Proximity labeling on phosphatidic acid-enriched membranes generated via optogenetic membrane editing

Cellular lipid metabolism is subject to strong homeostatic regulation, but players involved in and mechanisms underlying these pathways remain mostly uncharacterized. Here, we develop and exploit a “Feeding-Fishing” approach coupling membrane editing using optogenetic lipid-modifying enzymes (feeding) with organelle membrane proteomics via proximity labeling (fishing) to elucidate molecular players and pathways involved in homeostasis of phosphatidic acid (PA), a multifunctional lipid central to glycerolipid metabolism. By performing proximity biotinylation using a membrane tethered TurboID alongside membrane editing to selectively deliver PA to the same membrane, we identified numerous PA-metabolizing enzymes and lipid transfer proteins enriched in and depleted from PA-fed membranes. Subsequent mechanistic analysis revealed that PA homeostasis in the cytosolic leaflets of the plasma membrane and lysosomes is mediated by both local PA metabolism and the action of members of the lipid transfer protein families that carry out interorganelle lipid transport prior to additional metabolic steps. More broadly, the interfacing of membrane editing with organelle membrane proteomics using proximity labeling represents a strategy for revealing mechanisms governing lipid homeostasis.