Abstract
Cells tightly control the homeostatic levels and subcellular localizations of membrane phospholipids through the regulation of the activities of numerous lipid-metabolizing enzymes and lipid transfer proteins. Yet, the mechanisms by which lipid imbalances are sensed and corrected to establish and maintain homeostasis are, in most cases, unknown. Here we present an expanded view of plasma membrane (PM) phosphoinositide metabolism by revealing an unexpected metabolic connection between two key anionic lipids in this membrane, phosphatidylinositol 4-phosphate (PI4P) and phosphatidic acid (PA). PM pools of PI4P are generated by PI 4-kinase Type IIIα (PI4KIIIα/PI4KA), an essential enzyme whose partial dysfunction leads to numerous hereditary human diseases. We find that depletion of PI4P by pharmacological inhibition of PI4KA increases the activity of phospholipase Ds (PLDs) and the levels of their lipid product, PA, in the PM. Guided by RNA-seq analysis and proximity labeling proteomics, we elucidate how cells connect this PI4P decrease to a compensatory increase in PA levels. Loss of PM PI4P induces a concomitant decrease of phosphatidylserine (PS) levels, and this metabolic rewiring activates a reciprocal relationship between PS synthesis and PLD-mediated PA generation. These metabolic changes also lead to transcriptional and translational upregulation of the small GTPase RhoB, which enhances PLD-mediated PA synthesis and subsequent actin cytoskeletal remodeling. Our study reveals how disease-relevant perturbation of phosphoinositide synthesis induces an integrated response that ultimately boosts levels of PA, a key anionic lipid and metabolic intermediate in phosphoinositide resynthesis.