Lipid droplets (LDs) are intracellular organelles responsible for lipid storage, and they emerge from the endoplasmic reticulum (ER) upon the accumulation of neutral lipids, mostly triglycerides (TG), between the two leaflets of the ER membrane. LD biogenesis takes place at ER sites that are marked by the protein seipin, which subsequently recruits additional proteins to catalyze LD formation. Deletion of seipin, however, does not abolish LD biogenesis, and its precise role in controlling LD assembly remains unclear. Here, we use molecular dynamics simulations to investigate the molecular mechanism through which seipin promotes LD formation. We find that seipin clusters TG, as well as its precursor diacylglycerol, inside its unconventional ring-like oligomeric structure and that both its luminal and transmembrane regions contribute to this process. This mechanism is abolished upon mutations of polar residues involved in protein–TG interactions into hydrophobic residues. Our results suggest that seipin remodels the membrane of specific ER sites to prime them for LD biogenesis.

Cells store energy in the form of neutral lipids packaged into micrometer-sized organelles named lipid droplets (LD). These structures emerge from the endoplasmic reticulum (ER) at sites marked by the protein seipin, but the mechanisms regulating their biogenesis remain poorly understood. Using a combination of molecular simulations, yeast genetics and fluorescence microscopy, we show that interactions between lipids' acyl-chains modulate the propensity of neutral lipids to be stored in LD, in turn preventing or promoting their accumulation in the ER membrane. Our data suggest that diacylglycerol, that is enriched at sites of LD formation, promotes the packaging of neutral lipids into LDs, together with ER-abundant lipids, such as phosphatidylethanolamine. On the opposite end, short and saturated acyl-chains antagonize fat storage in LD and promote accumulation of neutral lipids in the ER. Our results provide a new conceptual understanding of LD biogenesis in the context of ER homeostasis and function.

Lipid droplets are found in most organisms where they serve to store energy in the form of neutral lipids. They are formed at the endoplasmic reticulum (ER) membrane where the neutral-lipidsynthesizing enzymes are located. Recent results indicate that lipid droplets remain functionally connected to the ER membrane in yeast and mammalian cells to allow the exchange of both lipids and integral membrane proteins between the two compartments. The precise nature of the interface between the ER membrane and lipid droplets, however, is still ill-defined. Here, we probe the topology of lipid droplet biogenesis by artificially targeting proteins that have high affinity for lipid droplets to inside the luminal compartment of the ER. Unexpectedly, these proteins still localize to lipid droplets in both yeast and mammalian cells, indicating that lipid droplets are accessible from within the ER lumen. These data are consistent with a model in which lipid droplets form a specialized domain in the ER membrane that is accessible from both the cytosolic and the ER luminal side.

Lipid droplets (LDs) are globular subcellular structures that store neutral lipids. LDs are closely associated with the endoplasmic reticulum (ER), and are limited by a phospholipid monolayer harboring a specific set of proteins. Most of these proteins associate with LDs through either an amphipathic helix or a membrane-embedded hairpin motif. Here we address the question whether integral membrane proteins could localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER residential proteins. The resulting fusion proteins localized to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, was due to a redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of an outer mitochondrial membrane protein, OM14. Expression of OM14-PLIN3 induced a close apposition between LDs and mitochondria. These data indicate that the ER-LD junction constitutes a barrier for ER-residential integral membrane proteins.

Cells store energy in the form of neutral lipids packaged into micrometer-sized organelles named lipid droplets (LD). These structures emerge from the endoplasmic reticulum (ER), but their biogenesis remains poorly understood. Using molecular simulations, we found that fat accumulation and LD formation are described by a liquid-liquid phase separation (LLPS) process. Within this framework, we could identify how ER membrane properties modulate LD formation, and we could directly test our computational predictions by combining yeast genetics with fluorescence microscopy. Our data suggest that the specific lipid composition of the ER together with its peculiar physical properties, such as low membrane tension and membrane curvature, promote the packaging of neutral lipids into LD, preventing their accumulation in the ER membrane. Our results provide a new conceptual understanding of LD biogenesis in the context of ER homeostasis and function.