Steric Repulsion Counteracts ER-to-Lipid Droplet Protein Movement

Abstract Lipid droplets (LDs) are uniquely shaped organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer, continuous with the cytosolic leaflet of the endoplasmic reticulum (ER). The dynamics and function of LDs are closely tied to their proteome composition, which is subject to dynamic remodeling. Key proteins essential for LD biology 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 vivo tool to quantify and classify ER proteins based on their affinity for LDs. We found a broad spectrum of ER-to-LD partitioning affinities. We identified steric hindrance as a key factor in regulating ER-to-LD protein transfer, where proteins with only slightly higher LD affinity can effectively displace those with lower affinity from the LD surface. Consistent with this model, we observed that differentiation of 3T3 pre-adipocytes into adipocytes involves extensive remodeling of ER proteins targeting LDs, with Plin1—a high-affinity LD protein—becoming predominantly recruited and excluding other ER proteins. These findings highlight lateral protein-protein exclusion as a fundamental mechanism in shaping the LD proteome, providing new insights into LD biogenesis and function. Competing Interest Statement The authors have declared no competing interest.