In vitro systems for Atg8 lipidation.
Bottom Line: 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.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.
Affiliation: Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9/3, 1030 Vienna, Austria.Show MeSH
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Mentions: The following lipid mix was used: 40% DOPC, 35% DOPS, 20% DOPE, 5% PI3P. 2% Marina Blue (Invitrogen) at the expense of DOPC was added to the membrane labeled GUVs shown in Fig. 4. All lipids were purchased from Avanti Polar Lipids Inc. Marina blue PE was from Invitrogen.
Affiliation: Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9/3, 1030 Vienna, Austria.