Lipid droplets are highly conversed intracellular organelles that store triglycerides and are essential for fatty acid trafficking. These dynamic organelles form associations with many organelles, including the mitochondria, and close lipid droplet-mitochondria interactions are proposed to facilitate efficient transfer of fatty acids between organelles, resulting in more efficient β-oxidation and reduced activation of signaling pathways associated with cellular ‘lipotoxicity’ and insulin resistance.
The primary aim of this study is to identify the proteins that regulate the interactions between lipid droplets and mitochondria in living cells. Lipid droplet and mitochondrial proteins were identified by proximity-dependent labeling using BioID, a novel technique that screens physiologically relevant protein interactions using the promiscuous protein BirA. Upon BirA labeling, proteins in close proximity to BirA can be biotinylated and later quantified by affinity purification and LC-MS/MS. Lipid droplet-associated proteins were identified using the fusion proteins HDS1-BirA or DGAT1-BirA and mitochondria-associated proteins were labeled with FIS1-BirA. We first showed that HDS1 and FIS1 localised to their respective organelles. Next, we identified 123 and 103 proteins associated with lipid droplets and mitochondria, respectively. Among these proteins, 33 proteins were enriched in both the lipid droplet and mitochondria pools. We are currently investigating which candidate proteins are involved in the tethering of these two organelles, and examining these interactions under varying metabolic states. Ongoing studies also aim to delineate the function of other lipid droplet-mitochondria associated proteins and their possible role in the protecting cells from ‘lipotoxic’ stress.