Mechanism of enhanced lipid transfer within a reconstituted autophagy membrane contact site

• Applicant: Dr. Alex Faesen
• Subject Area: Biochemistry ; Biophysics ; Structural Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545933338

Macro-autophagy (called autophagy hereafter) is a process of regulated degradation. It eliminates damaged and unnecessary cellular components. 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. Regulation of autophagy in space and time is key for cells to survive bouts of stress and starvation. When autophagy is initiated, the formation of autophagosomes is quick: bulk cytosol is taken up within minutes 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 formation of autophagosomes requires the generation 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 ist integrated activities are unknown. Unveiling the biochemical and structural organization of the contact site, as well as elucidating the molecular mechanism of ist (enhanced) lipid transfer, are the principal goals of this project. The last few years, we have, for the first time, biochemical reconstituted a stable autophagy initiation super-complex that integrates subcomplexes of the initiation- and growth machinery to regulate lipid flow. Among other things, this work showed a surprising acceleration of lipid transfer within this super-complex. This proposal explains how we will use a combination of biochemical reconstitution, lipid transfer and membrane binding assays, as well as structural biology to identify the minimal machinery for lipid transfer. We will characterize this complex to understand ist architecture and structure, as well as study ist lipid transfer mechanism following several working hypotheses. Due to ist 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