Ypk1 and Ypk2 kinases maintain Rho1 at the plasma membrane by flippase-dependent lipid remodeling after membrane stresses

The plasma membrane (PM) is frequently challenged by mechanical stresses. In budding yeast, TORC2-Ypk1/Ypk2 kinase cascade plays a crucial role in PM stress responses by reorganizing the actin cytoskeleton via Rho1 GTPase. However, the molecular mechanism by which TORC2-Ypk1/Ypk2 regulates Rho1 is not well defined. Here, we found that Ypk1/Ypk2 maintain PM localization of Rho1 under PM stress via spatial reorganization of the lipids including phosphatidylserine. Genetic evidence suggests that this process is mediated by the Lem3-containing lipid flippase.We propose that lipid remodeling mediated by the TORC2-Ypk1/Ypk2-Lem3 axis is a backup mechanism for PM anchoring of Rho1 after PM stressinduced acute degradation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂], which is responsible for Rho1 localization under normal conditions. Since all the signaling molecules studied here are conserved in higher eukaryotes, our findings might represent a general mechanism to cope with PM stress.