A Novel Role for RalA in Lipid Droplet Growth

The Ras-related small-GTPase RalA is involved in a number of cellular processes including membrane dynamics, vesicular trafficking, and filipodia formation. When membrane associated, RalA localizes to a number of cellular compartments including the plasma membrane, endosomes, mitochondria and lipid droplets. Unlike with the rest of these organelles, the functional relationship between RalA and lipid droplets remains unexplored. Lipid droplets are organelles that store excess cellular fatty acids (FAs) in the form of triacylglycerides and cholesterol esters. Far from being mere intracellular depots, lipid droplets are dynamic organelles and recent studies have highlighted their important role during nutrient deprivation. Amino acid starvation in MEFs and HeLa cells results in the induction of autophagy, a process that cells utilize to recycle their metabolic and structural building blocks. Autophagy liberates fatty acids from unwanted membranes for use as energy through β- oxidation. These fatty acids are cytotoxic if left in the cytosol and, therefore, are temporarily sequestered into lipid droplets that accumulate without harming the cell. Thus, lipid droplets play the critical role of protecting the cell from the cytotoxic effects of fatty acids while concurrently facilitating quick access to an energy source. Lipid droplets are found in most eukaryotic cells, all of which must be able to store excess FAs and utilize them on demand. However, the regulation of lipid droplets, including the mechanism of formation and growth is not fully understood.

The work in this dissertation identifies RalA as an important regulator of lipid droplets. RalA is required for lipid droplet growth during nutrient depletion. We find that RalA acts downstream of autophagy during nutrient starvation to directly facilitate lipid droplet growth. Mechanistically, RalA performs this function through phospholipase D1 (PLD1), an enzyme that converts phosphatidylcholine (PC) to phosphatidic acid (PA). We report that PLD1 is recruited to lysosomes during nutrient stress in a RalA-dependent fashion. Our data indicate that PLD1 is necessary for local PA production at the lysosome, a function necessary for lipid droplet growth during nutrient starvation. Additionally, we find that RalA promotes lipid droplet growth by facilitating the recruitment of the lipid droplet associated protein, perilipin 3, onto growing droplets. RalA and PLD1 inhibition prevents perilipin 3 recruitment onto lipid droplets, which associates with membranes in a PA-dependent fashion and is required for LD growth. Collectively, the data presented in this thesis support a model in which RalA recruits PLD1 to lysosomes during nutrient deprivation to promote the localized production of phosphatidic acid and the subsequent recruitment of perilipin 3 to expanding lipid droplets.