title: Assembly and dynamics of the autophagy-initiating Atg1 complex.
abstract: The autophagy-related 1 (Atg1) complex of Saccharomyces cerevisiae has a central role in the initiation of autophagy following starvation and TORC1 inactivation. The complex consists of the protein kinase Atg1, the TORC1 substrate Atg13, and the trimeric Atg17-Atg31-Atg29 scaffolding subcomplex. Autophagy is triggered when Atg1 and Atg13 assemble with the trimeric scaffold. Here we show by hydrogen-deuterium exchange coupled to mass spectrometry that the mutually interacting Atg1 early autophagy targeting/tethering domain and the Atg13 central domain are highly dynamic in isolation but together form a stable complex with ∼ 100-nM affinity. The Atg1-Atg13 complex in turn binds as a unit to the Atg17-Atg31-Atg29 scaffold with ∼ 10-μM affinity via Atg13. The resulting complex consists primarily of a dimer of pentamers in solution. These results lead to a model for autophagy initiation in which Atg1 and Atg13 are tightly associated with one another and assemble transiently into the pentameric Atg1 complex during starvation.
authors.affiliation: Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720; and.
authors.forename: Goran
authors.initials: G
authors.lastname: Stjepanovic
authors.affiliation: Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720; and.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Davies
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Robin E
authors.initials: RE
authors.lastname: Stanley
authors.affiliation: Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720; and Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Ragusa
authors.affiliation: Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720; and.
authors.forename: Do Jin
authors.initials: DJ
authors.lastname: Kim
authors.affiliation: Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720; and jimhurley@berkeley.edu.
authors.forename: James H
authors.initials: JH
authors.lastname: Hurley
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001343:Autophagy / Q000502:physiology*; D000071183:Autophagy-Related Proteins; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D017353:Gene Deletion; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C413216:ATG13 protein, S cerevisiae; C506770:ATG29 protein, S cerevisiae; D048868:Adaptor Proteins, Signal Transducing; C505602:Atg17 protein, S cerevisiae; C520043:Atg31 protein, S cerevisiae; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C477823:ATG1 protein, S cerevisiae
keywords: analytical ultracentrifugation; intrinsically disordered proteins; isothermal titration calorimetry; membrane tethering; protein structure