Deo, R.; Bhapkar, A.; Givan, E.; Li, C.; Haimov, E.; Binns, D.; Scherer, P. E.; Kozlov, M. M.; Toulmay, A.; Prinz, W.

Lipid droplets (LDs) are lipid storage organelles with critical roles in metabolism. Cells form LDs in response to various metabolic stresses (1). LD biogenesis occurs in the endoplasmic reticulum (ER) membrane and begins when triglyceride (TG) in the membrane phase separates (2-4). After nucleation, nascent LDs grow by absorbing TG from the membrane (5,6). How cells determine when TG in the ER membrane is added to existing LDs or is used to form new LDs or is not known. Here, we developed the first LD formation assay that allows the synthesis of TG in membranes, mimicking what occurs in cells. LDs arise stochastically after TG production begins. LD nucleation is affected by TG production rate and membrane phospholipid composition. Membrane-embedded LDs suppress nascent LD formation by sequestering TG and destabilizing nascent LDs by Ostwald ripening. We show that cells overcome this suppression in two ways. The ER membrane protein seipin accelerates LD nucleation where it localizes in a membrane, probably by increasing the kinetics of TG phase separation. The LD coat perilipin 3 inhibits nascent droplet dissipation by ripening. Together, our findings reveal a minimal LD formation machinery, and the mechanisms cells use to control whether TG in the ER drives new LD formation.