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Autophagy pp 119-133 | Cite as

Reconstituting Autophagy Initiation from Purified Components

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

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.

Key words

Autophagy Atg9 Atg1-kinase complex In vitro reconstitution Membrane tethering 

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Copyright information

© 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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