Highlights d Human SEIPIN exists as an undecamer d The oligomerization state of SEIPIN is critical for its physiological function d The lumenal domain of SEIPIN forms an eight-stranded b sandwich fold d Both full-length SEIPIN and its lumenal domain bind anionic phospholipids

SUMMARY Berardinelli-Seip congenital lipodystrophy 2 (BSCL2) is caused by loss-of-function mutations in SEIPIN, a protein implicated in both adipogenesis and lipid droplet expansion but whose molecular function remains obscure. Here, we identify physical and functional interactions between SEIPIN and microsomal isoforms of glycerol-3-phosphate acyltransferase (GPAT) in multiple organisms. Compared to controls, GPAT activity was elevated in SEIPIN-deficient cells and tissues and GPAT kinetic values were altered. Increased GPAT activity appears to underpin the block in adipogenesis and abnormal lipid droplet morphology associated with SEIPIN loss. Over-expression of Gpat3 blocked adipogenesis, and Gpat3 knockdown in SEIPIN-deficient preadipocytes partially restored differentiation. GPAT overexpression in yeast, preadipocytes, and fly salivary glands also formed supersized lipid droplets. Finally, pharmacological inhibition of GPAT in Seipin−/− mouse preadipocytes partially restored adipogenesis. These data identify SEIPIN as an evolutionarily conserved regulator of microsomal GPAT and suggest that GPAT inhibitors might be useful for the treatment of human BSCL2 patients.

TMEM41B and VMP1 are integral membrane proteins of the endoplasmic reticulum (ER) and regulate the formation of autophagosomes, lipid droplets (LDs), and lipoproteins. Recently, TMEM41B was identified as a crucial host factor for infection by all coronaviruses and flaviviruses. The molecular function of TMEM41B and VMP1, which belong to a large evolutionarily conserved family, remains elusive. Here, we show that TMEM41B and VMP1 are phospholipid scramblases whose deficiency impairs the normal cellular distribution of cholesterol and phosphatidylserine. Their mechanism of action on LD formation is likely to be different from that of seipin. Their role in maintaining cellular phosphatidylserine and cholesterol homeostasis may partially explain their requirement for viral infection. Our results suggest that the proper sorting and distribution of cellular lipids are essential for organelle biogenesis and viral infection.

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.

Oxysterol-binding protein (OSBP) and OSBPrelated proteins (ORPs) constitute a large family of proteins that mainly function in lipid transport and sensing. ORP5 is an endoplasmic reticulum (ER)-anchored protein implicated in lipid transfer at the contact sites between the ER and other membranes. Recent studies indicate that ORP5 is also involved in cancer cell invasion and tumor progression. However, the molecular mechanism underlying ORP5's involvement in cancer is unclear. Here, we report that ORP5 promotes cell proliferation and motility of HeLa cells, an effect that depends on its functional OSBP-related domain (ORD). We also found that ORP5 depletion or substitutions of key residues located within ORP5-ORD and responsible for interactions with lipids interfered with cell proliferation, migration and invasion. ORP5 interacted with the protein mechanistic target of rapamycin (mTOR), and this interaction also required ORP5-ORD. Of note, whereas ORP5 overexpression induced mTOR complex 1 (mTORC1) activity, ORP5 down-regulation had the opposite effect. Finally, ORP5-depleted cells exhibited impaired mTOR localization to lysosomes, which may have accounted for the blunted mTORC1 activation. Together, our results suggest that ORP5 expression is positively correlated with mTORC1 signaling and that ORP5 stimulates cell proliferation, at least in part, by activating mTORC1.