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Methods Enzymol ; 587: 227-246, 2017.
Article in English | MEDLINE | ID: mdl-28253958

ABSTRACT

Autophagosome formation and specific substrate recruitment during autophagy require ligation of the ubiquitin-like protein (UBL) Atg8 to the head group of the lipid phosphatidylethanolamine. Atg8 lipidation is mediated by distinctive UBL cascades involving autophagy-specific E1, E2, and E3 enzymes that differ substantially in sequence from components of other UBL conjugation cascades. Structural studies are important for elucidating the roles of Atg proteins that regulate multiple steps involved in autophagy. This chapter describes methods to prepare and crystallize selected proteins and complexes involved in autophagy UBL conjugation pathways, as a guide for strategies for structural and biochemical characterization of Atg proteins.


Subject(s)
Autophagy-Related Proteins/chemistry , Autophagy-Related Proteins/metabolism , Crystallography/methods , Autophagy , Autophagy-Related Protein 5/chemistry , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 5/isolation & purification , Autophagy-Related Protein 7/chemistry , Autophagy-Related Protein 7/metabolism , Autophagy-Related Proteins/isolation & purification , Crystallization/methods , Humans , Mutation , Protein Domains , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases
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