Regulated protein metamorphosis to initiate autophagy

• Applicants: Dr. Alex Faesen; Professor Dr. Björn Stork
• Subject Area: Biochemistry ; Structural Biology ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 542770124

Macro-autophagy (called autophagy hereafter) is a process of regulated degradation. It eliminates damaged and unnecessary cellular compo¬nents. These biomolecules are transported to lysosomes, where they are degraded and ready to be recycled. Autophagy has been proven to play a wide range of roles in cellular housekeeping, including removal of damaged or unneeded organelles, intracellular pathogens, and protein aggregates. It is an essential biological pathway that promotes organismal health, longevity and helps combat cancer and neurodegenerative diseases. A timely initiation of autophagy is key to maintain survival of cells under stress and starvation. In response to starvation, bulk cytosol is taken up within minutes by newly formed autophagosomes and transported to the lysosome. Where autophagosomes initially form, and the source of the proteins and lipids needed for autophagosome expansion, remains controversial. In multicellular organisms, the for¬mation of autophagosomes requires the genera¬tion of contact sites between a subdomain of the ER (‘omegasome’) and a cup¬shaped membrane (‘phagophore’). This phagophore will expand and eventually close to form the autophagosome. Conceptionally, these contact sites perform three distinct but highly integrated functions: 1) they assemble on-demand to create a dynamic interface for the growing autophagosome; 2) they tether the growing autophagosome to distinct ER sites; 3) they provide ways to flow lipids into the growing autophagosome. The composition of the contact site, how it is assembled and disassembled, and the molecular mechanism of its integrated activities are unknown. The last few years, the Faesen and Stork laboratories have closely collaborated in a biochemical reconstitution effort that resulted in the purification of a stable contact site super-complex that integrates subcomplexes of the initiation- and growth machinery to regulate lipid flow. Our work shows that the assembly of the ATG9-13-101 complex is a key event in the assembly and function of the contact site super- complex. We show that HORMA domain proteins ATG13 and ATG101 have the rare ability to slowly switch between distinct native folds, which creates an obligatory rate-limiting step in the assembly of the super-complex. This proposal explains that regulating their metamorphosis is likely to control contact site assembly in space and time. We aim to define the molecular details of the metamorphosis of ATG13 and ATG101 as regulatory mechanism of contact site assembly and disassembly, both in cells and with purified recombinant proteins. Due to its universal relevance, understanding the process of autophagy initiation is of primary interest for both the autophagy field, but also of the larger community aiming to understand dynamic contact sites and the biogenesis of membranous organelles.

• DFG Programme: Research Grants