Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly

The superoxide (O-2(.-))-generating NADPH oxidase complex of phagocytes con sists of a membrane-associated flavocytochrome (cytochrome b559) and four c ytosolic proteins, p47(phox) p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O-2 production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrom e b(559). This process can be reproduced in an in vitro system consisting o f phagocyte membranes, p47(phox) p67(phox), and Rac1 activated by an anioni c amphiphile. We now show that posttranslationally processed (prenylated) R ad initiates NADPH oxidase assembly, expressed in O-2, production, in a cel l-free system containing phagocyte membrane vesicles and p67(phox), in the absence of an activating amphiphile and of p47(phox). Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same conditions. Resu lts obtained with phagocyte membrane vesicles can be reproduced fully by re placing these with partially purified cytochrome b(559), incorporated in ph osphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds s pontaneously to phagocyte membrane vesicles and also to artificial, protein -free, phosphatidylcholine vesicles, a process counteracted by GDP dissocia tion inhibitor for Rho. Binding of prenylated Rac1 to membrane vesicles is accompanied by the recruitment of p67(phox) to the same location and the fo rmation of an assembled NADPH oxidase complex, producing O-2 upon the addit ion of NADPH. Amphiphile and p47(phox)-independent NADPH oxidase activation by prenylated Rad is inhibited by Rho GDP dissociation inhibitor and by ph osphatidylcholine vesicles, both competing with membrane for prenylated Rac 1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane i s sufficient for the induction of NADPH oxidase assembly, suggesting that t he principal or, possibly, the only role of Rac is to recruit cytosolic p67 (phox) to the membrane environment, to be followed by the interaction of p6 7(phox) with cytochrome b(559).