Mechanisms governing autophagosome biogenesis

Abstract

Autophagosomes are double-membrane vesicles newly formed during autophagy to engulf a wide range of intracellular material and transport this autophagic cargo to lysosomes (or vacuoles in yeasts and plants) for subsequent degradation. Autophagosome biogenesis responds to a plethora of signals and involves unique and dynamic membrane processes. Autophagy is an important cellular mechanism allowing the cell to meet various demands, and its disruption compromises homeostasis and leads to various diseases, including metabolic disorders, neurodegeneration and cancer. Thus, not surprisingly, the elucidation of the molecular mechanisms governing autophagosome biogenesis has attracted considerable interest. Key molecules and organelles involved in autophagosome biogenesis, including autophagy-related (ATG) proteins and the endoplasmic reticulum, have been discovered, and their roles and relationships have been investigated intensely. However, several fundamental questions, such as what supplies membranes/lipids to build the autophagosome and how the membrane nucleates, expands, bends into a spherical shape and finally closes, have proven difficult to address. Nonetheless, owing to recent studies with new approaches and technologies, we have begun to unveil the mechanisms underlying these processes on a molecular level. We now know that autophagosome biogenesis is a highly complex process, in which multiple proteins and lipids from various membrane sources, supported by the formation of membrane contact sites, cooperate with biophysical phenomena, including membrane shaping and liquid–liquid phase separation, to ensure seamless segregation of the autophagic cargo. Together, these studies pave the way to obtaining a holistic view of autophagosome biogenesis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Overview of the process of autophagic degradation.
Fig. 2: Core ATG proteins and their complexes.
Fig. 3: A model for the process of membrane nucleation in autophagosome biogenesis.
Fig. 4: Mechanisms of ER-associated membrane expansion in autophagosome biogenesis.
Fig. 5: Mechanisms of autophagosomal membrane shaping.
Fig. 6: Mechanism of autophagosomal pore closure.

References

  1. 1.

    Clark, S. L. Cellular differentiation in the kidneys of newborn mice studies with the electron microscope. J. Biophys. Biochem. Cytol. 3, 349–362 (1957).

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Novikoff, A. B. The proximal tubule cell in experimental hydronephrosis. J. Biophys. Biochem. Cytol. 6, 136–138 (1959).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Ashford, T. P. & Porter, K. R. Cytoplasmic components in hepatic cell lysosomes. J. Cell Biol. 12, 198–202 (1962).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Novikoff, A. B. & Essner, E. Cytolysomes and mitochondrial degeneration. J. Cell Biol. 15, 140–146 (1962).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    de Duve, C. & Wattiaux, R. Functions of lysosomes. Annu. Rev. Physiol. 28, 435–492 (1966).

    PubMed  Google Scholar 

  6. 6.

    Yang, Z. & Klionsky, D. J. Eaten alive: a history of macroautophagy. Nat. Cell Biol. 12, 814–822 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Klionsky, D. J. et al. A unified nomenclature for yeast autophagy-related genes. Dev. Cell 5, 539–545 (2003).

    CAS  PubMed  Google Scholar 

  8. 8.

    Nakatogawa, H., Suzuki, K., Kamada, Y. & Ohsumi, Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat. Rev. Mol. Cell Biol. 10, 458–467 (2009).

    CAS  PubMed  Google Scholar 

  9. 9.

    Ohsumi, Y. Historical landmarks of autophagy research. Cell Res. 24, 9–23 (2014).

    CAS  PubMed  Google Scholar 

  10. 10.

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

    CAS  PubMed  Google Scholar 

  11. 11.

    Dikic, I. & Elazar, Z. Mechanism and medical implications of mammalian autophagy. Nat. Rev. Mol. Cell Biol. 19, 349–364 (2018).

    CAS  PubMed  Google Scholar 

  12. 12.

    Levine, B. & Kroemer, G. Biological functions of autophagy genes: a disease perspective. Cell 176, 11–42 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1–222 (2016).

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    Mizushima, N. Physiological functions of autophagy. Curr. Top. Microbiol. Immunol. 335, 71–84 (2009).

    CAS  PubMed  Google Scholar 

  15. 15.

    Levine, B. & Klionsky, D. J. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev. Cell 6, 463–477 (2004).

    CAS  PubMed  Google Scholar 

  16. 16.

    Yang, Z., Huang, J., Geng, J., Nair, U. & Klionsky, D. J. Atg22 recycles amino acids to link the degradative and recycling functions of autophagy. Mol. Biol. Cell 17, 5094–5104 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Kawano-Kawada, M., Kakinuma, Y. & Sekito, T. Transport of amino acids across the vacuolar membrane of yeast: its mechanism and physiological role. Biol. Pharm. Bull. 41, 1496–1501 (2018).

    CAS  PubMed  Google Scholar 

  18. 18.

    Kirkin, V. History of the selective autophagy research: how did it begin and where does it stand today? J. Mol. Biol. 432, 3–27 (2019).

    PubMed  Google Scholar 

  19. 19.

    Johansen, T. & Lamark, T. Selective autophagy: ATG8 family proteins, LIR motifs and cargo receptors. J. Mol. Biol. 432, 80–103 (2019).

    PubMed  Google Scholar 

  20. 20.

    Gatica, D., Lahiri, V. & Klionsky, D. J. Cargo recognition and degradation by selective autophagy. Nat. Cell Biol. 20, 233–242 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Turco, E., Fracchiolla, D. & Martens, S. Recruitment and activation of the ULK1/Atg1 kinase complex in selective autophagy. J. Mol. Biol. 432, 123–134 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Kirkin, V. & Rogov, V. V. A diversity of selective autophagy receptors determines the specificity of the autophagy pathway. Mol. Cell 76, 268–285 (2019).

    CAS  PubMed  Google Scholar 

  23. 23.

    Matsuura, A., Tsukada, M., Wada, Y., & Ohsumi, Y. Apg1p, a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae. Gene 192, 245–250 (1997).

    CAS  PubMed  Google Scholar 

  24. 24.

    Noda, T. & Ohsumi, Y. Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast. J. Biol. Chem. 273, 3963–3966 (1998).

    CAS  PubMed  Google Scholar 

  25. 25.

    Ragusa, M. J., Stanley, R. E. & Hurley, J. H. Architecture of the Atg17 complex as a scaffold for autophagosome biogenesis. Cell 151, 1501–1512 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Stjepanovic, G. et al. Assembly and dynamics of the autophagy-initiating Atg1 complex. Proc. Natl Acad. Sci. USA 111, 12793–12798 (2014).

    CAS  PubMed  Google Scholar 

  27. 27.

    Chew, L. H., Setiaputra, D., Klionsky, D. J. & Yip, C. K. Structural characterization of the Saccharomyces cerevisiae autophagy regulatory complex Atg17-Atg31-Atg29. Autophagy 9, 1467–1474 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Chew, L. H. et al. Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms. Autophagy 11, 891–905 (2015).

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Kamada, Y. et al. Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J. Cell Biol. 150, 1507–1513 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Kamada, Y. et al. Tor directly controls the Atg1 kinase complex to regulate autophagy. Mol. Cell. Biol. 30, 1049–1058 (2010).

    CAS  PubMed  Google Scholar 

  31. 31.

    Fujioka, Y. et al. Structural basis of starvation-induced assembly of the autophagy initiation complex. Nat. Struct. Mol. Biol. 21, 513–521 (2014).

    CAS  PubMed  Google Scholar 

  32. 32.

    Yamamoto, H. et al. The intrinsically disordered protein Atg13 mediates supramolecular assembly of autophagy initiation complexes. Dev. Cell 38, 86–99 (2016).

    CAS  PubMed  Google Scholar 

  33. 33.

    Yorimitsu, T., He, C., Wang, K. & Klionsky, D. J. Tap42-associated protein phosphatase type 2A negatively regulates induction of autophagy. Autophagy 5, 616–624 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Yeasmin, A. M. S. T. et al. Orchestrated action of PP2A antagonizes Atg13 phosphorylation and promotes autophagy after the inactivation of TORC1. PLoS One 11, e0166636 (2016).

    PubMed  PubMed Central  Google Scholar 

  35. 35.

    Memisoglu, G., Eapen, V. V., Yang, Y., Klionsky, D. J. & Haber, J. E. PP2C phosphatases promote autophagy by dephosphorylation of the Atg1 complex. Proc. Natl Acad. Sci. USA 116, 1613–1620 (2019).

    CAS  PubMed  Google Scholar 

  36. 36.

    Jung, C. H. et al. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol. Biol. Cell 20, 1992–2003 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Hosokawa, N. et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol. Biol. Cell 20, 1981–1991 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Ganley, I. G. et al. ULK1·ATG13·FIP200 complex mediates mTOR signaling and is essential for autophagy. J. Biol. Chem. 284, 12297–12305 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Hara, T. et al. FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. J. Cell Biol. 181, 497–510 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Hosokawa, N. et al. Atg101, a novel mammalian autophagy protein interacting with Atg13. Autophagy 5, 973–979 (2009).

    CAS  PubMed  Google Scholar 

  41. 41.

    Mercer, C. A., Kaliappan, A. & Dennis, P. B. A novel, human Atg13 binding protein, Atg101, interacts with ULK1 and is essential for macroautophagy. Autophagy 5, 649–662 (2009).

    CAS  PubMed  Google Scholar 

  42. 42.

    Bach, M., Larance, M., James, D. E. & Ramm, G. The serine/threonine kinase ULK1 is a target of multiple phosphorylation events. Biochem. J. 440, 283–291 (2011).

    CAS  PubMed  Google Scholar 

  43. 43.

    Yeh, Y. Y., Wrasman, K. & Herman, P. K. Autophosphorylation within the Atg1 activation loop is required for both kinase activity and the induction of autophagy in Saccharomyces cerevisiae. Genetics 185, 871–882 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Kijanska, M. et al. Activation of Atg1 kinase in autophagy by regulated phosphorylation. Autophagy 6, 1168–1178 (2010).

    CAS  PubMed  Google Scholar 

  45. 45.

    Sánchez-Wandelmer, J. et al. Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation. Nat. Commun. 8, 295 (2017).

    PubMed  PubMed Central  Google Scholar 

  46. 46.

    Papinski, D. et al. Early steps in autophagy depend on direct phosphorylation of Atg9 by the Atg1 kinase. Mol. Cell 53, 471–483 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Hu, Z. et al. Multilayered control of protein turnover by TORC1 and Atg1. Cell Rep. 28, 3486–3496 (2019).

    CAS  PubMed  Google Scholar 

  48. 48.

    Pengo, N., Agrotis, A., Prak, K., Jones, J. & Ketteler, R. A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B. Nat. Commun. 8, 294 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Russell, R. C. et al. ULK1 induces autophagy by phosphorylating beclin-1 and activating VPS34 lipid kinase. Nat. Cell Biol. 15, 741–750 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Zhou, C. et al. Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy. Cell Res. 27, 184–201 (2017).

    CAS  PubMed  Google Scholar 

  51. 51.

    Wold, M. S., Lim, J., Lachance, V., Deng, Z. & Yue, Z. ULK1-mediated phosphorylation of ATG14 promotes autophagy and is impaired in Huntington’s disease models. Mol. Neurodegener. 11, 76 (2016).

    PubMed  PubMed Central  Google Scholar 

  52. 52.

    Park, J. M. et al. The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14. Autophagy 12, 547–564 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Egan, D. F. et al. Small molecule Inhibition of the autophagy kinase ULK1 and identification of ULK1 substrates. Mol. Cell 59, 285–297 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Di Bartolomeo, S. et al. The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy. J. Cell Biol. 191, 155–168 (2010).

    PubMed  PubMed Central  Google Scholar 

  55. 55.

    Jeong, Y. T. et al. The ULK1-FBXW5-SEC23B nexus controls autophagy. eLife 7, 1–25 (2018).

    Google Scholar 

  56. 56.

    Lin, S. Y. et al. GSK3-TIP60-ULK1 signaling pathway links growth factor deprivation to autophagy. Science 336, 477–481 (2012).

    CAS  PubMed  Google Scholar 

  57. 57.

    Nazio, F. et al. MTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6. Nat. Cell Biol. 15, 406–416 (2013).

    CAS  PubMed  Google Scholar 

  58. 58.

    Liu, C. C. et al. Cul3-KLHL20 ubiquitin ligase governs the turnover of ULK1 and VPS34 complexes to control autophagy termination. Mol. Cell 61, 84–97 (2016).

    CAS  PubMed  Google Scholar 

  59. 59.

    Nazio, F. et al. Fine-tuning of ULK1 mRNA and protein levels is required for autophagy oscillation. J. Cell Biol. 215, 841–856 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Li, J. et al. Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy. Autophagy 11, 1216–1229 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Yamamoto, H. et al. Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J. Cell Biol. 198, 219–233 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Mari, M. et al. An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis. J. Cell Biol. 190, 1005–1022 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Shirahama-Noda, K., Kira, S., Yoshimori, T. & Noda, T. TRAPPis responsible for vesicular transport from early endosomes to Golgi, facilitating Atg9 cycling in autophagy. J. Cell Sci. 126, 4963–4973 (2013).

    CAS  PubMed  Google Scholar 

  64. 64.

    Kakuta, S. et al. Atg9 vesicles recruit vesicle-tethering proteins Trs85 and Ypt1 to the autophagosome formation site. J. Biol. Chem. 287, 44261–44269 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Geng, J., Nair, U., Yasumura-Yorimitsu, K. & Klionsky, D. J. Post-Golgi Sec proteins are required for autophagy in Saccharomyces cerevisiae. Mol. Biol. Cell 21, 2257–2269 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Ohashi, Y. & Munro, S. Membrane delivery to the yeast autophagosome from the Golgi-endosomal system. Mol. Biol. Cell 21, 3998–4008 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Backues, S. K. et al. Atg23 and Atg27 act at the early stages of Atg9 trafficking in S. cerevisiae. Traffic 16, 172–190 (2015).

    CAS  PubMed  Google Scholar 

  68. 68.

    Yen, W. L., Legalds, J. E., Nair, U. & Klionsky, D. J. Atg27 is required for autophagy-dependent cycling of Atg9. Mol. Biol. Cell 18, 581–593 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Segarra, V. A., Boettner, D. R. & Lemmon, S. K. Atg27 tyrosine sorting motif is important for its trafficking and Atg9 localization. Traffic 16, 365–378 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Kakuta, S. et al. Small GTPase Rab1B is associated with ATG9A vesicles and regulates autophagosome formation. FASEB J. 31, 3757–3773 (2017).

    CAS  PubMed  Google Scholar 

  71. 71.

    Orsi, A. et al. Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol. Biol. Cell 23, 1860–1873 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. 72.

    Duke, E. M. H. et al. Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM). Ultramicroscopy 143, 77–87 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. 73.

    Lamb, C. A. et al. TBC 1D14 regulates autophagy via the TRAPP complex and ATG 9 traffic. EMBO J. 35, 281–301 (2016).

    CAS  PubMed  Google Scholar 

  74. 74.

    Longatti, A. et al. TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes. J. Cell Biol. 197, 659–675 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. 75.

    Popovic, D. & Dikic, I. TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy. EMBO Rep. 15, 392–401 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. 76.

    Takahashi, Y. et al. Bif-1 regulates Atg9 trafficking by mediating the fission of Golgi membranes during autophagy. Autophagy 7, 61–73 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. 77.

    Imai, K. et al. Atg9A trafficking through the recycling endosomes is required for autophagosome formation. J. Cell Sci. 129, 3781–3791 (2016).

    CAS  PubMed  Google Scholar 

  78. 78.

    Søreng, K. et al. SNX 18 regulates ATG 9A trafficking from recycling endosomes by recruiting Dynamin-2. EMBO Rep. 19, e44837 (2018).

    PubMed  PubMed Central  Google Scholar 

  79. 79.

    Puri, C., Renna, M., Bento, C. F., Moreau, K. & Rubinsztein, D. C. Diverse autophagosome membrane sources coalesce in recycling endosomes. Cell 154, 1285–1299 (2013). This work and Yamamoto et al. (2012) and Mari et al. (2010) together propose that Atg9/ATG9-containing vesicles contribute to the formation of autophagosome precursors.

    CAS  PubMed  PubMed Central  Google Scholar 

  80. 80.

    Kihara, A., Noda, T., Ishihara, N. & Ohsumi, Y. Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. J. Cell Biol. 152, 519–530 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 81.

    Araki, Y. et al. Atg38 is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity. J. Cell Biol. 203, 299–313 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. 82.

    Itakura, E., Kishi, C., Inoue, K. & Mizushima, N. Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. Mol. Biol. Cell 19, 5360–5372 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. 83.

    Lu, J. et al. NRBF2 regulates autophagy and prevents liver injury by modulating Atg14L-linked phosphatidylinositol-3 kinase III activity. Nat. Commun. 5, 4920 (2014).

    Google Scholar 

  84. 84.

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

    CAS  PubMed  Google Scholar 

  85. 85.

    Zhong, Y. et al. Distinct regulation of autophagic activity by Atg14L and Rubicon associated with beclin 1-phosphatidylinositol-3-kinase complex. Nat. Cell Biol. 11, 468–476 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. 86.

    Obara, K., Noda, T., Niimi, K. & Ohsumi, Y. Transport of phosphatidylinositol 3-phosphate into the vacuole via autophagic membranes in Saccharomyces cerevisiae. Genes. Cell 13, 537–547 (2008).

    CAS  Google Scholar 

  87. 87.

    Cheng, J. et al. Yeast and mammalian autophagosomes exhibit distinct phosphatidylinositol 3-phosphate asymmetries. Nat. Commun. 5, 3207 (2014).

    PubMed  Google Scholar 

  88. 88.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. 89.

    Ge, L., Zhang, M. & Schekman, R. Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER-Golgi intermediate compartment. eLife 3, 1–13 (2014).

    Google Scholar 

  90. 90.

    Ge, L. et al. Remodeling of ER-exit sites initiates a membrane supply pathway for autophagosome biogenesis. EMBO Rep. 18, e201744559 (2017).

    Google Scholar 

  91. 91.

    Ge, L., Melville, D., Zhang, M. & Schekman, R. The ER-Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis. eLife 2, e00947 (2013).

    PubMed  PubMed Central  Google Scholar 

  92. 92.

    Pattingre, S. et al. Bcl-2 antiapoptotic proteins inhibit beclin 1-dependent autophagy. Cell 122, 927–939 (2005).

    CAS  PubMed  Google Scholar 

  93. 93.

    Maiuri, M. C. et al. BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between beclin 1 and Bcl-2/Bcl-XL. Autophagy 3, 374–376 (2007).

    CAS  PubMed  Google Scholar 

  94. 94.

    Takahashi, Y. et al. Bif-1 interacts with beclin 1 through UVRAG and regulates autophagy and tumorigenesis. Nat. Cell Biol. 9, 1142–1151 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. 95.

    Maria Fimia, G. et al. Ambra1 regulates autophagy and development of the nervous system. Nature 447, 1121–1125 (2007).

    Google Scholar 

  96. 96.

    Barth, H., Meiling-Wesse, K., Epple, U. D. & Thumm, M. Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p. FEBS Lett. 508, 23–28 (2001).

    CAS  PubMed  Google Scholar 

  97. 97.

    Guan, J. et al. Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport, pexophagy, and autophagy in Saccharomyces cerevisiae and Pichia pastoris. Mol. Biol. Cell 12, 3821–3838 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  98. 98.

    Strømhaug, P. E., Reggiori, F., Guan, J., Wang, C.-W. & Klionsky, D. J. Atg21 is a phosphoinositide binding protein required for efficient lipidation and localization of Atg8 during uptake of aminopeptidase I by selective autophagy. Mol. Biol. Cell 15, 3553–3566 (2004).

    PubMed  PubMed Central  Google Scholar 

  99. 99.

    Meiling-Wesse, K. et al. Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway. J. Biol. Chem. 279, 37741–37750 (2004).

    CAS  PubMed  Google Scholar 

  100. 100.

    Proikas-Cezanne, T., Takacs, Z., Dönnes, P. & Kohlbacher, O. WIPI proteins: essential PtdIns3P effectors at the nascent autophagosome. J. Cell Sci. 128, 207–217 (2015).

    CAS  PubMed  Google Scholar 

  101. 101.

    Obara, K., Sekito, T., Niimi, K. & Ohsumi, Y. The Atg18-Atg2 complex is recruited to autophagic membranes via phosphatidylinositol 3-phosphate and exerts an essential function. J. Biol. Chem. 283, 23972–23980 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. 102.

    Chowdhury, S. et al. Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex. Proc. Natl Acad. Sci. USA 115, E9792–E9801 (2018).

    CAS  PubMed  Google Scholar 

  103. 103.

    Zheng, J. X. et al. Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation. Autophagy 8627, 1–14 (2017).

    Google Scholar 

  104. 104.

    Krick, R., Tolstrup, J., Appelles, A., Henke, S. & Thumm, M. The relevance of the phosphatidylinositolphosphat-binding motif FRRGT of Atg18 and Atg21 for the Cvt pathway and autophagy. FEBS Lett. 580, 4632–4638 (2006).

    CAS  PubMed  Google Scholar 

  105. 105.

    Nair, U., Cao, Y., Xie, Z. & Klionsky, D. J. Roles of the lipid-binding motifs of Atg18 and Atg21 in the cytoplasm to vacuole targeting pathway and autophagy. J. Biol. Chem. 285, 11476–11488 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. 106.

    Itakura, E. & Mizushima, N. Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 6, 764–776 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. 107.

    Velikkakath, A. K. G., Nishimura, T., Oita, E., Ishihara, N. & Mizushima, N. Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets. Mol. Biol. Cell 23, 896–909 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  108. 108.

    Mizushima, N. et al. A protein conjugation system essential for autophagy. Nature 395, 395–398 (1998).

    CAS  PubMed  Google Scholar 

  109. 109.

    Mizushima, N., Sugita, H., Yoshimori, T. & Ohsumi, Y. A new protein conjugation system in human. J. Biol. Chem. 273, 33889–33892 (1998).

    CAS  PubMed  Google Scholar 

  110. 110.

    Kuma, A., Mizushima, N., Ishihara, N. & Ohsumi, Y. Formation of the 350-kDa Apg12-Apg5·Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast. J. Biol. Chem. 227, 18619–18625 (2002).

    Google Scholar 

  111. 111.

    Mizushima, N. et al. Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate. J. Cell Sci. 116, 1679–1688 (2003).

    CAS  PubMed  Google Scholar 

  112. 112.

    Ishibashi, K. et al. Atg16L2, a novel isoform of mammalian Atg16L that is not essential for canonical autophagy despite forming an Atg12-5-16L2 complex. Autophagy 7, 1500–1513 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. 113.

    Fujioka, Y., Noda, N. N., Nakatogawa, H., Ohsumi, Y. & Inagaki, F. Dimeric coiled-coil structure of Saccharomyces cerevisiae Atg16 and its functional significance in autophagy. J. Biol. Chem. 285, 1508–1515 (2010).

    CAS  PubMed  Google Scholar 

  114. 114.

    Fujita, N. et al. Differential involvement of Atg16L1 in Crohn disease and canonical autophagy: analysis of the organization of the Atg16L1 complex in fibroblasts. J. Biol. Chem. 284, 32602–32609 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  115. 115.

    Dooley, H. C. et al. WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12-5-16L1. Mol. Cell 55, 238–252 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  116. 116.

    Juris, L. et al. PI 3P binding by Atg21 organises Atg8 lipidation. EMBO J. 34, 955–973 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  117. 117.

    Hanada, T. et al. The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J. Biol. Chem. 282, 37298–37302 (2007).

    CAS  PubMed  Google Scholar 

  118. 118.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  119. 119.

    Otomo, C., Metlagel, Z., Takaesu, G. & Otomo, T. Structure of the human ATG12ATG5 conjugate required for LC3 lipidation in autophagy. Nat. Struct. Mol. Biol. 20, 59–66 (2013).

    CAS  PubMed  Google Scholar 

  120. 120.

    Fujioka, Y. et al. In vitro reconstitution of plant Atg8 and Atg12 conjugation systems essential for autophagy. J. Biol. Chem. 283, 1921–1928 (2008).

    CAS  PubMed  Google Scholar 

  121. 121.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  122. 122.

    Kabeya, Y. et al. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J. Cell Sci. 117, 2805–2812 (2004).

    CAS  PubMed  Google Scholar 

  123. 123.

    Kirisako, T. et al. The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J. Cell Biol. 151, 263–276 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. 124.

    Ichimura, Y. et al. A ubiquitin-like system mediates protein lipidation. Nature 408, 488–492 (2000).

    CAS  PubMed  Google Scholar 

  125. 125.

    Sou, Y. S., Tanida, I., Komatsu, M., Ueno, T. & Kominami, E. Phosphatidylserine in addition to phosphatidylethanolamine is an in vitro target of the mammalian Atg8 modifiers, LC3, GABARAP, and GATE-16. J. Biol. Chem. 281, 3017–3024 (2006).

    CAS  PubMed  Google Scholar 

  126. 126.

    Nakatogawa, H. Two ubiquitin-like conjugation systems that mediate membrane formation during autophagy. Essays Biochem. 55, 39–50 (2013).

    CAS  PubMed  Google Scholar 

  127. 127.

    Wild, P., McEwan, D. G. & Dikic, I. The LC3 interactome at a glance. J. Cell Sci. 127, 3–9 (2014).

    CAS  PubMed  Google Scholar 

  128. 128.

    Slobodkin, M. R. & Elazar, Z. The Atg8 family: multifunctional ubiquitin-like key regulators of autophagy. Essays Biochem. 55, 51–64 (2013).

    CAS  PubMed  Google Scholar 

  129. 129.

    Nakatogawa, H., Ishii, J., Asai, E. & Ohsumi, Y. Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis. Autophagy 8, 177–186 (2012).

    CAS  PubMed  Google Scholar 

  130. 130.

    Yu, Z. Q. et al. Dual roles of Atg8-PE deconjugation by Atg4 in autophagy. Autophagy 8, 883–892 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  131. 131.

    Nair, U. et al. A role for Atg8-PE deconjugation in autophagosome biogenesis. Autophagy 8, 780–793 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  132. 132.

    Hirata, E., Ohya, Y. & Suzuki, K. Atg4 plays an important role in efficient expansion of autophagic isolation membranes by cleaving lipidated Atg8 in Saccharomyces cerevisiae. PLoS One 12, e0181047 (2017).

    PubMed  PubMed Central  Google Scholar 

  133. 133.

    Kauffman, K. J. et al. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases. Autophagy 14, 992–1010 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  134. 134.

    Abreu, S. et al. Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation. EMBO Rep. 18, 765–780 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  135. 135.

    He, C. & Klionsky, D. J. Regulation mechanisms and signaling pathways of autophagy. Annu. Rev. Genet. 43, 67–93 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  136. 136.

    Yang, Z. & Klionsky, D. J. Mammalian autophagy: core molecular machinery and signaling regulation. Curr. Opin. Cell Biol. 22, 124–131 (2010).

    CAS  PubMed  Google Scholar 

  137. 137.

    Corona Velazquez, A. F. & Jackson, W. T. So many roads: the multifaceted regulation of autophagy induction. Mol. Cell. Biol. 38, e00303–e00318 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  138. 138.

    Gross, A. & Graef, M. Mechanisms of autophagy in metabolic stress response. J. Mol. Biol. 432, 28–52 (2019).

    PubMed  Google Scholar 

  139. 139.

    Galluzzi, L., Pietrocola, F., Levine, B. & Kroemer, G. Metabolic control of autophagy. Cell 159, 1263–1276 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  140. 140.

    Loewith, R. & Hall, M. N. Target of rapamycin (TOR) in nutrient signaling and growth control. Genetics 189, 1177–1201 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. 141.

    Budovskaya, Y. V., Stephan, J. S., Deminoff, S. J. & Herman, P. K. An evolutionary proteomics approach identifies substrates of the cAMP-dependent protein kinase. Proc. Natl Acad. Sci. USA 102, 13933–13938 (2005).

    CAS  PubMed  Google Scholar 

  142. 142.

    Stephan, J. S., Yeh, Y. Y., Ramachandran, V., Deminoff, S. J. & Herman, P. K. The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy. Proc. Natl Acad. Sci. USA 106, 17049–17054 (2009).

    CAS  PubMed  Google Scholar 

  143. 143.

    Kim, J., Kundu, M., Viollet, B. & Guan, K. L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13, 132–141 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  144. 144.

    Egan, D. F. et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 331, 456–461 (2011).

    CAS  PubMed  Google Scholar 

  145. 145.

    Yi, C. et al. Formation of a Snf1-Mec1-Atg1 module on mitochondria governs energy deprivation-induced autophagy by regulating mitochondrial respiration. Dev. Cell 41, 59–71 (2017).

    CAS  PubMed  Google Scholar 

  146. 146.

    Kamber, R. A., Shoemaker, C. J. & Denic, V. Receptor-bound targets of selective autophagy use a scaffold protein to activate the Atg1 kinase. Mol. Cell 59, 372–381 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  147. 147.

    Vargas, J. N. S. et al. Spatiotemporal control of ULK1 activation by NDP52 and TBK1 during selective autophagy. Mol. Cell 74, 347–362 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  148. 148.

    Ravenhill, B. J. et al. The cargo receptor NDP52 initiates selective autophagy by recruiting the ULK complex to cytosol-invading bacteria. Mol. Cell 74, 320–329 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  149. 149.

    Turco, E. et al. FIP200 claw domain binding to p62 promotes autophagosome formation at ubiquitin condensates. Mol. Cell 74, 330–346 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  150. 150.

    Torggler, R. et al. Two independent pathways within selective autophagy converge to activate Atg1 kinase at the vacuole. Mol. Cell 64, 221–235 (2016).

    CAS  PubMed  Google Scholar 

  151. 151.

    Zientara-Rytter, K. & Subramani, S. Mechanistic insights into the role of Atg11 in selective autophagy. J. Mol. Biol. 432, 104–222 (2019).

    PubMed  Google Scholar 

  152. 152.

    Suzuki, K. et al. The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. EMBO J. 20, 5971–5981 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  153. 153.

    Suzuki, K., Akioka, M., Kondo-Kakuta, C., Yamamoto, H. & Ohsumi, Y. Fine mapping of autophagy-related proteins during autophagosome formation in Saccharomyces cerevisiae. J. Cell Sci. 126, 2534–2544 (2013).

    CAS  PubMed  Google Scholar 

  154. 154.

    Graef, M., Friedman, J. R., Graham, C., Babu, M. & Nunnari, J. ER exit sites are physical and functional core autophagosome biogenesis components. Mol. Biol. Cell 24, 2918–2931 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  155. 155.

    Hollenstein, D. M. et al. Vac8 spatially confines autophagosome formation at the vacuole in S. cerevisiae. J. Cell Sci. 132, jcs235002 (2019). This study describes tethering of the Atg1 complex to the vacuolar membrane by Vac8.

    CAS  PubMed  PubMed Central  Google Scholar 

  156. 156.

    Hamasaki, M. et al. Autophagosomes form at ER-mitochondria contact sites. Nature 495, 389–393 (2013).

    CAS  PubMed  Google Scholar 

  157. 157.

    Cheong, H., Nair, U., Geng, J. & Klionsky, D. J. The Atg1 kinase complex is involved in the regulation of protein recruitment to initiate sequestering vesicle formation for nonspecific autophagy in Saccharomyces cerevisiae. Mol. Biol. Cell 19, 433–476 (2008).

    Google Scholar 

  158. 158.

    Kawamata, T., Kamada, Y., Kabeya, Y., Sekito, T. & Ohsumi, Y. Organization of the pre-autophagosomal structure responsible for autophagosome formation. Mol. Biol. Cell 19, 2039–2050 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  159. 159.

    Koyama-Honda, I., Itakura, E., Fujiwara, T. K. & Mizushima, N. Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site. Autophagy 9, 1491–1499 (2013).

    CAS  PubMed  Google Scholar 

  160. 160.

    Geng, J., Baba, M., Nair, U. & Klionsky, D. J. Quantitative analysis of autophagy-related protein stoichiometry by fluorescence microscopy. J. Cell Biol. 182, 129–140 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  161. 161.

    Fujioka, Y. et al. Phase separation organizes the site of autophagosome formation. Nature 578, 301–305 (2020). This study and Fujioka et al. (2014), Yamamoto et al. (2016), Kamber et al. (2015) and Torggler et al. (2016) together show how the Atg1/ULK complex is formed and how multiple copies of the complex are further assembled to form a platform for the initiation of autophagosome formation.

    CAS  PubMed  Google Scholar 

  162. 162.

    Zhang, G., Wang, Z., Du, Z. & Zhang, H. mTOR regulates phase separation of PGL granules to modulate their autophagic degradation. Cell 174, 1492–1506 (2018).

    CAS  PubMed  Google Scholar 

  163. 163.

    Yamasaki, A. et al. Liquidity is a critical determinant for selective autophagy of protein condensates. Mol. Cell 77, 1163–1175 (2020).

    CAS  PubMed  Google Scholar 

  164. 164.

    Sun, D., Wu, R., Zheng, J., Li, P. & Yu, L. Polyubiquitin chain-induced p62 phase separation drives autophagic cargo segregation. Cell Res. 28, 405–415 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  165. 165.

    Yang, Y. et al. Cytoplasmic DAXX drives SQSTM1/p62 phase condensation to activate Nrf2-mediated stress response. Nat. Commun. 10, 3759 (2019).

    PubMed  PubMed Central  Google Scholar 

  166. 166.

    Sánchez-Martín, P. et al. NBR 1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system. EMBO Rep. 21, e48902 (2020).

    PubMed  Google Scholar 

  167. 167.

    You, Z. et al. Requirement for p62 acetylation in the aggregation of ubiquitylated proteins under nutrient stress. Nat. Commun. 10, 5792 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  168. 168.

    Cloer, E. W. et al. p62-dependent phase separation of patient-derived KEAP1 mutations and NRF2. Mol. Cell. Biol. 38, e00644-17 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  169. 169.

    Nishimura, T. et al. Autophagosome formation is initiated at phosphatidylinositol synthase-enriched ER subdomains. EMBO J. 36, 1719–1735 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  170. 170.

    Morita, K. et al. Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation. J. Cell Biol. 217, 3817–3828 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  171. 171.

    Moretti, F. et al. TMEM 41B is a novel regulator of autophagy and lipid mobilization. EMBO Rep. 19, e45889 (2018).

    PubMed  PubMed Central  Google Scholar 

  172. 172.

    Shoemaker, C. J. et al. CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor. PLoS Biol. 17, e2007044 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  173. 173.

    Zhao, Y. G. et al. The ER contact proteins VAPA/B Interact with multiple autophagy proteins to modulate autophagosome biogenesis. Curr. Biol. 28, 1234–1245 (2018).

    CAS  PubMed  Google Scholar 

  174. 174.

    Zhao, Y. G. et al. Regulates SERCA activity to control ER-isolation membrane contacts for autophagosome formation article the ER-localized transmembrane protein EPG-3 / VMP1 regulates SERCA activity to control ER-isolation membrane contacts for autophagosome formation. Mol. Cell 67, 974–989.e6 (2017).

    CAS  PubMed  Google Scholar 

  175. 175.

    Bodemann, B. O. et al. RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly. Cell 144, 253–267 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  176. 176.

    Suzuki, S. W. et al. Atg13 HORMA domain recruits Atg9 vesicles during autophagosome formation. Proc. Natl Acad. Sci. USA 112, 3350–3355 (2015).

    CAS  PubMed  Google Scholar 

  177. 177.

    Sekito, T., Kawamata, T., Ichikawa, R., Suzuki, K. & Ohsumi, Y. Atg17 recruits Atg9 to organize the pre-autophagosomal structure. Genes. Cell 14, 525–538 (2009).

    CAS  Google Scholar 

  178. 178.

    He, C. et al. Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast. J. Cell Biol. 175, 925–935 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  179. 179.

    Itakura, E., Kishi-Itakura, C., Koyama-Honda, I. & Mizushima, N. Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy. J. Cell Sci. 125, 1488–1499 (2012).

    CAS  PubMed  Google Scholar 

  180. 180.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  181. 181.

    Ravikumar, B., Moreau, K., Jahreiss, L., Puri, C. & Rubinsztein, D. C. Plasma membrane contributes to the formation of pre-autophagosomal structures. Nat. Cell Biol. 12, 747–757 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  182. 182.

    Moreau, K., Ravikumar, B., Renna, M., Puri, C. & Rubinsztein, D. C. Autophagosome precursor maturation requires homotypic fusion. Cell 146, 303–317 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  183. 183.

    Knævelsrud, H. et al. Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. J. Cell Biol. 202, 331–349 (2013).

    PubMed  PubMed Central  Google Scholar 

  184. 184.

    Judith, D. et al. ATG9A shapes the forming autophagosome through Arfaptin 2 and phosphatidylinositol 4-kinase IIIβ. J. Cell Biol. 218, 1634–1652 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  185. 185.

    Suzuki, K., Kubota, Y., Sekito, T. & Ohsumi, Y. Hierarchy of Atg proteins in pre-autophagosomal structure organization. Genes. Cell 12, 209–218 (2007).

    CAS  Google Scholar 

  186. 186.

    Puri, C. et al. The RAB11A-positive compartment is a primary platform for autophagosome assembly mediated by WIPI2 recognition of PI3P-RAB11A. Dev. Cell 45, 114–131 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  187. 187.

    Sakoh-Nakatogawa, M. et al. Atg12-Atg5 conjugate enhances E2 activity of Atg3 by rearranging its catalytic site. Nat. Struct. Mol. Biol. 20, 433–439 (2013).

    CAS  PubMed  Google Scholar 

  188. 188.

    Fujita, N. et al. Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin. J. Cell Biol. 203, 115–128 (2013).

    PubMed  PubMed Central  Google Scholar 

  189. 189.

    Nishimura, T. et al. FIP200 regulates targeting of Atg16L1 to the isolation membrane. EMBO Rep. 14, 284–291 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  190. 190.

    Harada, K. et al. Two distinct mechanisms target the autophagy-related E3 complex to the pre-autophagosomal structure. eLife 8, e43088 (2019).

    PubMed  PubMed Central  Google Scholar 

  191. 191.

    Kaminska, J. et al. Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress. Int. J. Biochem. Cell Biol. 79, 494–504 (2016).

    CAS  PubMed  Google Scholar 

  192. 192.

    Gómez-Sánchez, R. et al. Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores. J. Cell Biol. 217, 2743–2763 (2018).

    PubMed  PubMed Central  Google Scholar 

  193. 193.

    Tang, Z. et al. TOM40 targets Atg2 to mitochondria-associated ER membranes for phagophore expansion. Cell Rep. 28, 1744–1757 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  194. 194.

    Lin, M. G., Schöneberg, J., Davies, C. W., Ren, X. & Hurley, J. H. The dynamic Atg13-free conformation of the Atg1 EAT domain is required for phagophore expansion. Mol. Biol. Cell 29, 1228–1237 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  195. 195.

    Stanga, D. et al. TRAPPC11 functions in autophagy by recruiting ATG2B-WIPI4/WDR45 to preautophagosomal membranes. Traffic 20, 325–345 (2019).

    CAS  PubMed  Google Scholar 

  196. 196.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  197. 197.

    Nakatogawa, H., Ichimura, Y. & Ohsumi, Y. Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. Cell 130, 165–178 (2007).

    CAS  PubMed  Google Scholar 

  198. 198.

    Weidberg, H. et al. LC3 and GATE-16 N termini mediate membrane fusion processes required for autophagosome biogenesis. Dev. Cell 20, 444–454 (2011).

    CAS  PubMed  Google Scholar 

  199. 199.

    Wu, F. et al. Structural basis of the differential function of the two C. elegans Atg8 homologs, LGG-1 and LGG-2, in autophagy. Mol. Cell 60, 914–929 (2015).

    CAS  PubMed  Google Scholar 

  200. 200.

    Xie, Z., Nair, U. & Klionsky, D. J. Atg8 controls phagophore expansion during autophagosome formation. Mol. Biol. Cell 19, 3290–3298 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  201. 201.

    Nair, U. et al. SNARE proteins are required for macroautophagy. Cell 146, 290–302 (2012).

    Google Scholar 

  202. 202.

    Kraft, C. et al. Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy. EMBO J. 31, 3691–3703 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  203. 203.

    Nakatogawa, H. et al. The autophagy-related protein kinase Atg1 interacts with the ubiquitin-like protein Atg8 via the Atg8 family interacting motif to facilitate autophagosome formation. J. Biol. Chem. 287, 28503–28507 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  204. 204.

    Alemu, E. A. et al. ATG8 family proteins act as scaffolds for assembly of the ULK complex: sequence requirements for LC3-interacting region (LIR) motifs. J. Biol. Chem. 287, 39275–39290 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  205. 205.

    Herhaus, L. et al. TBK1-mediated phosphorylation of LC3C and GABARAP-L2 controls autophagosome shedding by ATG4 protease. EMBO Rep. 21, e48317 (2020).

    CAS  PubMed  Google Scholar 

  206. 206.

    Scherz-Shouval, R. et al. Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO J. 26, 1749–1760 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  207. 207.

    Sakoh-Nakatogawa, M., Kirisako, H., Nakatogawa, H. & Ohsumi, Y. Localization of Atg3 to autophagy-related membranes and its enhancement by the Atg8-family interacting motif to promote expansion of the membranes. FEBS Lett. 589, 744–749 (2015).

    CAS  PubMed  Google Scholar 

  208. 208.

    Ngu, M., Hirata, E. & Suzuki, K. Visualization of Atg3 during autophagosome formation in Saccharomyces cerevisiae. J. Biol. Chem. 290, 8146–8153 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  209. 209.

    Weidberg, H. et al. LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis. EMBO J. 29, 1792–1802 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  210. 210.

    Sou, Y. et al. The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice. Mol. Biol. Cell 19, 4762–4775 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  211. 211.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  212. 212.

    Tsuboyama, K. et al. The ATG conjugation systems are important for degradation of the inner autophagosomal membrane. Science 354, 1036–1041 (2016).

    CAS  PubMed  Google Scholar 

  213. 213.

    Nguyen, T. N. et al. Atg8 family LC3/GAB ARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation. J. Cell Biol. 215, 857–874 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  214. 214.

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

    CAS  PubMed  Google Scholar 

  215. 215.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  216. 216.

    Lamb, C. A., Yoshimori, T. & Tooze, S. A. The autophagosome: origins unknown, biogenesis complex. Nat. Rev. Mol. Cell Biol. 14, 759–774 (2013).

    CAS  PubMed  Google Scholar 

  217. 217.

    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).

    PubMed  Google Scholar 

  218. 218.

    Uemura, T. et al. A cluster of thin tubular structures mediates transformation of the endoplasmic reticulum to autophagic isolation membrane. Mol. Cell. Biol. 34, 1695–1706 (2014). This work and Axe et al. (2008), Hayashi-Nishino et al. (2009) and Ylä-Anttila et al. (2009) together reveal a direct connection of the isolation membrane to the ER via the omegasome/IMATs.

    PubMed  PubMed Central  Google Scholar 

  219. 219.

    Baba, M. et al. A nuclear membrane-derived structure associated with Atg8 is involved in the sequestration of selective cargo, the Cvt complex, during autophagosome formation in yeast. Autophagy 15, 423–437 (2019).

    CAS  PubMed  Google Scholar 

  220. 220.

    Kotani, T., Kirisako, H., Koizumi, M., Ohsumi, Y. & Nakatogawa, H. The Atg2-Atg18 complex tethers pre-autophagosomal membranes to the endoplasmic reticulum for autophagosome formation. Proc. Natl Acad. Sci. USA 115, 10363–10368 (2018).

    CAS  PubMed  Google Scholar 

  221. 221.

    Valverde, D. P. et al. ATG2 transports lipids to promote autophagosome biogenesis. J. Cell Biol. 218, 1787–1798 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  222. 222.

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

    CAS  PubMed  Google Scholar 

  223. 223.

    Tamura, N. et al. Differential requirement for ATG2A domains for localization to autophagic membranes and lipid droplets. FEBS Lett. 591, 3819–3830 (2017).

    CAS  PubMed  Google Scholar 

  224. 224.

    Maeda, S., Otomo, C. & Otomo, T. The autophagic membrane tether ATG2A transfers lipids between membranes. eLife 8, e45777 (2019). This work and Chowdhury et al. (2018), Gómez-Sánchez et al. (2018), Kotani et al. (2018), Valverde et al. (2019) and Osawa et al. (2019) together reveal the membrane-tethering and lipid transfer functions of Atg2/ATG2.

    PubMed  PubMed Central  Google Scholar 

  225. 225.

    Osawa, T., Ishii, Y. & Noda, N. N. Human ATG2B possesses a lipid transfer activity which is accelerated by negatively charged lipids and WIPI4. Genes to Cells 25, 65–70 (2020).

    CAS  PubMed  Google Scholar 

  226. 226.

    Baba, M., Ohsumi, Y. & Osumi, M. Analysis of the membrane structures involved in autophagy in yeast by freeze-replica method. Cell Struct. Funct. 20, 465–471 (1995).

    CAS  PubMed  Google Scholar 

  227. 227.

    Fengsrud, M., Erichsen, E. S., Berg, T. O., Raiborg, C. & Seglen, P. O. Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy. Eur. J. Cell Biol. 79, 871–882 (2000).

    CAS  PubMed  Google Scholar 

  228. 228.

    Ishihara, N. et al. Autophagosome requires specific early Sec proteins for its formation and NSF/SNARE for vacuolar fusion. Mol. Biol. Cell 12, 3690–3702 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  229. 229.

    Shima, T., Kirisako, H. & Nakatogawa, H. COPII vesicles contribute to autophagosomal membranes. J. Cell Biol. 218, 1503–1510 (2019). This work and Ge et al. (2017), Suzuki et al. (2013) and Graef et al. (2013) report the involvement of COPII vesicles in autophagosome biogenesis.

    CAS  PubMed  PubMed Central  Google Scholar 

  230. 230.

    Lynch-Day, M. A. et al. Trs85 directs a Ypt1 GEF, TRAPPIII, to the phagophore to promote autophagy. Proc. Natl Acad. Sci. USA 107, 7811–7816 (2010).

    CAS  PubMed  Google Scholar 

  231. 231.

    Wang, J. et al. Ypt1 recruits the Atg1 kinase to the preautophagosomal structure. Proc. Natl Acad. Sci. USA 110, 9800–9805 (2013).

    CAS  PubMed  Google Scholar 

  232. 232.

    Lipatova, Z. et al. Regulation of selective autophagy onset by a Ypt/Rab GTPase module. Proc. Natl Acad. Sci. USA 109, 6981–6986 (2012).

    CAS  PubMed  Google Scholar 

  233. 233.

    Davis, S. et al. Sec24 phosphorylation regulates autophagosome abundance during nutrient deprivation. eLife 5, 1–22 (2016).

    Google Scholar 

  234. 234.

    Wang, J. et al. Ypt1/Rab1 regulates Hrr25/CK1δ kinase activity in ER-Golgi traffic and macroautophagy. J. Cell Biol. 210, 273–285 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  235. 235.

    Tan, D. et al. The EM structure of the TRAPPIII complex leads to the identification of a requirement for COPII vesicles on the macroautophagy pathway. Proc. Natl Acad. Sci. USA 110, 19432–19437 (2013).

    CAS  PubMed  Google Scholar 

  236. 236.

    Abada, A., Levin-Zaidman, S., Porat, Z., Dadosh, T. & Elazar, Z. SNARE priming is essential for maturation of autophagosomes but not for their formation. Proc. Natl Acad. Sci. USA 114, 12749–12754 (2017).

    CAS  PubMed  Google Scholar 

  237. 237.

    Ogasawara, Y. et al. Stearoyl-CoA desaturase 1 activity is required for autophagosome formation. J. Biol. Chem. 289, 23938–23950 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  238. 238.

    Ogasawara, Y., Kira, S., Mukai, Y., Noda, T. & Yamamoto, A. Ole1, fatty acid desaturase, is required for Atg9 delivery and isolation membrane expansion during autophagy in Saccharomyces cerevisiae. Biol. Open 6, 35–40 (2017).

    CAS  PubMed  Google Scholar 

  239. 239.

    Andrejeva, G. et al. De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy. Autophagy https://doi.org/10.1080/15548627.2019.1659608 (2019).

    Article  PubMed  Google Scholar 

  240. 240.

    Schütter, M., Giavalisco, P., Brodesser, S. & Graef, M. Local fatty acid channeling into phospholipid synthesis drives phagophore expansion during autophagy. Cell 180, 135–149 (2020).

    PubMed  Google Scholar 

  241. 241.

    Biazik, J., Ylä-Anttila, P., Vihinen, H., Jokitalo, E. & Eskelinen, E. L. Ultrastructural relationship of the phagophore with surrounding organelles. Autophagy 11, 439–451 (2015).

    PubMed  PubMed Central  Google Scholar 

  242. 242.

    Shpilka, T. et al. Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis. EMBO J. 34, 2117–2131 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  243. 243.

    Li, D. et al. Storage lipid synthesis is necessary for autophagy induced by nitrogen starvation. FEBS Lett. 589, 269–276 (2015).

    CAS  PubMed  Google Scholar 

  244. 244.

    Velázquez, A. P., Tatsuta, T., Ghillebert, R., Drescher, I. & Graef, M. Lipid droplet-mediated ER homeostasis regulates autophagy and cell survival during starvation. J. Cell Biol. 212, 621–631 (2016).

    PubMed  PubMed Central  Google Scholar 

  245. 245.

    Nguyen, N., Shteyn, V. & Melia, T. J. Sensing membrane curvature in macroautophagy. J. Mol. Biol. 429, 457–472 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  246. 246.

    Romanov, J. et al. Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation. EMBO J. 31, 4304–4317 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  247. 247.

    Kaufmann, A., Beier, V., Franquelim, H. G. & Wollert, T. Molecular mechanism of autophagic membrane-scaffold assembly and disassembly. Cell 156, 469–481 (2014).

    CAS  PubMed  Google Scholar 

  248. 248.

    Knorr, R. L. et al. Membrane morphology is actively transformed by covalent binding of the protein Atg8 to PE-lipids. PLoS One 9, e115357 (2014).

    PubMed  PubMed Central  Google Scholar 

  249. 249.

    Nath, S. et al. Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3. Nat. Cell Biol. 16, 415–424 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  250. 250.

    Monastyrska, I., Rieter, E., Klionsky, D. J. & Reggiori, F. Multiple roles of the cytoskeleton in autophagy. Biol. Rev. 84, 431–448 (2009).

    PubMed  Google Scholar 

  251. 251.

    Mi, N. et al. CapZ regulates autophagosomal membrane shaping by promoting actin assembly inside the isolation membrane. Nat. Cell Biol. 17, 1112–1123 (2015).

    CAS  PubMed  Google Scholar 

  252. 252.

    Kast, D. J., Zajac, A. L., Holzbaur, E. L. F., Ostap, E. M. & Dominguez, R. WHAMM directs the Arp2/3 complex to the ER for autophagosome biogenesis through an actin comet tail mechanism. Curr. Biol. 25, 1791–1797 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  253. 253.

    Kaksonen, M., Toret, C. P. & Drubin, D. G. Harnessing actin dynamics for clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 7, 404–414 (2006).

    CAS  PubMed  Google Scholar 

  254. 254.

    Knorr, R. L., Dimova, R. & Lipowsky, R. Curvature of double-membrane organelles generated by changes in membrane size and composition. PLoS One 7, e32753 (2012). This study proposes that the expanding isolation membrane bends into a spherical shape on the basis of the physical properties of the lipid bilayer.

    CAS  PubMed  PubMed Central  Google Scholar 

  255. 255.

    Nice, D. C., Sato, T. K., Stromhaug, P. E., Emr, S. D. & Klionsky, D. J. Cooperative binding of the cytoplasm to vacuole targeting pathway proteins, Cvt13 and Cvt20, to phosphatidylinositol 3-phosphate at the pre-autophagosomal structure is required for selective autophagy. J. Biol. Chem. 277, 30198–30207 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  256. 256.

    Zhao, D. et al. Atg20- and Atg24-family proteins promote organelle autophagy in fission yeast. J. Cell Sci. 129, 4289–4304 (2016).

    CAS  PubMed  Google Scholar 

  257. 257.

    Kanki, T. & Klionsky, D. J. Mitophagy in yeast occurs through a selective mechanism. J. Biol. Chem. 283, 32386–32393 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  258. 258.

    Knorr, R. L., Lipowsky, R. & Dimova, R. Autophagosome closure requires membrane scission. Autophagy 11, 2134–2137 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  259. 259.

    Vietri, M., Radulovic, M. & Stenmark, H. The many functions of ESCRTs. Nat. Rev. Mol. Cell Biol. 21, 25–42 (2019).

    PubMed  Google Scholar 

  260. 260.

    Takahashi, Y. et al. An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure. Nat. Commun. 9, 2855 (2018).

    PubMed  PubMed Central  Google Scholar 

  261. 261.

    Zhen, Y. et al. ESCRT-mediated phagophore sealing during mitophagy. Autophagy 16, 826–841 (2019).

    PubMed  PubMed Central  Google Scholar 

  262. 262.

    Takahashi, Y. et al. VPS37A directs ESCRT recruitment for phagophore closure. J. Cell Biol. 218, 3336–3354 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  263. 263.

    Zhou, F. et al. Rab5-dependent autophagosome closure by ESCRT. J. Cell Biol. 218, 1908–1927 (2019). This work and Takahashi et al. (2018), Zhen et al. (2019) and Takahashi et al. (2019) propose that the ESCRT machinery is involved in isolation membrane pore closure.

    CAS  PubMed  PubMed Central  Google Scholar 

  264. 264.

    Backues, S. K., Chen, D., Ruan, J., Xie, Z. & Klionsky, D. J. Estimating the size and number of autophagic bodies by electron microscopy. Autophagy 10, 155–164 (2014).

    CAS  PubMed  Google Scholar 

  265. 265.

    Lamb, C. A., Longatti, A. & Tooze, S. A. Rabs and GAPs in starvation-induced autophagy. Small GTPases 7, 265–269 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  266. 266.

    Itoh, T. & Fukuda, M. Roles of Rab-GAPs in regulating autophagy. in Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging (ed. Hayat, M. A.) Ch. 6, 143–157 (Elsevier, 2017).

  267. 267.

    Martens, S., Nakamura, S. & Yoshimori, T. Phospholipids in autophagosome formation and fusion. J. Mol. Biol. 428, 4819–4827 (2016).

    CAS  Google Scholar 

  268. 268.

    Dall’Armi, C., Devereaux, K. A. & Di Paolo, G. The role of lipids in the control of autophagy. Curr. Biol. 23, R33–R45 (2013).

    PubMed  PubMed Central  Google Scholar 

  269. 269.

    Itakura, E., Kishi-Itakura, C. & Mizushima, N. The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell 151, 1256–1269 (2012).

    CAS  PubMed  Google Scholar 

  270. 270.

    Matsui, T. et al. Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17. J. Cell Biol. 217, 2633–2645 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  271. 271.

    Gao, J., Reggiori, F. & Ungermann, C. A novel in vitro assay reveals SNARE topology and the role of Ykt6 in autophagosome fusion with vacuoles. J. Cell Biol. 217, 3670–3682 (2018).

    PubMed  PubMed Central  Google Scholar 

  272. 272.

    Licheva, M. et al. Reconstitution reveals Ykt6 as the autophagosomal SNARE in autophagosome–vacuole fusion. J. Cell Biol. 217, 3656–3669 (2018).

    PubMed  PubMed Central  Google Scholar 

  273. 273.

    Kimura, S., Noda, T. & Yoshimori, T. Dynein-dependent movement of autophagosomes mediates efficient encounters with lysosomes. Cell Struct. Funct. 33, 109–122 (2008).

    CAS  PubMed  Google Scholar 

  274. 274.

    Johansson, M. et al. Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-p150Glued, ORP1L, and the receptor βIII spectrin. J. Cell Biol. 176, 459–471 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  275. 275.

    Jordens, I. et al. The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors. Curr. Biol. 11, 1680–1685 (2001).

    CAS  PubMed  Google Scholar 

  276. 276.

    Wijdeven, R. H. et al. Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway. Nat. Commun. 7, 11808 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  277. 277.

    Takáts, S. et al. Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila. Mol. Biol. Cell 25, 1338–1354 (2014).

    PubMed  PubMed Central  Google Scholar 

  278. 278.

    Takáts, S. et al. Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion. PLoS Genet. 14, e1007359 (2018).

    PubMed  PubMed Central  Google Scholar 

  279. 279.

    Bas, L. et al. Reconstitution reveals Ykt6 as the autophagosomal SNARE in autophagosome-vacuole fusion. J. Cell Biol. 217, 3656–3669 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  280. 280.

    Jiang, P. et al. The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin 17. Mol. Biol. Cell 25, 1327–1337 (2014).

    PubMed  PubMed Central  Google Scholar 

  281. 281.

    Wang, C. W., Stromhaug, P. E., Kauffman, E. J., Weisman, L. S. & Klionsky, D. J. Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during the tethering/docking stage. J. Cell Biol. 163, 973–985 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  282. 282.

    Gao, J., Langemeyer, L., Kümmel, D., Reggiori, F. & Ungermann, C. Molecular mechanism to target the endosomal Mon1-Ccz1 GEF complex to the pre-autophagosomal structure. eLife 7, e31145 (2018).

    PubMed  PubMed Central  Google Scholar 

  283. 283.

    McEwan, D. G. et al. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins. Mol. Cell 57, 39–54 (2015).

    CAS  PubMed  Google Scholar 

  284. 284.

    Tabata, K. et al. Rubicon and PLEKHM1 negatively regulate the endocytic/autophagic pathway via a novel Rab7-binding domain. Mol. Biol. Cell 21, 4162–4172 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  285. 285.

    Wang, Z. et al. The vici syndrome protein EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. Mol. Cell 63, 781–795 (2016).

    CAS  PubMed  Google Scholar 

  286. 286.

    Diao, J. et al. ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes. Nature 520, 563–566 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  287. 287.

    Chen, D. et al. A mammalian autophagosome maturation mechanism mediated by TECPR1 and the Atg12-Atg5 conjugate. Mol. Cell 45, 629–641 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  288. 288.

    Liu, X. et al. The Atg17-Atg31-Atg29 complex coordinates with Atg11 to recruit the Vam7 SNARE and mediate autophagosome-Vacuole fusion. Curr. Biol. 26, 150–160 (2016).

    PubMed  PubMed Central  Google Scholar 

  289. 289.

    Yu, L. et al. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 465, 942–946 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  290. 290.

    Rong, Y. et al. Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvation. Proc. Natl Acad. Sci. USA 108, 7826–7831 (2011).

    CAS  PubMed  Google Scholar 

  291. 291.

    Rong, Y. et al. Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation. Nat. Cell Biol. 14, 924–934 (2012).

    CAS  PubMed  Google Scholar 

  292. 292.

    Du, W. et al. Kinesin 1 drives autolysosome tubulation. Dev. Cell 37, 326–336 (2016).

    CAS  PubMed  Google Scholar 

  293. 293.

    Fernández, Á. F. et al. Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice. Nature 558, 136–140 (2018).

    PubMed  PubMed Central  Google Scholar 

  294. 294.

    Nakamura, S. et al. Suppression of autophagic activity by Rubicon is a signature of aging. Nat. Commun. 10, 847 (2019).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The author thanks several colleagues for helpful comments and apologizes to those whose work could not be cited due to lack of space. Research in the author’s group is supported in part by KAKENHI Grants-in-Aid for Scientific Research 17H01430 and 19H05708 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, JST CREST grant number JPMJCR13M7 and a STAR grant funded by the Tokyo Institute of Technology Foundation.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Hitoshi Nakatogawa.

Ethics declarations

Competing interests

The author declares no competing interests.

Additional information

Peer review information

Nature Reviews Molecular Cell Biology thanks F. Reggiori and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Glossary

Target of rapamycin complex 1

(TORC1). A serine/threonine kinase complex that controls various cellular activities, including autophagy, according to nutrient availability.

Recycling endosomes

Organelles that receive proteins from early endosomes and mediate their recycling to the plasma membrane.

Ubiquitin-like protein

Small protein similar to ubiquitin that is conjugated to the amino group of target molecules at their C termini.

E1 enzyme

Enzyme that activates the C-terminal carboxyl group of ubiquitin or ubiquitin-like proteins using ATP.

E2 enzyme

Enzyme that receives ubiquitin/ubiquitin-like proteins from E1 enzymes and conjugates them to target molecules directly or via E3 enzymes.

E3 enzyme

Enzyme that determines target specificity and stimulates or mediates the conjugation reaction by E2 enzymes.

cAMP-dependent protein kinase A

A protein kinase that is activated on increase in cAMP levels and regulates various metabolic enzymes.

AMP-activated protein kinase

(AMPK). A protein kinase that is activated in response to low ATP levels and acts to maintain cellular energy homeostasis.

Contact sites

Sites where two different organelles contact with each other for interorganellar communication, including transfer of molecules such as lipids.

Intrinsically disordered region

Protein region that does not adopt a defined 3D structure even under physiological conditions.

Exocyst complex

A multimeric protein complex that tethers secretory vesicles to the plasma membrane in exocytosis.

Early endosomes

Organelles that serve as a sorting platform for proteins endocytosed from the plasma membrane or transported from the Golgi apparatus.

SNARE

A family of proteins that mediate most membrane fusion events within cells. For fusion, SNAREs on two opposed membranes tightly associate with each other, bring the membranes in close proximity and induce their fusion.

Sorting nexin

A group of proteins that contain the PX and BAR domains and function in membrane trafficking.

ER exit sites

(ERES). A domain in the endoplasmic reticulum where COPII proteins are assembled to form COPII vesicles.

ER–Golgi intermediate compartment

(ERGIC). A membrane compartment that mediates vesicle transport between the endoplasmic reticulum and the Golgi apparatus.

Acyl-CoA

Coenzyme that functions in fatty acid metabolism; it serves as an acyl chain donor in phospholipid synthesis.

Giant unilamellar vesicles

(GUVs). Artificial micrometre-sized vesicles bound by a single membrane that are used as a model for analysis of cellular membranes.

Actin-capping protein

Protein that binds to the end of actin filaments and blocks the polymerization and depolymerization of the filaments at the end.

Multivesicular bodies

Endosome-related organelles that contain lumenal vesicles that mediate lysosomal degradation of membrane proteins or secrete these vesicles as exosomes.

ESCRT

(Endosomal sorting complexes required for transport). Protein complexes (ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III) that mediate protein sorting and intralumenal vesicle formation in the endosome and are also responsible for different membrane fission events. Their main activity is driving membrane constriction, and hence they can be involved in different processes requiring membrane shaping in the cell.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nakatogawa, H. Mechanisms governing autophagosome biogenesis. Nat Rev Mol Cell Biol 21, 439–458 (2020). https://doi.org/10.1038/s41580-020-0241-0

Download citation

Further reading