Mechanism of lipid droplet formation at the endoplasmic reticulum
Biochemistry
Final Report Abstract
To balance fluctuations in energy availability and expenditure, eukaryotes store energy-rich neutral lipids, such as triacylglycerol (TG), in intracellular lipid droplets (LDs). LDs have a unique architecture consisting of a hydrophobic core of neutral lipids surrounded by a phospholipid monolayer. LDs are versatile organelles that are connected to many cellular functions. These functions include many reactions of metabolism, membrane synthesis, viral replication and protein storage. Notably, the molecular machinery that orchestrates LD biogenesis, homeostasis and degradation are only beginning to be understood. The overarching question of my research is to determine how LDs are formed, a key unresolved mystery in cell biology. Our current model posits that in the first step, TG is synthetized in the ER, TG then accumulates to form a lipid lens at specific sites and grows in size. In the third step, LDs bud and eventually separate from the ER to form a mature LD. LD biogenesis is a key aspect of cell biology since it determines the location and number of LDs in cells. However, despite its importance, the current formation model has not been tested and insights into the molecular mechanisms are still lacking. A major question in the field is how delocalized TG synthesis in the ER bilayer leads to formation of LDs at specific sites. By combining cell biological, biochemical and structural biology approaches, I studied two proteins that are involved in this process, the transmembrane protein seipin and the soluble Rab GTPase Rab18. Rab18 was shown to act in LD formation by tethering of LDs to the ER in concert with known protein complexes in the ER. We tested this idea directly in human cells and were not able to establish a direct role for this protein in LD biogenesis, or turnover. Using single particle cryoEM studies of fly and yeast seipin, we showed that it forms a ringshaped oligomer at sites of LD formation. Furthermore, seipin can detect packing defects in the bilayer induced by phase separation of TG. Genetic and biochemical evidence supports a role for the transmembrane domains in complex formation in addition to its luminal domain. These new information on the seipin complex architecture raise several new hypotheses on seipin function that will guide future research. These hypotheses range from regulation of LD protein targeting to neutral lipid binding or lipid lens formation or phase separation in the complex itself. Analysis of protein function in cells can be challenging, especially in the complex environment of the ER. To circumvent these problems, we established an in vitro reconstitution assay of LD formation using purified membranes that recapitulates neutral lipid compartmentalization and LD protein targeting in vitro. We used this assay to identify a novel cytosolic protein factor acting in LD biogenesis and will use this assay in the future to analyze specific protein functions and the ultrastructure of LD formation in the future. This ongoing investigation addresses LD biogenesis as a fundamental aspect of cell biology. Mechanistic insights on LD formation will reveal the biology underlying severe human diseases caused by over-accumulation of LDs, such as type-2 diabetes, atherosclerosis and fatty liver disease, or lipodystrophy-related diseases that are caused by problems in LD accumulation.
Publications
- (2018) Cryo-electron microscopy structure of the lipid droplet-formation protein seipin. J Cell Biol, 217, 4080
Sui X., Arlt, H., Brock K.P., Lai Z.W., DiMaio F., Marks D.S., Liao M., Farese, Jr. R.V., and Walther T.C.
(See online at https://doi.org/10.1083/jcb.201809067) - (2018) Rab18 is not necessary for lipid droplet biogenesis or turnover in human mammary carcinoma cells. Mol Biol Cell, 29, 2045-2054
Jayson C.B.K., Arlt, H., Fischer A.W., Lai Z.W., Farese, Jr. R.V., and Walther T.C.
(See online at https://doi.org/10.1091/mbc.E18-05-0282)
