Mechanism of NADPH oxidase activation by the Rac/Rho-GDI complex

The low molecular weight GTP binding protein Rac is essential to the activa tion of the NADPH oxidase complex, involved in pathogen killing during phag ocytosis. In resting cells, Rac exists as a heterodimeric complex with Rho GDP dissociation inhibitor (Rho-GDI). Two types of interactions exist betwe en Rac and Rho-GDI: a protein-lipid interaction, implicating the polyisopre ne of the GTPase, as well as protein-protein interactions. Using the two-hy brid system, we show that nonprenylated Rac1 interacts very weakly with Rho -GDI, pointing to the predominant role of protein-isoprene interaction in c omplex formation. In the absence of this strong interaction, we demonstrate that three sites of protein-protein interaction, Arg66(Rac)-Leu67(Rac), Hi s103(Rac), and the C-terminal polybasic region Arg183R(ac)-Lys188(Rac), are involved and cooperate in complex formation. When Rac1 mutants are prenyla ted by expression in insect cells, they all interact with Rho-GDI. Rho-GDI is able to exert an inhibitory effect on the GDP/GTP exchange reaction exce pt in the complex in which Rac1 has a deletion of the polybasic region (Arg 183Rac-LysI88Rac). This complex is, most likely, held together through prot ein-lipid interaction only. Although able to function as GTPases, the mutan ts of Rac1 that failed to interact with Rho-GDI also failed to activate the NADPH oxidase in a cell-free assay after loading with GTP. Mutant Leu119Ra c1Gln could both interact with Rho-GDI and activate the NADPH oxidase. The Racl/Rho-GDI and Rac1(Leu119Gln)/Rho-GDI complexes, in which the GTPases we re bound to GDP, were found to activate the oxidase efficiently. These data suggest that Rho-GDI stabilizes Rac in an active conformation, even in the GDP-bound state, and presents it to its effector, the p67phox component of the NADPH oxidase.