Abstract
The autophagy-initiating human ULK complex consists of the kinase ULK1/2, FIP200, ATG13, and ATG101. Hydrogen-deuterium exchange mass spectrometry was used to map their mutual interactions. The N-terminal 640 residues (NTD) of FIP200 interact with the C-terminal IDR of ATG13. Mutations in these regions abolish their interaction. Negative stain EM and multiangle light scattering showed that FIP200 is a dimer, while a single molecule each of the other subunits is present. The FIP200NTD is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C ∼20 nm across. The ULK1 EAT domain interacts loosely with the NTD dimer, while the ATG13:ATG101 HORMA dimer does not contact the NTD. Cryo-EM of the NTD dimer revealed a structural similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy-initiating TBK1 kinase and the ULK1 kinase complex.
Highlights
Macroautophagy is the conserved eukaryotic cellular process responsible for replenishment of biosynthetic precursors during starvation (Wen and Klionsky, 2016) and engulfment and degradation of molecular aggregates, organelles, intracellular pathogens, and many other cellular substrates (Anding and Baehrecke, 2017; Gomes and Dikic, 2014; Zaffagnini and Martens, 2016)
FIP200NTD assembles with unc-51–like autophagy activating kinase 1 (ULK1), ATG13, and ATG101 Given that family kinase-interacting protein of 200 kD (FIP200) is essential for autophagy and is the largest protein in the ULK1 complex, we began with the hypothesis that some part of FIP200 was likely to be the main hub organizing the complex
Purified FIP200NTD–MBP and FIP200 C-terminal domain (CTD)– MBP were used in a pulldown assay with the ternary ULK1:ATG13: ATG101 complex
Summary
Macroautophagy (henceforward, autophagy) is the conserved eukaryotic cellular process responsible for replenishment of biosynthetic precursors during starvation (Wen and Klionsky, 2016) and engulfment and degradation of molecular aggregates, organelles, intracellular pathogens, and many other cellular substrates (Anding and Baehrecke, 2017; Gomes and Dikic, 2014; Zaffagnini and Martens, 2016). Autophagy proceeds by the de novo formation of a cup-shaped double membrane known as the phagophore or isolation membrane. The phagophore double membrane grows such that it engulfs and isolates its substrates. Upon sealing of the double membrane, the mature structure is referred to as an autophagosome. The mechanisms by which these protein complexes orchestrate autophagosome initiation, growth, closure, and delivery to the lysosome are being actively sought (Hurley and Young, 2017; Mercer et al, 2018)
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