@article{d7be48da1d3340ab80ce3e330ee7bc9a,
title = "Structure, lipid scrambling activity and role in autophagosome formation of ATG9A",
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.",
author = "Shintaro Maeda and Hayashi Yamamoto and Kinch, {Lisa N.} and Garza, {Christina M.} and Satoru Takahashi and Chinatsu Otomo and Grishin, {Nick V.} and Stefano Forli and Noboru Mizushima and Takanori Otomo",
note = "Funding Information: 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. Publisher Copyright: {\textcopyright} 2020, The Author(s), under exclusive licence to Springer Nature America, Inc.",
year = "2020",
month = dec,
doi = "10.1038/s41594-020-00520-2",
language = "English (US)",
volume = "27",
pages = "1194--1201",
journal = "Nature Structural Biology",
issn = "1545-9993",
publisher = "Nature Publishing Group",
number = "12",
}