Abstact

Rho family small GTPases are essential for cytokinesis completion. RacGAP1, a dimeric multidomain protein with a lipid-binding C1 domain and a GTPase-activating protein (GAP) domain, is a major regulator in this process. However, despite many cellular and biochemical studies, whether RhoA or Rac1 is the actual substrate inactivated by RacGAP1 has remained a matter of debate. Rho family GTPases are inactivated by their GAPs on membranes, but the activity and specificity of RacGAP1 have only been studied biochemically in solution. Here, we reconstituted RacGAP1 in a membrane environment, using liposomes and highly purified proteins. Our study reveals that PS is a major lipid required for RacGAP1 membrane-binding in addition to PIP(2), and that the GAP domain cooperates with the C1 domain for membrane-binding. Consistently, fluorescence-based kinetics show that membranes potentiate the activity of RacGAP1 and of the C1GAP and GAP constructs towards Rac1. However, membranes do not modify RacGAP1 marked specificity for Rac1, identifying it as a Rac1-selective GAP. The Rac1-GDP-Pi crystal structure and mutagenesis identify the switch 1 and the insert region as important determinants for this specificity. Together, our results suggest a structural model in which the Rac1-RacGAP1 complex associates snugly with the membrane to efficiently remove Rac1 from the division plane.