title: In Vitro Reconstitution of Atg8 Conjugation and Deconjugation.
abstract: Macroautophagy, hereafter autophagy, is a major degradation pathway in eukaryotic systems that allows the removal of large intracellular structures such as entire organelles or protein aggregates, thus contributing to the homeostasis of cells and tissues. Autophagy entails the de novo formation of an organelle termed autophagosome, where a cup-shaped structure called isolation membrane nucleates in proximity of a cytoplasmic cargo material. Upon elongation and closure of isolation membranes, the mature autophagosome delivers the sequestered cargo into the lysosomal system for degradation. Among the factors for autophagosome formation are the autophagy-related (Atg) proteins belonging to the Atg8 conjugation system. In this system, the ubiquitin-like Atg8 protein is conjugated to the membrane lipid phosphatidylethanolamine present in autophagosomal membranes. Atg8 can also be removed from membranes by Atg4-mediated deconjugation. Here, we describe in vitro systems that recapitulate the enzymatic reactions occurring in vivo by presenting expression and purification strategies for all the components of the Saccharomyces cerevisiae Atg8 conjugation system. We also present protocols for in vitro Atg8 conjugation and deconjugation reactions employing small and giant unilamellar vesicles.
authors.affiliation: Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Vienna, Austria.
authors.forename: D
authors.initials: D
authors.lastname: Fracchiolla
authors.affiliation: Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Vienna, Austria.
authors.forename: B
authors.initials: B
authors.lastname: Zens
authors.affiliation: Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Vienna, Austria. Electronic address: sascha.martens@univie.ac.at.
authors.forename: S
authors.initials: S
authors.lastname: Martens
mesh_terms: D001343:Autophagy; D000071192:Autophagy-Related Protein 12 / Q000378:metabolism; D000071187:Autophagy-Related Protein 5 / Q000378:metabolism; D000071193:Autophagy-Related Protein 7 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000071190:Autophagy-Related Protein 8 Family / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071183:Autophagy-Related Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D046912:Multiprotein Complexes; D010714:Phosphatidylethanolamines / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000737:chemistry* / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D000071192:Autophagy-Related Protein 12; D000071187:Autophagy-Related Protein 5; D000071193:Autophagy-Related Protein 7; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D010714:Phosphatidylethanolamines; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D053835:Unilamellar Liposomes; D000097573:Red Fluorescent Protein; C491969:ATG12 protein, S cerevisiae; C498649:ATG16 protein, S cerevisiae; C102268:ATG5 protein, S cerevisiae; C114830:ATG7 protein, S cerevisiae; C113757:ATG8 protein, S cerevisiae; C113755:Atg4 protein, S cerevisiae; C483858:phosphatidylethanolamine; C104235:ATG3 protein, S cerevisiae
keywords: Atg8 lipidation; Autophagy; GUVs; Isolation membrane; Phosphatidylethanolamine; Reconstitution; SUVs; Ubiquitin-like system