Regulation of Rac translocation and activation by membrane domains and their boundaries

Abstract

Activation of Rac1 and related Rho GTPases involves dissociation from RhoGDI and translocation to membranes, where they bind effectors. Previous studies suggested that Rac membrane binding requires and co-localizes with cholesterol-rich, liquid-ordered (lo) membrane domains, called lipid rafts. Here, we develop a fluorescence resonance energy transfer (FRET) assay that robustly detects Rac membrane targeting in living cells. Surprisingly, FRET with acceptor constructs targeted to either raft or non-raft regions indicated Rac was present in both regions. Functional studies showed that Rac localization to non-raft regions decreased GTP loading due to inactivation by GAPs. In vitro, Rac translocation to supported lipid bilayers also required lo domains, yet Rac was concentrated in the liquid-disordered (ld) phase. Single molecule analysis demonstrated that translocation occurred preferentially at lo-ld boundaries. These results therefore suggest that Rac translocates to the membrane at domain boundaries, then diffuses into raft and non-raft domains, which controls interactions. These findings resolve discrepancies in our understanding of Rac biology and identify novel mechanisms by which lipid rafts modulate Rho GTPase signaling.