A Conserved Family of Proteins Facilitates Nascent Lipid Droplet Budding from the ER

Abstract

Lipid droplets (LDs) are found in almost all cells and play central roles in energy and lipid metabolism; it is also becoming clear that LDs have additional functions, including important roles in protein degradation, ER stress, and viral replication. Unlike all other membrane‐bound organelles, LDs are surrounded by a phospholipid monolayer rather than a bilayer. How this unique structure is formed has remained an important unsolved mystery for many years. Quite a number of models of LD biogenesis have been proposed but the simplest and arguably most popular is one that has sometimes been termed the “lens” model. Neutral lipids, triacylglycerols (TAG) and steryl esters (SE) which form the core of all LDs, are synthesized in the ER and it is thought that as these lipids accumulate in the ER bilayer they form a lens (or blister) in the ER that can grow and bud. We visualized nascent LD formation in yeast using electron microscopy (EM)‐tomography and found that the lens model is correct. Lens of neutral lipid approximately 30–60 nm wide were found between the two leaflets of the ER bilayer. This is the first time the beginnings of LD biogenesis have been visualized and resolves a long‐standing question in LD biogenesis. We next asked whether proteins are necessary to facilitate nascent LD budding from the ER and focused on a conserved family of proteins, called fat storage‐inducing transmembrane (FIT) proteins. Yeast has two FITs (called Fit2a and Fit2b) and we found that in cells missing both most LDs are wrapped by the ER membrane and that this is caused by LD budding into the ER lumen rather than the cytosol. The wrapping membrane is not derived from the phagophore or related to autophagy since we found that wrapping still occurred in mutants lacking the FIT proteins and various proteins required for autophagy. The role of FIT proteins in LD budding is conserved; knock down of FIT2 in 3T3 fibroblasts and in C.elegans caused LDs to become wrapped in the ER. Interestingly deletion of the single FIT protein in C. elegans is lethal, suggesting that LD budding is an essential process in this organism. Our findings indicate that nascent LDs form lenses in the ER and that FIT proteins are necessary to promote proper budding of LDs from the ER. The mechanism of how FIT proteins control the directionality of LD budding remains to be investigated.Support or Funding InformationThis work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases. Vineet Choudhary is supported by fellowship from Swiss National Science Foundation (Grant Nr; PA00P3_145358, P300P3_158454).