Lipid droplets (LDs) are evolutionarily conserved organelles that play critical roles in mammalian lipid storage and metabolism. However, the molecular mechanisms governing the biogenesis and growth of LDs remain poorly understood. Phosphatidic acid (PA) is a precursor of phospholipids and triacylglycerols and substrate of CDP-diacylglycerol (CDP-DAG) synthase 1 (CDS1) and CDS2, which catalyze the formation of CDP-DAG. Here, using siRNA-based gene knockdowns and CRISPR/Cas9-mediated gene knockouts, along with immunological, molecular, and fluorescence microscopy approaches, we examined the role of CDS1 and CDS2 in LD biogenesis and growth. Knockdown of eitherorexpression resulted in the formation of giant or supersized LDs in cultured mammalian cells. Interestingly, down-regulation of cell death-inducing DFF45-like effector C (), encoding a prominent regulator of LD growth in adipocytes, restored LD size in- but not in-deficient cells. On the other hand, reducing expression of two enzymes responsible for triacylglycerol synthesis, diacylglycerol-acyltransferase 2 () and glycerol-3-phosphate acyltransferase 4 (), rescued the LD phenotype in-deficient, but not-deficient, cells. Moreover,deficiency, but notdeficiency, promoted the LD association of DGAT2 and GPAT4 and impaired initial LD maturation. Finally, although both CDS1 and CDS2 appeared to regulate PA levels on the LD surface, CDS2 had a stronger effect. We conclude that CDS1 and CDS2 regulate LD dynamics through distinct mechanisms. CDS1CDS2CIDEC CDS1CDS2O DGAT2GPAT4CDS2CDS1CDS2CDS1
