Autophagy pp 119-133 | Cite as

Reconstituting Autophagy Initiation from Purified Components

  • Peter Mayrhofer
  • Thomas WollertEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)


The hallmark of macroautophagy is the de novo generation of a membrane structure that collects cytoplasmic material and delivers it to lysosomes for degradation. The nucleation of this precursor membrane, termed phagophore, involves the coordinated assembly of the Atg1-kinase complex and the recruitment of Atg9 vesicles. The latter represents one important membrane source in order to produce phagophores in vivo. We explain how the process of phagophore nucleation can be reconstituted from purified components in vitro. We describe the assembly of the ~500 kDa pentameric Atg1-kinase complex from its purified subunits. We also explain how Atg9-donor vesicles are generated in vitro to study the interaction of Atg9 and Atg1-kinase complexes by floatation experiments.

Key words

Autophagy Atg9 Atg1-kinase complex In vitro reconstitution Membrane tethering 


  1. 1.
    Reggiori F, Klionsky DJ (2005) Autophagosomes: biogenesis from scratch? Curr Opin Cell Biol 17(4):415–422CrossRefGoogle Scholar
  2. 2.
    Yang Z, Klionsky DJ (2010) Eaten alive: a history of macroautophagy. Nat Cell Biol 12(9):814–822. Scholar
  3. 3.
    Feng Y, He D, Yao Z, Klionsky DJ (2014) The machinery of macroautophagy. Cell Res 24(1):24–41. Scholar
  4. 4.
    Suzuki K, Kubota Y, Sekito T, Ohsumi Y (2007) Hierarchy of Atg proteins in pre- autophagosomal structure organization. Genes Cells 12(2):209–218. Scholar
  5. 5.
    Kawamata T, Kamada Y, Kabeya Y, Sekito T, Ohsumi Y (2008) Organization of the pre-autophagosomal structure responsible for autophagosome formation. Mol Biol Cell 19(5):2039–2050. Scholar
  6. 6.
    Sekito T, Kawamata T, Ichikawa R, Suzuki K, Ohsumi Y (2009) Atg17 recruits Atg9 to organize the pre-autophagosomal structure. Genes Cells 14(5):525–538. Scholar
  7. 7.
    Kabeya Y, Noda NN, Fujioka Y, Suzuki K, Inagaki F, Ohsumi Y (2009) Characterization of the Atg17-Atg29-Atg31 complex specifically required for starvation- induced autophagy in Saccharomyces cerevisiae. Biochem Biophys Res Commun 389(4):612–615. Scholar
  8. 8.
    Kamada Y, Yoshino K-I, Kondo C, Kawamata T, Oshiro N, Yonezawa K, Ohsumi Y (2010) Tor directly controls the Atg1 kinase complex to regulate autophagy. Mol Cell Biol 30(4):1049–1058. Scholar
  9. 9.
    Kabeya Y, Kamada Y, Baba M, Takikawa H, Sasaki M, Ohsumi Y (2005) Atg17 functions in cooperation with Atg1 and Atg13 in yeast autophagy. Mol Biol Cell 16(5):2544–2553. Scholar
  10. 10.
    Kamada Y (2010) Prime-numbered Atg proteins act at the primary step in autophagy: unphosphorylatable Atg13 can induce autophagy without TOR inactivation. Autophagy 6(3):415–416CrossRefGoogle Scholar
  11. 11.
    Fujioka Y, Suzuki SW, Yamamoto H, Kondo-Kakuta C, Kimura Y, Hirano H et al (2014) Structural basis of starvation-induced assembly of the autophagy initiation complex. Nat Struct Mol Biol 21(6):513–521. Scholar
  12. 12.
    Mari M, Griffith J, Rieter E, Krishnappa L, Klionsky DJ, Reggiori F (2010) An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis. J Cell Biol 190(6):1005–1022. Scholar
  13. 13.
    Yamamoto H, Kakuta S, Watanabe TM, Kitamura A, Sekito T, Kondo-Kakuta C et al (2012) Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J Cell Biol 198(2):219–233. Scholar
  14. 14.
    Nair U, Jotwani A, Geng J, Gammoh N, Richerson D, Yen WL et al (2011) SNARE proteins are required for macroautophagy. Cell 146(2):290–302. Scholar
  15. 15.
    Rao Y, Perna MG, Hofmann B, Beier V, Wollert T (2016b) The Atg1-kinase complex tethers Atg9-vesicles to initiate autophagy. Nat Commun 7:10338. Scholar
  16. 16.
    Matscheko N, Mayrhofer P, Wollert T (2017) Passing membranes to autophagy: unconventional membrane tethering by Atg17. Autophagy 13(3):629–630. Scholar
  17. 17.
    Rao Y, Matscheko N, Wollert T (2016a) Autophagy in the test tube in vitro reconstitution of aspects of autophagosome biogenesis. FEBS J 283(11):2034–2043. Scholar
  18. 18.
    Tan S (2001) A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli. Protein Expr Purif 21(1):224–234. Scholar
  19. 19.
    Scholz J, Besir H, Strasser C, Suppmann S (2013) A new method to customize protein expression vectors for fast, efficient and background free parallel cloning. BMC Biotechnol 13(1):12. Scholar
  20. 20.
    Kijanska M, Dohnal I, Reiter W, Kaspar S, Stoffel I, Ammerer G et al (2010) Activation of Atg1 kinase in autophagy by regulated phosphorylation. Autophagy 6(8):1168–1178. Scholar
  21. 21.
    Bieniossek C, Richmond TJ, Berger I (2001) MultiBac: multigene baculovirus-based eukaryotic protein complex production, vol 21. John Wiley & Sons, Inc., Hoboken, NJ, pp 5.20.1–5.20.26. Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Unit Membrane Biochemistry and TransportInstitut PasteurParisFrance

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