title: SDS-PAGE techniques to study ubiquitin-like conjugation systems in yeast autophagy.
abstract: Autophagy is a bulk degradation system highly conserved among eukaryotic cells and plays crucial roles in a wide range of physiological and pathological situations. Remarkably, this process involves two ubiquitin-like (Ubl) conjugation systems. Here, we describe two sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques to analyze these systems: one that allows separation of the Ubl protein Atg8 conjugated to the lipid phosphatidylethanolamine from its unlipidated form, and the other by which otherwise labile thioester intermediates between Atg8 and either the E1 enzyme Atg7 or the E2 enzyme Atg3 are stably preserved during electrophoresis, and thus easily detected by following protein visualization. Especially, the latter technique is also ubiquitously applicable for studies on conjugation reactions of ubiquitin (Ub) and other Ubl proteins.
authors.affiliation: Frontier Research Center, Tokyo Institute of Technology, Yokohama, Japan. hnakatogawa@iri.titech.ac.jp
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nakatogawa
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Ohsumi
mesh_terms: D001343:Autophagy; D000071193:Autophagy-Related Protein 7; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism
chemical_list: C114830:ATG7 protein, S cerevisiae; C113757:ATG8 protein, S cerevisiae; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D008869:Microtubule-Associated Proteins; D010714:Phosphatidylethanolamines; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D014452:Ubiquitins; C483858:phosphatidylethanolamine; D044763:Ubiquitin-Conjugating Enzymes; D000071193:Autophagy-Related Protein 7; C104235:ATG3 protein, S cerevisiae