Abstract
Autophagy is a tightly regulated intracellular bulk degradation/recycling system that has fundamental roles in cellular homeostasis1. Autophagy is initiated by isolation membranes, which form and elongate as they engulf portions of the cytoplasm and organelles. Eventually isolation membranes close to form double membrane-bound autophagosomes and fuse with lysosomes to degrade their contents. The physiological role of autophagy has been determined since its discovery, but the origin of autophagosomal membranes has remained unclear. At present, there is much controversy about the organelle from which the membranes originate—the endoplasmic reticulum (ER), mitochondria and plasma membrane1,2. Here we show that autophagosomes form at the ER–mitochondria contact site in mammalian cells. Imaging data reveal that the pre-autophagosome/autophagosome marker ATG14 (also known as ATG14L) relocalizes to the ER–mitochondria contact site after starvation, and the autophagosome-formation marker ATG5 also localizes at the site until formation is complete. Subcellular fractionation showed that ATG14 co-fractionates in the mitochondria-associated ER membrane3,4,5 fraction under starvation conditions. Disruption of the ER–mitochondria contact site prevents the formation of ATG14 puncta. The ER-resident SNARE protein syntaxin 17 (STX17) binds ATG14 and recruits it to the ER–mitochondria contact site. These results provide new insight into organelle biogenesis by demonstrating that the ER–mitochondria contact site is important in autophagosome formation.
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Acknowledgements
We thank all members of the Amano and Yoshimori laboratories for discussions. We thank O. Kunitaki and K. Miura for continuous help throughout the research. We also thank P. Karagiannis for proofreading. This research was supported by grants-in-aid for Scientific Research (A) and (B) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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M.H., N. Furuta, T.H., Y.H., N. Fujita, T.N., T.Y. and A.A. designed the experiments. M.H. performed image analysis, including confocal and time-lapse microscopic analysis. A.M. performed image analysis on live-cell imaging. A.Y., M.H. and A.N. preformed immunoelectron microscopy. M.H. and A.N. performed conventional electron microscopy. H.O. performed correlative light and electron microscopy. N. Furuta performed the remaining experiments. M.H., N. Furuta, T.Y. and A.A. wrote the manuscript.
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This file contains Supplementary Figures 1-10. (PDF 3432 kb)
Example of an autophagosome formation site in relation to ER-mitochondria contact sites in a COS7 cell stably expressing GFP-Atg5 and transiently expressing RFP-Sec61β (an ER marker) and TFP-mito (a mitochondia marker) under starved conditions
Images were taken every 30 seconds and are shown at 2 frames per second. (AVI 1679 kb)
Example of an autophagosome formation site in relation to ER-mitochondria contact sites in a COS7 cell stably expressing GFP-Atg5 and transiently expressing RFP-Sec61β (an ER marker) and TFP-mito (a mitochondia marker) under starved conditions
Images were taken every 30 seconds and are shown at 2 frames per second. (AVI 1522 kb)
Example of an autophagosome formation site in relation to ER-mitochondria contact sites in a COS7 cell stably expressing GFP-Atg5 and transiently expressing RFP-Sec61β (an ER marker) and TFP-mito (a mitochondia marker) under starved conditions
Images were taken every 30 seconds and are shown at 2 frames per second. (AVI 1898 kb)
Example of an autophagosome formation site in relation to VDAC1 in a Hela cell stably expressing GFP-Atg5 and transiently expressing Cherry-VDAC1 under starved conditions
Images were taken every 30 seconds and are shown at 2 frames per second. (AVI 3076 kb)
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Hamasaki, M., Furuta, N., Matsuda, A. et al. Autophagosomes form at ER–mitochondria contact sites. Nature 495, 389–393 (2013). https://doi.org/10.1038/nature11910
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DOI: https://doi.org/10.1038/nature11910
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