Deciphering phosphatidic acid homeostasis and signaling using optogenetic membrane editors

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 method development and mechanistic understanding of 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 elucidate
roles for PA in mediating crosstalk between two major cell signaling pathways and discover new regulators of
PA homeostasis. Second, we will elucidate roles for a new player implicated in the interorganelle 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.

Grant Number: 5R01GM151682-03
NIH Institute/Center: NIH
Principal Investigator: Jeremy Baskin