Project Summary/Abstract Macroautophagy (hereafter autophagy) is a cellular degradative process that is intimately connected to the process of aging. Autophagy maintains cell health and homeostasis through the delivery of potentially cytotoxic cargo to the lysosome through the de novo formation of the double membrane autophagosome. This process is impaired with increased age. Furthermore, deletion of the core autophagy genes has been shown in multiple organisms to decrease lifespan, while rescue experiments conversely restore a full lifespan. Autophagy is especially critical in maintaining the health of long-lived neurons, and defects in autophagy result in various neurodegenerative diseases, including Parkinson’s Disease, Alzheimer’s Disease, and amyotrophic lateral sclerosis. Autophagy protein ATG2 is essential to this process and tissue-specific ablation of this protein results in decreased lifetime, motility, and age-related tissue deterioration. We recently demonstrated that ATG2 is a lipid transfer protein with a novel structure that allows for bulk lipid delivery. This activity is essential for autophagosome biogenesis. I predict that ATG2 delivers lipids directly into the nascent autophagosome to expand the growing membrane. As-of-yet, the identity of the lipid-donating organelle is not known. Intriguingly, a mechanism to deliver a net transfer of lipids in one direction is without clear precedent in mammalian biology. As this mechanism likely depends on the biophysical properties of both membranes, I propose to formally identify the donor membrane from which ATG2 extracts lipids. This project is designed to provide the training necessary to achieve a future career in independent research. Furthermore, as this project will elucidate the membrane source for this critical age-delaying cell biological process, this body of research will identify key regulators of autophagy that represent additional methods of age-related therapeutic intervention. In this proposal, I seek to understand the mechanism by which ATG2 delivers a net transfer of lipids into the autophagosome by addressing three main questions. First, what is the donor membrane for autophagosome biogenesis? I propose to leverage the non-biased and high resolution APEX strategy of proximity labeling with live cell fluorescent microscopy to formally identify the donor membrane. Strikingly, preliminary data reveal that ATG2 resides at ER exit sites (ERES) during autophagosome biogenesis, a highly specialized subdomain of the ER. Second, does ATG2 lipid transfer possess intrinsic directionality, or does it rely upon local membrane energetics? I have developed two in vitro lipid transfer assays to mimic the ERES-autophagosome contact site, through which I can assess the directionality and specificity of lipid transfer through bulk-ensemble measurement and by visual examination. Third, how is ATG2 recruited to the donor membrane? Following identification of the donor membrane, I can now s...