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Proc Natl Acad Sci U S A. 2014 Sep 2;111(35):12793-8. doi: 10.1073/pnas.1407214111. Epub 2014 Aug 19.

Assembly and dynamics of the autophagy-initiating Atg1 complex.

Author information

1
Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720; and.
2
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
3
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.
4
Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720; and jimhurley@berkeley.edu.

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.

KEYWORDS:

analytical ultracentrifugation; intrinsically disordered proteins; isothermal titration calorimetry; membrane tethering; protein structure

PMID:
25139988
PMCID:
PMC4156731
DOI:
10.1073/pnas.1407214111
[Indexed for MEDLINE]
Free PMC Article

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