Mechanisms of endosomal trafficking in lipid droplet catabolism

Project Summary/Abstract Lipids provide a rich source of energy to fuel fundamental cellular processes including migration, differentiation, and survival through periods of low nutrient availability. The storage and utilization of lipid fuel relies on lipid droplets (LD). These unique organelles are surrounded by peripheral phospholipid monolayer that encapsulate a core containing neutral lipids triacylglycerol (TAG) and cholesterol ester (CE). LDs possess a unique proteome of adapters, enzymes, and structural proteins that interact directly with the LD monolayer and regulate trafficking for biogenesis and catabolism. However, detailed mechanistic knowledge regarding the relationship between the LD monolayer and LD trafficking machinery is lacking. This knowledge is especially critical for the process of lipophagy, whereby subpopulations of LDs are selectively targeted for lysosomal degradation. Our preliminary data suggest that LDs possess heterogenous lipid signatures at the level of the LD monolayer, and this heterogeneity influences the fate of certain small LD subpopulations toward lipophagy. Furthermore, we postulate that lipids of the LD monolayer including diacylglycerol (DAG) and phosphatidtyliositol (PI) are modulated by LD catabolic enzymes to influence their trafficking. In Project 1, we will determine the role of adipose triglyceride lipase (ATGL) in generating DAG on the LD surface via TAG hydrolysis, and explore the impact of DAG on the recruitment and activation of protein kinase C (PKC) on the LD surface. In Project 2, we will investigate Rab5 in recruiting PI 3-Kinases to the LD surface to phosphorylate PI, define the role of ESCRT proteins in the trafficking of LDs for lipophagy in mammalian cells. We will also explore an unexpected role for certain ESCRTs in LD biogenesis. The results gained from the proposed research will provide a mechanistic understanding of lipid droplet trafficking and the modulation of the LD monolayer.