How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation—the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.

Lipid droplets (LDs) form from the endoplasmic reticulum (ER) and grow in size by obtaining triacylglycerols (TG). Triacylglycerol hydrolase (TGH), a lipase residing in the ER, is involved in the mobilization of TG stored in LDs for the secretion of very-low-density lipoproteins. In this study, we investigated TGH-mediated changes in cytosolic LD dynamics. We have found that TGH deficiency resulted in decreased size and increased number of LDs in hepatocytes. Using fluorescent fatty acid analogues to trace LD formation, we observed that TGH deficiency did not affect the formation of nascent LDs on the ER. However, the rate of lipid transfer into preformed LDs was significantly slower in the absence of TGH. Absence of TGH expression resulted in increased levels of membrane diacylglycerol and augmented phospholipid synthesis, which may be responsible for the delayed lipid transfer. Therefore, altered maturation (growth) rather than nascent formation (de novo synthesis) may be responsible for the observed morphological changes of LDs in TGH-deficient hepatocytes.

The assembly of very low density lipoproteins involves the formation of a primordial, poorly lipidated apoB-containing particle in the endoplasmic reticulum, followed by the addition of neutral lipid from luminal lipid droplets (LLD). However, the lipid and protein compositions of LLD have not been determined. We have isolated LLD from mouse liver microsomes and analyzed their lipid and protein compositions. LLD are variably sized particles relatively poor in triacylglycerol (TG) content when compared with the lipid composition of cytosolic lipid droplets (CLD). They are devoid of apoB, adipophilin, and albumin but contain numerous proteins different from those found on CLD, including TG hydrolase (TGH), carboxylesterase 1 (Ces1), microsomal triglyceride transfer protein (MTP), and apoE. Ectopic expression of TGH in McArdle RH7777 hepatoma cells resulted in decreased cellular TG levels, demonstrating a role for TGH in the mobilization of hepatic neutral lipid stores. The isolation and characterization of LLD provide new supporting evidence for the two-step assembly of very low density lipoproteins.