Project Summary/Abstract Lipid droplets (LDs) are cellular energy reservoirs in the form of triglycerides and steryl esters and are central to the maintenance of membrane structure and energy homeostasis. They serve as the primary organelle for energy storage both in cells and in organisms (as adipose tissue). To regulate cellular and organismal energetics, lipids must be trafficked to other organelles where they are consumed by fatty acid oxidation or enzymatically altered to maintain membrane structure. Dysregulation of cellular lipid metabolism is a common feature of metabolic disorders such as obesity, diabetes, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). The VPS13 or repeating beta groove (RBG) family of proteins- containing ATG2A/B- comprises proteins thought to partake in pipe-like, bulk lipid transport between two membranes. Deletion of ATG2 and two of its known binding partners lead to massive accumulation of LDs, defects in lipoprotein biogenesis, and full-blown NASH. ATG2 localizes primarily to LDs, and our group has recently demonstrated that the protein can facilitate bulk lipid transfer in vitro. Loss of ATG2 blocks fluorescent fatty acid movement from the LD to mitochondria through yet undefined mechanisms. However, whether ATG2 directly participates in lipid transport at the LD and how other organelles and protein machinery partake in this process is not known. In this proposal, I outline a strategy to directly test ATG2 mediated lipid transport at the LD, identify the membranes that participate in lipid transport at the LD, and mechanistically define the proteins that cooperate in lipid transport. This project is structured to maximize progress toward my training goals in in vitro biochemistry, assay design, and quantitative image analysis, thereby equipping me with a full set of technical and intellectual skills to generate mechanistic explanations to complex biological questions. Accordingly, I place a strong focus on in vitro biochemistry, cell-based assays, and automated image analysis. In aim 1, I draw heavily on the expertise of the Melia and Reinisch labs to directly test for lipid transport activity of ATG2 at the LD using a combination of newly published and newly engineered in vitro lipid transport assays and cell-based assays of LD accumulation. In aim 2, I build on an APEX proximity labeling approach validated in the Melia lab to identify proteins and membranes that ATG2 links to the LD. In aim 3, I employ a wide range of cell-based assays, binding assays, and automated image analysis to determine which proteins mechanistically cooperate with ATG2 to move lipids at the LD. This project will pioneer new approaches to test lipid transport in VPS13/RBG family proteins and it will elucidate novel mechanisms of lipid transfer at the LD.