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Atg2 mediates direct lipid transfer between membranes for autophagosome formation


A key event in autophagy is autophagosome formation, whereby the newly synthesized isolation membrane (IM) expands to form a complete autophagosome using endomembrane-derived lipids. Atg2 physically links the edge of the expanding IM with the endoplasmic reticulum (ER), a role that is essential for autophagosome formation. However, the molecular function of Atg2 during ER–IM contact remains unclear, as does the mechanism of lipid delivery to the IM. Here we show that the conserved amino-terminal region of Schizosaccharomyces pombe Atg2 includes a lipid-transfer-protein-like hydrophobic cavity that accommodates phospholipid acyl chains. Atg2 bridges highly curved liposomes, thereby facilitating efficient phospholipid transfer in vitro, a function that is inhibited by mutations that impair autophagosome formation in vivo. These results suggest that Atg2 acts as a lipid-transfer protein that supplies phospholipids for autophagosome formation.

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Fig. 1: Crystal Structure of SpAtg2NR.
Fig. 2: Structural basis of phospholipid recognition by Atg2NR.
Fig. 3: Atg2 tethers membranes and facilitates phospholipid transfer in vitro.
Fig. 4: The LT and MT activities of Atg2 are important for autophagy.
Fig. 5: Model of Atg2-mediated supply of phospholipids for autophagosome formation.

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Data availability

Coordinates and structure factors of apo- and PE-bound SpAtg2NR have been deposited in the Protein Data Bank under accession codes 6A9E and 6A9J, respectively. Source data for Figs. 3e,h and 4c are available with the paper online. All other data are available from the corresponding authors upon reasonable request.


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We thank Y. Ishii for her help with protein expression, C. Kondo-Kakuta and Y. Kamada (National Institute for Basic Biology, Japan) for generation of the pCK6 plasmid, S. Kawano and T. Endo (Kyoto Sangyo University) for providing the plasmid for the Mmm1–Mdm12 complex, H. Tachikawa (University of Tokyo) for providing the cDNA of Vps13, and A. I. May for proofreading the manuscript. S. pombe Atg2 cDNA was provided by the RIKEN BioResource Research Center (BRC) through the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and Agency for Medical Research and Development (AMED), Japan. The synchrotron radiation experiments were performed using beamlines BL-1A and BL-17A at Photon Factory (KEK, Japan). This work was supported by the Japan Science and Technology Agency (JST) Core Research for Evolutionary Science and Technology (CREST, Grant Number JPMJCR13M7 to N.N.N. and H.N.), the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research 25111004 and 18H03989 (to N.N.N.), 25111003 (to H.N.), 16H06375 (to Y.O.), 18J13429 (to E.H.), 16H06280 and 18H04853 (to K.S.), and the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from MEXT and AMED.

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Authors and Affiliations



T.O. and N.N.N. conceived the project. T.O. carried out protein expression, purification, crystallization, structure determination, and in vitro analyses of Atg2 and Atg18. N.N.N. performed microscopy of liposomes. T. Kawaoka, E.H., K.S., T. Kotani, H.N., and Y.O. performed the yeast experiments. T.O. and N.N.N. wrote the manuscript with input from other authors.

Corresponding author

Correspondence to Nobuo N. Noda.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Structural study of SpAtg2NR.

a, Secondary structure prediction of Chorein_N from several species. Chorein_N seems to conserve a fold quite similar to a part of SpAtg2NR. b, An artificial dimer found in SpAtg2NR crystals. SpAtg2NR dimerizes through formation of intermolecular antiparallel β-sheet.

Supplementary Figure 2 Structural study of PE-bound SpAtg2NR.

a, Structural superposition of SpAtg2NR and its PE-bound form. Both structures are represented by Cα-trace. b, Interaction between sn-2 acyl chains within the dimer found in crystal packing.

Supplementary Figure 3 Mutational analyses of ScAtg2.

a, SDS-PAGE of purified recombinant proteins used for in vitro analyses. Protein bands were stained with Coomassie Brilliant Blue. Uncropped gel images are shown in Supplementary Data Set 1. b, The effect of ATP on phospholipid transfer mediated by Atg2. c, Comparison of the MT activity between Atg2 and the Vps13 fragment analyzed by dynamic light scattering. d, MT activities of Atg2 as assessed by fluorescence microscopy of NBD-PE and Rhod-PE liposomes. Scale bar indicates 5 μm. e, Bulk autophagic activity measured by the ALP assay. In e and f, data are presented as mean±s.d. from n = 3 independent experiments. N.S. means not statistically significant (two-tailed Student’s test). f, Maturation of a selective autophagy cargo aminopeptidase I (Ape1) via autophagy monitored by western blotting. Uncropped blot image is shown in Supplementary Data Set 1. g, LT and MT activities of the K108E R216E mutant. h, Confocal microscopic analysis of liposomes mixed with the 3D mutant of ScAtg2. i, Circular dichroism spectra of wild-type and 3D mutant of MBP-fused SpAtg2NR.

Supplementary Figure 4 Structural feature of Atg2 supporting its lipid transfer activity.

a, Left, Displacement of the H4 helix creates a hydrophobic groove connecting the hydrophobic cavity and the membrane bound to the basic residues of Atg2NR. Arrows indicate a possible path for a phospholipid from the membrane to the cavity. Right, Superimposition of the structure of Vps13NR (PDB 6CBC) onto that of Atg2NR indicates the displacement of H4 in Vps13NR. b, Secondary structure prediction of ScAtg2 indicates that Atg2 contains repeated structures downstream of the N-terminal region that are abundant with β-strands. Boundaries of the repeated structures are roughly defined. The fifth repeated structure contains ATG2_CAD.

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Supplementary Information

Supplementary Figures 1–4, Supplementary Tables 1 and 2 and Supplementary Dataset 1

Reporting Summary

Supplementary Video 1

Effect of protease treatment on tethering of liposomes. Moving puncta of liposomes induced by Atg2 treatment as in Fig. 3h were subjected to proteinase K treatment, and NBD signals were monitored by fluorescence microscopy.

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Osawa, T., Kotani, T., Kawaoka, T. et al. Atg2 mediates direct lipid transfer between membranes for autophagosome formation. Nat Struct Mol Biol 26, 281–288 (2019).

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