Acyl-chain saturation drives lipid sorting to model lipid droplets

Lipid droplets (LDs) form from the endoplasmic reticulum (ER), involving lipid exchange between the ER bilayer and the emerging LD monolayer. However, how lipids naturally partition based on their chemistry remains unclear. In this study, we use model systems to examine how saturated and monounsaturated phospholipids distribute between the bilayer and the droplet monolayer. Our findings show that lipid saturation influences partitioning, depending on the neutral lipid composition of the droplet. Saturated phospholipids generally disfavor artificial LDs (aLDs) more than monounsaturated lipids. Adding cholesteryl oleate to triolein significantly reduces phospholipid levels in aLDs, especially saturated phospholipids. Additionally, we observe that membranes with liquid-disordered regions tend to contain more neutral lipids, particularly triolein. Conversely, membranes with liquid-ordered regions, rich in saturated lipids and cholesterol, tend to exclude them. Molecular dynamics simulations support this observation. As a result, triolein-based aLDs consistently localize to the liquid-disordered regions, which serve as the main interface for neutral lipid exchange. Our study sheds light on how lipid saturation and neutral lipid chemistry interact to influence lipid composition and distribution within both the bilayer and an aLD, raising important questions about how cells regulate spontaneous lipid sorting in the ER and LDs.