Project Summary/Abstract Phosphatidic acid (PA) is a multifunctional signaling lipid and central biosynthetic intermediate that is subject to strong homeostatic regulation, with its levels tightly controlled in space and time. Though many PA- metabolizing enzymes and PA transporters are characterized, it is not well understood how cells sense changes in PA levels and how homeostasis is achieved. To both elucidate mechanisms underlying the spatiotemporal regulation of PA metabolism and reveal a broader spectrum of effector proteins that propagate PA signaling, we posit that new strategies to rapidly perturb PA levels with organelle-level precision are required. We have begun to develop precision “membrane editing” tools for the rapid installation of physiologically active pools of PA on target organelles. An optogenetic phospholipase D (optoPLD) uses blue light to recruit a bacterial PLD to desired organelle membranes, where it generates transient pools of PA via phosphatidylcholine hydrolysis, and recent directed evolution efforts have yielded second-generation, super-active optoPLDs (superPLDs). The combination of superPLD-mediated membrane editing and organelle membrane proteomics via proximity biotinylation using a membrane tethered TurboID, which we term a “feeding and fishing” (F+F) strategy, has afforded us a global view of rapid changes to the integral and peripheral membrane proteomes of the plasma membrane during conditions when its lipidome is edited using superPLD to transiently elevate PA levels. Beyond detecting known regulators of PA metabolism, we identified and validated new candidate proteins for sensing, transporting, and signaling the presence of PA in these membranes. Yet, several critical issues remain unaddressed, related to both methods development and mechanistic understanding of preliminary hits from our screens. The overall objective of this proposal is to deploy new optogenetic and proteomics tools to understand how cells establish and maintain functionally distinct PA pools in different locations to balance biosynthetic and signaling needs. First, we will develop ultralow-background, next-generation optogenetic PLDs and apply them to control PA-dependent signaling and discover new regulators of PA homeostasis. Second, we will elucidate roles for a new player implicated in the inter-organelle transport of PA using a combination of cellular and in vitro studies. Third, we will elucidate the molecular details and functional importance of the interaction of PA with a newly discovered PA-binding protein whose mutation causes a heritable musculoskeletal disease. Collectively, our studies will yield widely useful tools for membrane editing and deciphering PA signaling and establish a mechanistic framework for understanding how cells exert spatiotemporal control over the levels and bioactivity of a pleiotropic lipid to maintain homeostasis and direct specific physiological and signaling events.