Lipid Droplet Proteome Dynamics and Lipotoxicity

Abstract

Lipid droplets are endoplasmic reticulum (ER)-derived neutral lipid storage organelles that act as central hubs of cellular lipid and energy homeostasis. Lipid droplets consist of a neutral lipid core (e.g. triacylglycerol) that is encircled by a phospholipid monolayer. A unique set of integral and peripheral proteins (i.e. the lipid droplet proteome) associates with the bounding monolayer and regulates lipid droplet functions. Employing an APEX2 proximity labeling proteomic approach, we defined high confidence lipid droplet proteomes in multiple human cell lines. These studies identified novel lipid droplet proteins such as FSP1, which we recently discovered is a CoQ oxidoreductase that suppresses oxidative lipid damage and ferroptosis. Coupling our proteomic methods with inhibitors of the AAA ATPase p97/VCP allowed us to trap and identify a Class I lipid droplet protein as a substrate for proteasomal clearance by ER-associated degradation (ERAD), uncovering a role for ERAD in regulating the lipid droplet proteome. To further understand the mechanisms of lipid droplet protein degradation, we engineered fluorescence-based reporter cell lines and performed a series of genetic screens using genome-wide and ubiquitination-focused CRISPR knockout libraries. These screens identified ER-resident ubiquitination factors as candidate regulators of PLIN2 proteasomal clearance during lipolytic lipid droplet turnover. Our results suggest that the lipid droplet-localized PLIN2 traffics to the ER for clearance via ERAD during lipolysis, underscoring the high degree of crosstalk between the ER and lipid droplets throughout the lipid droplet life cycle. Together, our findings highlight the utility of global discovery approaches for characterizing the mechanisms of lipid droplet proteome dynamics and protein degradation pathways.