Structure/function studies of lipid droplet-associated proteins perilipin A and CGI-58

In mammalian cells, proteins coat the surfaces of intracellular lipid droplets. The goal of this dissertation was to study the structure-function relationships of two lipid droplet associated proteins, perilipin A and CGI-58. Perilipin A is a major lipid droplet associated protein in adipocytes that plays a key role in regulating the storage and hydrolysis of triacylglycerol. Three moderately hydrophobic sequences present within the central region of perilipin A in conjunction with either an amino terminal sequence predicted to form amphipathic beta-strands or a central acidic region are important for targeting perilipin A to lipid droplets. These moderately hydrophobic sequences also mediate the anchoring of perilipin A to lipid droplets. Other sequences within perilipin A likely interact with cellular proteins; CGI-58 requires perilipin A for localization to lipid droplets. Both endogenous and GFP-tagged ectopic CGI-58 localize to perilipin coated lipid droplets in 3T3-L1 adipocytes and in preadipocytes expressing ectopic perilipin A. In adipocytes under basal conditions, CGI-58 localizes to perilipin-coated lipid droplets, however, upon stimulation of beta-adrenergic receptors, CGI-58 disperses off of lipid droplets and shows diffuse cytoplasmic localization, suggesting that the interaction between CGI-58 and perilipin A is sensitive to metabolic status. We mapped the site of interaction of CGI-58 to a 48-amino acid sequence within the carboxyl terminus of perilipin A. Mutations in CGI-58 cause NLSD, which is characterized by accumulation of triacylglycerol-rich lipid droplets in many tissues; however, the function of CGI-58 in triacylglycerol metabolism is unknown. Expression of ectopic CGI-58 in NLSD fibroblasts clears the excessive triacylglycerol without altering the triacylglycerol lipase activity of cell lysates. In contrast, expression of ectopic hormone-sensitive lipase decreases triacylglycerol accumulation in NLSD fibroblasts lacking CGI-58 by increasing cellular triacylglycerol lipase activity, suggesting that CGI-58 is not required for this pathway of lipolysis. In conclusion, three central hydrophobic sequences target and anchor perilipin A to lipid droplets, whereas a 48-amino acid sequence within the carboxyl terminus of perilipin A mediates an interaction with CGI-58. In addition, CGI58 facilitates the clearance of triacylglycerol in human skin fibroblasts without altering triacylglycerol lipase activity, and CGI-58 is not a component of all lipolytic pathways.