When the neutrophil
NADPH oxidase is activated to generate
superoxide, the cytosolic components, p47phox,
p67phox, and the
GTP-binding protein Rac, become stably associated with the plasma membrane. The translocation of p47phox and
p67phox is dependent on the presence in the membrane of the
flavocytochrome b558, itself composed of two subunits, gp91phox and p22phox. In this study we have quantitated Rac1 and Rac2 in human neutrophils and show that > 96% of the Rac
protein is Rac2 and that
chronic granulomatous disease (CGD) neutrophils contain normal levels of the
protein. We used a cell-free translocation system as well as intact normal and CGD neutrophils to determine whether the translocation of Rac2 is dependent upon the presence of the other
oxidase components. When cell-free reactions contained any combination of normal, p47phox-deficient or p67phox-deficient cytosol and normal or flavocytochrome b558-deficient membranes, the
GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-dependent association of Rac2 with the re-isolated membranes was not significantly different from the control mixture of normal membranes and cytosol. In intact CGD neutrophils lacking p47phox or
p67phox and stimulated with
phorbol myristate acetate the translocation of Rac2 was also normal, and we conclude that Rac translocation can occur independently of the cytosolic
NADPH oxidase components. In contrast, in the absence of
flavocytochrome b558 from intact X-chromosome linked CGD neutrophils, Rac2 translocation was reduced to only 25% of the control value. On the basis of these observations we propose that while Rac2 can bind to a site distinct from either gp91phox or p22phox, it depends upon an interaction with the
flavocytochrome b558 for maximal stability in the membrane.