CDP-DAG synthase 1 and 2 regulate lipid droplet growth through distinct mechanisms

Abstract

Lipiddroplets(LDs) are evolutionarily conserved organellesthat play critical roles in mammalian lipid storage and metabolism. However,themolecularmechanisms governingthe biogenesis and growthofLDs remainpoorlyunderstood.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 siRNAbased 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 either CDS1 or CDS2 expression resulted in the formation of giant or supersized LDs in cultured mammalian cells. Interestingly, down-regulation of cell death-inducing DFF45-like effector C (CIDEC), encoding a prominent regulator of LD growth in adipocytes, restored LD size inCDS1-butnotinCDS2-deficient cells.Ontheotherhand, reducing expression of two enzymes responsible for triacylglycerol synthesis, diacylglycerol O-acyltransferase 2 (DGAT2) and glycerol-3- phosphateacyltransferase4(GPAT4), rescuedtheLDphenotypein CDS2-deficient, but not CDS1-deficient, cells. Moreover, CDS2 deficiency, but not CDS1 deficiency, 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.