POPE:PI:PE MCC=50:30:10:10 (mol%). Experiments were repeated independently 5 times with similar results for wild-type, whereas it was performed once for each mutant. Scale bar, 10 μm. c, Change in membrane area of GUV upon mCherry-Atg8 lipidation. The results of the other 5 experiments related to Fig.1g are shown. Source data for graphs are available online.

Source data

Extended Data Fig. 2 Reconstitution of Atg8-PE onto lipid bilayer nanodiscs.

a, Atg8–PE formation on nanodisc incorporating various amounts of PE. Lipidation reaction mixtures containing Atg8, Atg7, Atg3, nanodiscs incorporating 50–95% POPC and 5–50 mol% POPE and MgATP are incubated for 1 hr in the presence and absence of Atg12–Atg5–Atg16N and subjected to urea SDS–PAGE. The gel is stained by Coomassie brilliant blue. At lower concentration of PE, Atg8–MSP1D1 conjugate is produced as a byproduct. The experiment was performed once. b, Quantification of Atg8 lipidation in (a). The population of Atg8-PE is calculated from the band brightness in (a) measured by ImageJ software. c, Purification of Atg8–PE reconstituted nanodiscs incorporating 70 mol% POPC and 30 mol% POPE by a cation exchange column. The ratio of MSP1D1 and Atg8–PE in the band brightness is approximately 2, indicating that one Atg8–PE molecule is reconstituted per nanodisc on average. d, Delipidation of Atg8–PE by Atg4. Atg8-PE is incubated with Atg4 and delipidation is confirmed by urea SDS–PAGE as shown in the right panel. e, 1H-15N HSQC spectrum of [u-15N]-labeled Atg8-PE on nanodiscs. Few amide signals are observed. f, Size exclusion chromatography of Atg8-PE (blue) and empty (black) nanodiscs. SDS-PAGE analysis of the elution peak of Atg8-PE nanodiscs is shown in the right panel. Source data for graphs are available online. Experiments were repeated once (a) or three times independently (c,d,f) with similar results.

Source data

Extended Data Fig. 3 NMR spectral changes of Atg8 upon lipidation and delipidation.

a, Schematic representation of NMR sample preparation. Atg8–PE is produced onto lipid bilayer nanodiscs incorporating 70 mol% POPC and 30 mol% POPE via a lipidation reaction mixture containing Atg7, Atg3, Atg12–Atg5–Atg16N, and MgATP. Resultant Atg8–PE is purified to remove conjugation machinery. Delipidation of Atg8–PE is achieved by the addition of Atg4. b, 1H-13C HMQC spectra of {u-2H, Met-[13CH3], Alaβ-[13CH3], Ileδ1-[13CH3], Leu/Val-[13CH3,12CD3]}-labeled Atg8, Atg8-PE and delipidated Atg8. Each signal is assigned based on the data deposited in the Biological Magnetic Resonance Data Bank (BMRB entry:16835). For methyl groups in Leu and Val residues, methyl signals with lower and higher 1H chemical shift are denoted as residue name with a and b, respectively. NMR signals derived from phospholipids in nanodiscs are colored in brown. Lipidation and delipidation are confirmed by urea SDS-PAGE (Extended Data Fig. 2). c, Overlay of 1H-13C HMQC spectra of Atg8 and Atg8-PE. Minor signals observed in Atg8-PE and delipidated Atg8 are labeled with asterisk.

Extended Data Fig. 4 Modeling of Atg8-PE nanodisc using HADDOCK.

a, HADDOCK scores of final 200 docked structures of Atg8-PE nanodisc. 10 structures with the lowest HADDOCK scores are colored in blue. b, Superposition of 10 docked structures with the lowest HADDOCK scores. Atg8 and MSP1D1 are shown as ribbon model. POPC lipids are shown as stick model. c, Orientations of Atg8-PE on membranes. Carbon atoms of NMR-probed methyl groups are shown as sphere colored according to Fig. 2. The docked structure with the lowest HADDOCK score shown in Fig. 2 belongs to Orientation IV. Source data for graphs are available online.

Source data

Extended Data Fig. 5 Change in membrane area upon lipidation of Atg8 mutants.

a,b, Changes in membrane area of GUV upon lipidation of mCherry–Atg8F77A (a) and mCherry–Atg8F79A (b). The results of the other experiments related to Fig. 3 are shown. Source data for graphs are available online.

Source data

Extended Data Fig. 6 Role of Atg8 aromatic residues on autophagosome formation.

a, Electron micrographs of autophagosomes accumulated in cytosol. Autophagy in atg8Δypt7Δcells expressing Atg8 variant is induced by rapamycin treatment for 4 hr and subjected to rapid freezing and freeze-substitution fixation. Arrow heads indicate autophagosomes. Scale bars, 1 μm. The experiment was performed once. b,c, The number (b) and size (c) of autophagosome in atg8Δypt7Δcells expressing Atg8 variant. Fourteen cells were analyzed for Atg8 and Atg8F77A. Twelve cells were analyzed for Atg8F79A. Total numbers of autophagosomes were 71 for Atg8, 44 for Atg8F77A, and 36 for Atg8F79A. The center of the box represents the median; the top and the bottom edges of the box represent the third and first quartiles, respectively. Whiskers represent minima and maxima. ****P = 4.7 × 10−11 (Size, Atg8F77A), 8.0 × 10−15 (Size, Atg8F79A); **P = 2.7 × 10−3 (Number, Atg8F77A), 4.1 × 10−3 (Number, Atg8F79A); n.s., not significant, two-sided Dunn’s multiple comparisons test.

Extended Data Fig. 7 Comparison of Atg8 family proteins.

a, Sequence alignment of Atg8 from Saccharomyces cerevisiae, LC3B from homo sapiens, GABARAP from homo sapiens, LGG–1, LGG–2 from Caenorhabditis elegans is performed by CLASTALW server. Residue number of Atg8 is denoted above sequence. Residues corresponding to Phe77 and Phe79 in Atg8 are colored in magenta or orange and are shown as stick in the crystal structures in the same orientation. Each structure (Atg8, PDB: 2ZPN; LC3B, PDB: 3VTU; GABARAP, PDB: 1GNU; LGG-1, PDB: 5AZF; LGG-2, PDB: 5E6N) is shown as ribbon model colored from blue to red from the N- to the C-terminus, prepared by CCP4MG software. b, Membrane deformation of GUVs with a prolate shape upon lipidation of Atg8 mutants. F79L, F79Y and F79W mutants of Atg8 were analyzed in the same manner as Fig. 1. Experiments were repeated independently 3 times with similar results. Scale bar, 10 μm. Source data for graphs are available online.

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Maruyama, T., Alam, J.M., Fukuda, T. et al. Membrane perturbation by lipidated Atg8 underlies autophagosome biogenesis. Nat Struct Mol Biol 28, 583–593 (2021). https://doi.org/10.1038/s41594-021-00614-5

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