Timely Control of Autophagy in Humans: Identification of Rate-Limiting Steps and Acceleration Nodes

Autophagy is a conserved process of regulated degradation in eukaryotic cells. Initiation of autophagy is marked by the prompt convergence of ‘ATG’ initiation proteins in spatially defined autophagic hubs. To understand the initiation cascade in detail, in vitro reconstitution of key autophagy complexes was performed. A small protein named ATG101 is essential for initiating autophagy in mammalian cells, but its function was unknown. To elucidate the function of ATG101, recombinant ATG101 was purified from insect cells. Its interaction kinetics with other ATG proteins were monitored using a fluorescence polarisation-based assay. Extremely slow interaction rates were observed. Finally, a combination of biophysical techniques, cross-linking mass spectrometry, and structure prediction methods identified a previously unidentified dimerisation step in the autophagy initiation cascade. This dimerisation, in turn, led to acceleration of the slow interactions, providing a way to dictate the assembly rate of autophagy initiation.