The study investigates the molecular mechanisms behind the heterogeneity of lipid droplets (LDs) in differentiating adipocytes. The authors identify that perilipin 1 (PLIN1), an integral membrane protein, plays a key role in this process. PLIN1 contains an unconventional integral membrane segment (iMS) that mediates its insertion into the endoplasmic reticulum (ER) and facilitates high-affinity binding to LDs. This high affinity excludes other PLINs from the initial LD population, leading to the formation of distinct LD classes. The study also reveals that PLIN1's movement between LDs and the ER is likely gated, and that perturbing this process can lead to changes in LD class specificity. The findings provide insights into how differences in organelle targeting and lipid affinity contribute to LD heterogeneity, which is crucial for understanding metabolic diseases such as lipodystrophy, fatty liver disease, and type 2 diabetes.The study investigates the molecular mechanisms behind the heterogeneity of lipid droplets (LDs) in differentiating adipocytes. The authors identify that perilipin 1 (PLIN1), an integral membrane protein, plays a key role in this process. PLIN1 contains an unconventional integral membrane segment (iMS) that mediates its insertion into the endoplasmic reticulum (ER) and facilitates high-affinity binding to LDs. This high affinity excludes other PLINs from the initial LD population, leading to the formation of distinct LD classes. The study also reveals that PLIN1's movement between LDs and the ER is likely gated, and that perturbing this process can lead to changes in LD class specificity. The findings provide insights into how differences in organelle targeting and lipid affinity contribute to LD heterogeneity, which is crucial for understanding metabolic diseases such as lipodystrophy, fatty liver disease, and type 2 diabetes.