Steric Repulsion Counteracts ER-to-Lipid Droplet Protein Movement

Lipid droplets (LDs) are organelles with a neutral lipid core surrounded by a phospholipid monolayer continuous with the endoplasmic reticulum’s (ER) cytosolic leaflet. LDs' dynamics and function relate closely to their constantly remodeling proteome composition. Key proteins relocate from the ER to LDs, yet the mechanisms governing their movement and accumulation in LDs remain poorly understood. Here, we developed an innovative ex cellulo tool to classify ER proteins based on their affinity for LDs. We identified steric hindrance as a prime factor regulating ER-to-LD protein transfer, where proteins with higher LD affinity can effectively displace those with lower affinity from the LD surface. Consistent with this model, we observed that the differentiation of 3T3 pre-adipoc tes into adipocytes involves extensive remodeling of ER proteins targeting LDs, with Plin1—a high-affinity LD protein—becoming predominantly recruited, correlating with a reduction in the recruitment of other ER proteins. These findings highlight lateral protein-protein exclusion as a fundamental mechanism in shaping the LD proteome.