A palmitoylation switch mechanism regulates Rac1 function and membrane organization

The small GTPase Rac1 plays important roles in many processes, including cytoskeletal reorganization, cell migration, cell-cycle progression and gene expression. The initiation of Rac1 signalling requires at least two mechanisms: GTP loading via the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle, and targeting to cholesterol-rich liquid-ordered plasma membrane microdomains. Little is known about the molecular mechanisms governing this specific compartmentalization. We show that Rac1 can incorporate palmitate at cysteine 178 and that this post-translational modification targets Rac1 for stabilization at actin cytoskeleton-linked ordered membrane regions. Palmitoylation of Rac1 requires its prior prenylation and the intact C-terminal polybasic region and is regulated by the triproline-rich motif. Non-palmitoylated Rac1 shows decreased GTP loading and lower association with detergent-resistant (liquid-ordered) membranes (DRMs). Cells expressing no Rac1 or a palmitoylation-deficient mutant have an increased content of disordered membrane domains, and markers of ordered membranes isolated from Rac1-deficient cells do not correctly partition in DRMs. Importantly, cells lacking Rac1 palmitoylation show spreading and migration defects. These data identify palmitoylation as a mechanism for Rac1 function in actin cytoskeleton remodelling by controlling its membrane partitioning, which in turn regulates membrane organization.