A primary role of adipose tissue is to store fat in lipid droplets. Storage of triacylglycerol (TAG) and steryl esters, the main components of stored fat, not only provides energy in times of caloric deprivation;it also protects the organism from lipotoxicity. However, adipose tissue has a limited capacity for fat storage, and many obese individuals have exceeded it, leading to fatty liver disease and a variety of cardiovascular, immunological and metabolic imbalances with serious negative health outcomes and great cost to society. In contrast, some individuals cannot store any fat properly, a condition known as lipodystrophy;health outcomes of these patients are even more serious and often lead to death at an early age. The most severe form of lipodystrophy is the fault of mutations in the gene that encodes seipin. Considering the negative outcomes and the costs associated with poor fat storage, it is surprising that there are huge gaps in our basic knowledge of this process. Lipid droplets are not just coalesced neutral lipids in the cytoplasm but are surrounded by a monolayer of phospholipids into which are integrated a constellation of specific proteins;they emanate from the endoplasmic reticulum (ER) and may always be associated with it. To understand fat storage the mechanism of lipid droplet assembly must be known. The key enzymes that produce neutral lipid have been identified but the details of droplet assembly are murky. Fortunately, droplets are made in virtually all eukaryotic cells and can be studied in model organisms. Bakers yeast, Saccharomyces cerevisiae is a great system for this research, since the cells can robustly store lipid in droplets and the organism provides facile genetic and cell biological approaches. We reported the identification of yeast seipin in 2007;it is now clear that it plays an important role in fat storage in yeast as well as humans. We propose to study three basic aspects of droplet assembly using reagents derived from seipin as springboards to understand fundamental biology of fat storage: (1) we have developed a yeast strain in which storage of TAG is exquisitely sensitive to seipin. We shall interrogate this system to understand the molecular mechanism by which seipin catalyzes packaging of TAG and we shall determine whether TAG normally flows between droplets and the connecting ER. (2) We have identified a seipin mutant that apparently causes an imbalance of TAG vs. phospholipid packaging into droplets. We shall determine whether seipin balances the packaging of neutral lipid and phospholipids into the organelle. We shall also screen for suppressors of seipin mutations and use whole genome approaches to uncover novel proteins in droplet assembly. (3) Many lipases reside on lipid droplets where they mobilize fat for energy and export. We have found conditions in which the lipase Tgl3p and others require seipin for localization to droplets. The mechanism by which lipases traffic to droplets and the role of seipin in this process will be determined. Overall, our proposed work will increase our understanding of droplet assembly, the basis of obesity and lipodystrophy, at its most basic level.
Excess caloric input is stored as fat, eventually resulting in obesity. At a cell level, fat is stored in organelles called lipid droplets. This proposal addresse basic mechanisms of lipid droplet assembly, and what goes wrong in human disease when fat storage does not function properly.
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