Activation of the superoxide-generating NADPH oxidase by chimeric proteinsconsisting of segments of the cytosolic component p67(phox) and the small GTPase Rac1

Activation of the superoxide (O-2(-))-generating NADPH oxidase of phagocyte s is the consequence of the assembly of a membrane-associated flavocytochro me b(559) with the cytosolic proteins p47(phox) and p67(phox) and the small GTPase Rac (1 or 2). We proposed that Rac I serves as a membrane-targeting molecule for p67(phox). This hypothesis was tested by constructing recombi nant chimeric proteins, joining various functional domains of p67(phox) and Rac 1, and expressing these in Escherichia coli. Chimeras were assayed for the ability to support O-2(-) production by phagocyte membranes in an amph iphile-activated cell-free system in the presence or absence of p47phox. A chimera consisting of p67(phox) truncated at residue 212 and fused to a ful l-length Rac1 [p67(phox)(1-212)-Rac1(1-192)] was a potent NADPH oxidase act ivator. A p67(phox)(1-212)-Rac1(178-192) chimera, to which Rac1 contributed only the C-terminal polybasic domain, was a weaker but consistent activato r. Chimeras comprising the full length of Rae I bound GTP/ GDP, like bona f ide GTPases, The activity of p67(phox)-Rac1 chimeras was dependent on the p resence of the tetratricopeptide repeat and activation domains, in the p67( phox) segment, and on an intact polybasic region, at the C terminus of the Rac1 segment, but not on the insert region of Rac1. Partial activation by c himeras, in the GTP-bound form, was also possible in the absence of p47(pho x). Evidence is offered in support of the proposal that the GTP- and GDP-bo und forms of chimera p67phox(1 -212)-Rac1 (1 - 192) have distinct conformat ions, corresponding to the presence and absence of intrachimeric bonds, res pectively.