Phosphatidylserine and RhoB connect PI4P and PA metabolism to maintain plasma membrane identity.

Proper functions of cellular organelles require tight control of membrane phospholipid composition, yet the mechanisms by which lipid imbalances are sensed and corrected remain largely unknown. Here, we present evidence of an unexpected metabolic connection between plasma membrane (PM) phosphoinositide metabolism and two key anionic lipids, phosphatidylserine (PS) and phosphatidic acid (PA). Prolonged depletion of PM phosphatidylinositol 4-phosphate (PI4P) by pharmacological inhibition of PI 4-kinase IIIα (PI4KIIIα/PI4KA) increases phospholipase D (PLD) activity and PA levels in the PM. Using lipidomics, RNA-seq, and proximity proteomics, we find that PI4P loss induces a concomitant decrease in PS, activating a reciprocal relationship between PS synthesis and PLD-mediated PA generation. These changes also drive transcriptional and translational upregulation of the small GTPase RhoB, which enhances PLD-mediated PA synthesis and actin cytoskeletal remodeling. Because reduced PI4KA activity underlies numerous hereditary diseases, our studies reveal how perturbation of PM phosphoinositide synthesis triggers an integrated response that maintains the anionic character and structural integrity of the PM.