title: Detection of Saccharomyces cerevisiae Atg13 by western blot.
abstract: The proteins that comprise the Atg1 kinase complex constitute a key set of components that participate in macroautophagy (hereafter autophagy). Among these proteins, Atg13 plays a particularly important, although as yet undefined role, in that it is critical for the proper localization of Atg1 to the phagophore assembly site (PAS) and its efficient kinase activity. Atg13 is hyperphosphorylated in vegetative conditions when autophagy occurs at a basal level, and is largely dephosphorylated upon the induction of autophagy. Inhibitory phosphorylation of Atg13 reflects the activity of TOR complex 1 (TORC1) and protein kinase A. Accordingly, monitoring the phosphorylation state of Atg13 provides a convenient way to follow early steps of autophagy induction as well as the activity of some of the upstream nutrient-sensing kinases. However, the detection of Atg13 by western blot can be problematic. Here, we present a detailed protocol for sample preparation and detection of the Atg13 protein from yeast.
authors.affiliation: Cell and Molecular Neuroscience Unit; Instituto de Medicina Molecular; Lisboa, Portugal.
authors.forename: Leonor
authors.initials: L
authors.lastname: Miller-Fleming
authors.affiliation: Department of Cancer Biology and Genetics; Memorial Sloan-Kettering Cancer Center; New York, NY USA.
authors.forename: Heesun
authors.initials: H
authors.lastname: Cheong
authors.affiliation: Cell and Molecular Neuroscience Unit; Instituto de Medicina Molecular; Lisboa, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Antas
authors.affiliation: Life Sciences Institute and Departments of Molecular, Cellular, and Developmental Biology, and Biological Chemistry; University of Michigan; Ann Arbor, MI USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Klionsky
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D001343:Autophagy / Q000502:physiology*; D000071183:Autophagy-Related Proteins; D015153:Blotting, Western; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: C413216:ATG13 protein, S cerevisiae; D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; D029701:Saccharomyces cerevisiae Proteins; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: Atg13; TORC1; autophagy; vacuole; yeast