Steric repulsion counteracts ER-to-lipid droplet protein movement.

Lipid droplets (LDs) are organelles with a neutral lipid core surrounded by a phospholipid monolayer, which is continuous with the cytoplasmic leaflet of the endoplasmic reticulum (ER). LD function depends on a highly dynamic LD surface proteome. Key proteins continuously exchange between the ER and LDs; however, the mechanisms governing the interorganelle movement and accumulation on the LD surface remain poorly understood. Here, we developed an ex cellulo tool introducing a classification of ER-derived proteins based on their different affinity for LDs. We find that proteins with higher LD affinity can effectively displace those with lower affinity from the LD surface, identifying steric hindrance as a key mechanism in regulating ER-to-LD protein transfer. Consistent with this model, we show that, during adipocyte differentiation Plin1-an adipocyte-specific high-affinity LD protein-reduces the recruitment of ER proteins with lower affinity by displacing them from the LD surface. These findings highlight lateral protein-protein exclusion as a fundamental mechanism in shaping the LD proteome.