Triglyceride lipolysis triggers liquid crystalline phases in lipid droplets and alters the LD proteome.

Lipid droplets (LDs) are reservoirs for triglycerides (TGs) and sterol-esters (SEs), but how these lipids are organized within LDs and influence their proteome remain unclear. Using in situ cryo-electron tomography, we show that glucose restriction triggers lipid phase transitions within LDs generating liquid crystalline lattices inside them. Mechanistically this requires TG lipolysis, which decreases the LD's TG:SE ratio, promoting SE transition to a liquid crystalline phase. Molecular dynamics simulations reveal TG depletion promotes spontaneous TG and SE demixing in LDs, additionally altering the lipid packing of the PL monolayer surface. Fluorescence imaging and proteomics further reveal that liquid crystalline phases are associated with selective remodeling of the LD proteome. Some canonical LD proteins, including Erg6, relocalize to the ER network, whereas others remain LD-associated. Model peptide LiveDrop also redistributes from LDs to the ER, suggesting liquid crystalline phases influence ER-LD interorganelle transport. Our data suggests glucose restriction drives TG mobilization, which alters the phase properties of LD lipids and selectively remodels the LD proteome.

ORP5 and ORP8 orchestrate lipid droplet biogenesis and maintenance at ER-mitochondria contact sites.

Lipid droplets (LDs) are the primary organelles of lipid storage, buffering energy fluctuations of the cell. They store neutral lipids in their core that is surrounded by a protein-decorated phospholipid monolayer. LDs arise from the endoplasmic reticulum (ER). The ER protein seipin, localizing at ER-LD junctions, controls LD nucleation and growth. However, how LD biogenesis is spatially and temporally coordinated remains elusive. Here, we show that the lipid transfer proteins ORP5 and ORP8 control LD biogenesis at mitochondria-associated ER membrane (MAM) subdomains, enriched in phosphatidic acid. We found that ORP5/8 regulates seipin recruitment to these MAM-LD contacts, and their loss impairs LD biogenesis. Importantly, the integrity of ER-mitochondria contact sites is crucial for ORP5/8 function in regulating seipin-mediated LD biogenesis. Our study uncovers an unprecedented ORP5/8 role in orchestrating LD biogenesis and maturation at MAMs and brings novel insights into the metabolic crosstalk between mitochondria, ER, and LDs at the membrane contact sites.