Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Reggiori F, Klionsky DJ (2005) Autophagosomes: biogenesis from scratch? Curr Opin Cell Biol 17(4):415–422
Yang Z, Klionsky DJ (2010) Eaten alive: a history of macroautophagy. Nat Cell Biol 12(9):814–822. https://doi.org/10.1038/ncb0910-814
Feng Y, He D, Yao Z, Klionsky DJ (2014) The machinery of macroautophagy. Cell Res 24(1):24–41. https://doi.org/10.1038/cr.2013.168
Suzuki K, Kubota Y, Sekito T, Ohsumi Y (2007) Hierarchy of Atg proteins in pre- autophagosomal structure organization. Genes Cells 12(2):209–218. https://doi.org/10.1111/j.1365-2443.2007.01050.x
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. https://doi.org/10.1091/mbc.E07-10-1048
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. https://doi.org/10.1111/j.1365-2443.2009.01299.x
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. https://doi.org/10.1016/j.bbrc.2009.09.034
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. https://doi.org/10.1128/MCB.01344-09
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. https://doi.org/10.1091/mbc.E04-08-0669
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):415–416
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. https://doi.org/10.1038/nsmb.2822
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. https://doi.org/10.1083/jcb.200912089
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. https://doi.org/10.1083/jcb.201202061
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. https://doi.org/10.1016/j.cell.2011.06.022
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. https://doi.org/10.1038/ncomms10338
Matscheko N, Mayrhofer P, Wollert T (2017) Passing membranes to autophagy: unconventional membrane tethering by Atg17. Autophagy 13(3):629–630. https://doi.org/10.1080/15548627.2016.1276678
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. https://doi.org/10.1111/febs.13661
Tan S (2001) A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli. Protein Expr Purif 21(1):224–234. https://doi.org/10.1006/prep.2000.1363
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. https://doi.org/10.1186/1472-6750-13-12
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. https://doi.org/10.4161/auto.6.8.13849
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. https://doi.org/10.1002/0471140864.ps0520s51
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Mayrhofer, P., Wollert, T. (2019). Reconstituting Autophagy Initiation from Purified Components. In: Ktistakis, N., Florey, O. (eds) Autophagy. Methods in Molecular Biology, vol 1880. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8873-0_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8873-0_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8872-3
Online ISBN: 978-1-4939-8873-0
eBook Packages: Springer Protocols