Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Autophagosomes form at ER–mitochondria contact sites

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: ATG14 assembles at the ER–mitochondria contact site.
Figure 2: Isolation membrane forms at ER–mitochondria contact sites.
Figure 3: STX17 regulates the relocalization of ATG14 to MAM.
Figure 4: Functional autophagosomes do not form in STX17 knockdown cells.

Similar content being viewed by others

References

  1. Mizushima, N., Yoshimori, T. & Ohsumi, Y. The role of Atg proteins in autophagosome formation. Annu. Rev. Cell Dev. Biol. 27, 107–132 (2011)

    Article  CAS  Google Scholar 

  2. Tooze, S. A. & Yoshimori, T. The origin of the autophagosomal membrane. Nature Cell Biol. 12, 831–835 (2010)

    Article  CAS  Google Scholar 

  3. Vance, J. E. Phospholipid synthesis in a membrane fraction associated with mitochondria. J. Biol. Chem. 265, 7248–7256 (1990)

    CAS  PubMed  Google Scholar 

  4. Rizzuto, R. et al. Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280, 1763–1766 (1998)

    Article  ADS  CAS  Google Scholar 

  5. Hayashi, T., Rizzuto, R., Hajnoczky, G. & Su, T.-P. MAM: more than just a housekeeper. Trends Cell Biol. 19, 81–88 (2009)

    Article  CAS  Google Scholar 

  6. Matsunaga, K. et al. Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nature Cell Biol. 11, 385–396 (2009)

    Article  CAS  Google Scholar 

  7. Matsunaga, K. et al. Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L. J. Cell Biol. 190, 511–521 (2010)

    Article  CAS  Google Scholar 

  8. Hayashi-Nishino, M. et al. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nature Cell Biol. 11, 1433–1437 (2009)

    Article  CAS  Google Scholar 

  9. Ylä-Anttila, P., Vihinen, H., Jokitalo, E. & Eskelinen, E.-L. 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 5, 1180–1185 (2009)

    Article  Google Scholar 

  10. Axe, E. L. et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J. Cell Biol. 182, 685–701 (2008)

    Article  CAS  Google Scholar 

  11. Hayashi, T. & Su, T.-P. Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca2+ signaling and cell survival. Cell 131, 596–610 (2007)

    Article  CAS  Google Scholar 

  12. Simmen, T. et al. PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis. EMBO J. 24, 717–729 (2005)

    Article  CAS  Google Scholar 

  13. Künkele, K. P. et al. The preprotein translocation channel of the outer membrane of mitochondria. Cell 93, 1009–1019 (1998)

    Article  Google Scholar 

  14. Mizushima, N. et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J. Cell Biol. 152, 657–668 (2001)

    Article  CAS  Google Scholar 

  15. Szabadkai, G. et al. Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. J. Cell Biol. 175, 901–911 (2006)

    Article  CAS  Google Scholar 

  16. Hailey, D. W. et al. Mitochondria supply membranes for autophagosome biogenesis during starvation. Cell 141, 656–667 (2010)

    Article  CAS  Google Scholar 

  17. de Brito, O. M. & Scorrano, L. Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456, 605–610 (2008)

    Article  ADS  Google Scholar 

  18. Kabeya, Y. et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720–5728 (2000)

    Article  CAS  Google Scholar 

  19. Furuta, N., Fujita, N., Noda, T., Yoshimori, T. & Amano, A. Combinational soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins VAMP8 and Vti1b mediate fusion of antimicrobial and canonical autophagosomes with lysosomes. Mol. Biol. Cell 21, 1001–1010 (2010)

    Article  CAS  Google Scholar 

  20. Steegmaier, M., Oorschot, V., Klumperman, J. & Scheller, R. H. Syntaxin 17 is abundant in steroidogenic cells and implicated in smooth endoplasmic reticulum membrane dynamics. Mol. Biol. Cell 11, 2719–2731 (2000)

    Article  CAS  Google Scholar 

  21. Fujita, N. et al. An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure. Mol. Biol. Cell 19, 4651–4659 (2008)

    Article  CAS  Google Scholar 

  22. Bjørkøy, G. et al. Monitoring autophagic degradation of p62/SQSTM1. Methods Enzymol. 452, 181–197 (2009)

    Article  Google Scholar 

  23. Mizushima, N. & Yoshimori, T. How to interpret LC3 immunoblotting. Autophagy 3, 542–545 (2007)

    Article  CAS  Google Scholar 

  24. Kimura, S., Noda, T. & Yoshimori, T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3, 452–460 (2007)

    Article  CAS  Google Scholar 

  25. Yamamoto, A. & Masaki, R. Pre-embedding nanogold silver and gold intensification. Methods Mol. Biol. 657, 225–235 (2010)

    Article  CAS  Google Scholar 

  26. Kageyama, S. et al. The LC3 recruitment mechanism is separate from Atg9L1-dependent membrane formation in the autophagic response against Salmonella. Mol. Biol. Cell 22, 2290–2300 (2011)

    Article  CAS  Google Scholar 

  27. Kamimoto, T. et al. Intracellular inclusions containing mutant α1-antitrypsin Z are propagated in the absence of autophagic activity. J. Biol. Chem. 281, 4467–4476 (2006)

    Article  CAS  Google Scholar 

  28. Saitoh, T. et al. Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response. Proc. Natl Acad. Sci. USA 106, 20842–20846 (2009)

    Article  ADS  CAS  Google Scholar 

  29. Kawai, S., Yamauchi, M., Wakisaka, S., Ooshima, T. & Amano, A. Zinc-finger transcription factor odd-skipped related 2 is one of the regulators in osteoblast proliferation and bone formation. J. Bone Miner. Res. 22, 1362–1372 (2007)

    Article  CAS  Google Scholar 

  30. Nakagawa, I. et al. Autophagy defends cells against invading group A Streptococcus. Science 306, 1037–1040 (2004)

    Article  ADS  CAS  Google Scholar 

  31. Fujita, N. et al. The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. Mol. Biol. Cell 19, 2092–2100 (2008)

    Article  CAS  Google Scholar 

  32. Slot, J. W. & Geuze, H. J. Cryosectioning and immunolabeling. Nature Protocols 2, 2480–2491 (2007)

    Article  CAS  Google Scholar 

  33. Griffiths, G., McDowall, A., Back, R. & Dubochet, J. On the preparation of cryosections for immunocytochemistry. J. Ultrastruct. Res. 89, 65–78 (1984)

    Article  CAS  Google Scholar 

  34. Boulanger, J., Kervrann, C. & Bouthemy, P. A simulation and estimation framework for intracellular dynamics and trafficking in video-microscopy and fluorescence imagery. Med. Image Anal. 13, 132–142 (2009)

    Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Atsuo Amano or Tamotsu Yoshimori.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

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)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11910

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing