Mechanisms of Lipid Droplet Formation

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PROJECT SUMMARY Lipid droplets (LDs) are ubiquitous monolayer-bound organelles that function in cellular lipid storage (for metabolic energy or membrane synthesis). LDs form from the ER, but how LDs are formed remains unknown and is a central question for the field. The current model indicates that neutral lipids, such as triacylglycerols (TG), are synthesized in the ER and released into the bilayer. At a critical concentration, TGs de-mix from the phospholipid bilayer in a phase transition that forms nascent LDs that bud toward the cytosol. We hypothesize that proteins are essential to ensure this process occurs in a defined manner and to prevent the formation of “ectopic” and potentially dysfunctional LDs, disrupting ER and cell function. Specifically, two ER proteins – seipin and lipid droplet assembly factor 1 (LDAF1) – operate in the lipid droplet assembly complex (LDACs) in the ER to form LDs. Both proteins form an oligomeric assembly with seipin forming a ring of 10-12 subunits and an equal number of LDAF1 occupying the middle of the ring. While we have identified components of the LD formation machinery and gained some insight into their structures, how these proteins function to facilitate LD formation remains mostly a mystery. Here we propose to utilize the latest tools and approaches, including biochemistry, structural biology, molecular simulations, and cell biology, to address the following questions: How and where is TG made relative to LDACs? What are the molecular structures of the seipin/LDAF1 LDACs? How do these oligomeric complexes assemble/disassemble? Where do LDACs localize in cells? How do they function to organize LD formation? We will address these questions by completing four specific aims. Aim 1 will address the mechanism of TG synthesis in the ER by the DGAT1 enzyme. We will expand on our recent elucidation of the molecular structure of human DGAT1, combining molecular dynamics and biochemical experiments to elucidate the precise mechanism of TG generation and determine how TG is released into the ER membrane for LD formation. Aim 2 will determine how and where LD assembly complexes assemble in cells to form LDs. We will determine the relationship of TG synthesis to LDACs, whether seipin/LDAF1 LDACs localize to ER tubules and how they assemble. Aim 3 will focus on elucidating the molecular structure of the seipin/LDAF1 LDAC in vitro and in cells. We will utilize cell and structural biology approaches, including cryo-EM and cryo-ET to test the hypothesis that seipin and LDAF1 form a ring structure with LDAF1 in center and that these LDACs form at areas of membrane curvature (tubules) where the structure may adopt dynamic conformations and activate of the complex. Aim 4 will determine the molecular function of the seipin/LDAF1 LDAC in vitro and in molecular dynamics simulations. We will reconstitute LD formation to test the hypothesis that the seipin/LDAF1 LDAC catalyzes phase transition of TG in the membrane, ensuring LDs form at these designated formation sites. Successful completion of these aims will advance the molecular understanding of a fundamental process central to energy metabolism and provide information on the mechanistic underpinning of many metabolic diseases, such as obesity, atherosclerosis, and fatty liver disease.