title: In vitro systems for Atg8 lipidation.
abstract: Macroautophagy is a major bulk degradation pathway for cytoplasmic material in eukaryotic cells. During macroautophagy, double membrane-bound organelles called autophagosomes are formed in a de novo manner. In the course of their formation autophagosomes capture cytoplasmic material, which is subsequently degraded upon fusion with the lysosomal system in complex eukaryotes or the vacuole in yeast. Several proteins are required for autophagosome formation. Among these are the components of two ubiquitin-like conjugation reactions that collectively mediate the conjugation of the ubiquitin-like Atg12 to the Atg5 protein and of the ubiquitin-like protein Atg8 to the headgroup of the membrane lipid phosphatidylethanolamine. The lipidated form of Atg8 is membrane-bound and marks the growing autophagosomal membrane as well as the completed autophagosome. Here we describe assays for the in vitro reconstitution of the Atg8 lipidation reaction using recombinantly expressed and purified proteins derived from Saccharomycescerevisiae in combination with small and giant unilamellar vesicles. The assays enable the study of the biochemical mechanisms of action of the Atg8 lipidation machinery and to analyze the impact of mutations and post-translational modifications of the conjugation machinery on Atg8 lipidation.
authors.affiliation: Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9/3, 1030 Vienna, Austria.
authors.forename: Bettina
authors.initials: B
authors.lastname: Zens
authors.affiliation: Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9/3, 1030 Vienna, Austria.
authors.forename: Justyna
authors.initials: J
authors.lastname: Sawa-Makarska
authors.affiliation: Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9/3, 1030 Vienna, Austria. Electronic address: sascha.martens@univie.ac.at.
authors.forename: Sascha
authors.initials: S
authors.lastname: Martens
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D001343:Autophagy / Q000235:genetics*; D000071190:Autophagy-Related Protein 8 Family; D002462:Cell Membrane / Q000378:metabolism; D066298:In Vitro Techniques; D050356:Lipid Metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D009154:Mutation; D010588:Phagosomes / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin
chemical_list: C113757:ATG8 protein, S cerevisiae; D048868:Adaptor Proteins, Signal Transducing; D000071190:Autophagy-Related Protein 8 Family; C498698:GABARAPL2 protein, human; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin
keywords: Atg12; Atg4; Atg8; Autophagosome; Autophagy; Protein conjugation