Open Collections

Structural characterization of the Atg1 kinase complex by single particle electron microscopy

University of British Columbia

In eukaryotes, autophagy is an evolutionarily conserved and essential “self-degradative” process used to maintain cellular homeostasis. Central to autophagy is the formation of double-membrane vesicles termed autophagosomes. The process of autophagosome formation is coordinated by over 35 autophagy-related (Atg) proteins. The Atg1 kinase complex constitutes one group of proteins required for the initial induction step of autophagosome formation. The Atg1 kinase complex is composed of the kinase Atg1, a regulatory phosphoprotein Atg13, and a protein scaffold Atg17 that forms a ternary complex with Atg31 and Atg29. In this study, we have determined the structure of the Saccharomyces cerevisiae Atg17-Atg31-Atg29 ternary complex by single-particle electron microscopy. The complex is an “S-shaped” dimer exhibiting an elongated architecture with an end-to-end distance of 345Å. Atg17 was found to form the central scaffold while Atg31 and Atg29 formed two globular densities tethered to the arcs formed by Atg17. Further analysis of purified Atg17 dimers showed that Atg17 mediated dimerization of the complex while Atg31 and Atg29 had a structural role in maintaining the distinct curvature of the complex. We further studied Atg1 kinase complex assembly by co-expressing a minimal pentameric assembly consisting of Atg1 CTD (residues 589-897) and Atg13 CTD (residues 384-738) with Atg17-Atg31-Atg29. Structural analysis localized Atg1 CTD and Atg13 CTD to the terminal regions of the ternary complex supporting that the N-terminus of Atg17 likely mediates complex assembly. Finally, we structurally characterized an important Atg1 kinase complex interacting partner, Atg11. Purified Atg11 exhibited an elongated architecture supporting its role as a coiled-coil protein scaffold.

Text

2015-06-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/45666

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/