In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47 phox , p67 phox , p40 phox , and Rac) translocate and associate with the membrane-spanning flavocytochrome b 558 , leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40 phox that regulate its translocation to membranes upon stimulation. GFP-p40 phox translocates to early endosomes, whereas GFP-p47 phox translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67 phox does not translocate to membranes when expressed alone, but it is dependent on p40 phox and p47 phox for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40 phox or GFP-p47 phox to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67 phox translocation to either membrane is abolished by mutations that disrupt its interaction with p40 phox or p47 phox . Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40 phox in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40 phox and p47 phox function as diverse p67 phox "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms. INTRODUCTIONIn phagocytic cells, reactive oxygen species (ROS) are produced by NADPH oxidase (Nox2 system). The enzyme is a multiprotein complex assembled from a membrane-spanning flavocytochrome b 558 (composed of gp91 phox [Nox2] and p22 phox ) and four cytoplasmic components (p47 phox , p67 phox , p40 phox , and Rac) (Leto, 1999;Nauseef, 2004;Quinn and Gauss, 2004). In unstimulated phagocytes, the oxidase is dissociated and inactive: the flavocytochrome b 558 is stored on the membranes of intracellular granules (Jesaitis et al., 1990), Rac is maintained in a GDP-bound cytoplasmic complex dimerized with Rho-guanine nucleotide dissociation inhibitor (GDI) , and the other phox proteins associate in a separate ternary cytoplasmic complex (p47 phox -p67 phox -p40 phox ) in a dephosphorylated state (Bolscher et al., 1989;Rotrosen and Leto, 1990;Kuribayashi et al., 2002;Lapouge et al., 2002). During phagocyte activation, intracellular granules containing flavocytochrome b 558 fuse with phagosomes; p47 phox is phosphorylated, thereby inducing conformational changes in p47 phox that promote the interaction of the cytoplasmic complex with the flavocytochrome b 558 ; and Rac dissociates from Rho-GDI and translocates independently to the membrane after exchange of GDP for GTP (Heyworth et al., 1994;Zhao et al., 2003), resulting in generation of superoxide anion by the transfer of electrons from cytoplasmic NADPH to molecular oxygen.Chronic granulomatous disease...

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