Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 111, Issue 35, Pages 12793-12798Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1407214111
Keywords
analytical ultracentrifugation; isothermal titration calorimetry; membrane tethering; intrinsically disordered proteins; protein structure
Categories
Funding
- National Institutes of Health [GM111730]
- Ruth Kirschstein National Research Service Award [GM112301, GM099319]
- Damon Runyon Cancer Research Fellowship
- L'Oreal USA Women in Science Fellowship
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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 similar to 100-nM affinity. The Atg1-Atg13 complex in turn binds as a unit to the Atg17-Atg31-Atg29 scaffold with similar to 10-mu M 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.
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