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© Thomas Wollert
Publication : Methods in molecular biology (Clifton, N.J.)

Reconstituting Autophagy Initiation from Purified Components.

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Methods in molecular biology (Clifton, N.J.) - 01 Jan 2019

Mayrhofer P, Wollert T,

Link to Pubmed [PMID] – 30610692

Link to DOI – 10.1007/978-1-4939-8873-0_6

Methods Mol Biol 2019 ; 1880(): 119-133

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.

https://pubmed.ncbi.nlm.nih.gov/30610692