Activation of the Superoxide-Generating NADPH Oxidase by Chimeric Proteins Consisting of Segments of the Cytosolic Component p67phoxand the Small GTPase Rac1

Abstract

Activation of the superoxide (O₂^-)-generating NADPH oxidase of phagocytes is the consequence of the assembly of a membrane-associated flavocytochrome b₅₅₉ with the cytosolic proteins p47^phox and p67^phox and the small GTPase Rac (1 or 2). We proposed that Rac1 serves as a membrane-targeting molecule for p67^phox. This hypothesis was tested by constructing recombinant chimeric proteins, joining various functional domains of p67^phox and Rac1, and expressing these in Escherichia coli. Chimeras were assayed for the ability to support O₂^- production by phagocyte membranes in an amphiphile-activated cell-free system in the presence or absence of p47^phox. A chimera consisting of p67^phox truncated at residue 212 and fused to a full-length Rac1 [p67^phox(1−212)−Rac1(1−192)] was a potent NADPH oxidase activator. A p67^phox(1−212)−Rac1(178−192) chimera, to which Rac1 contributed only the C-terminal polybasic domain, was a weaker but consistent activator. Chimeras comprising the full length of Rac1 bound GTP/GDP, like bona fide GTPases. The activity of p67^phox−Rac1 chimeras was dependent on the presence 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 chimeras, in the GTP-bound form, was also possible in the absence of p47^phox. Evidence is offered in support of the proposal that the GTP- and GDP-bound forms of chimera p67^phox(1−212)−Rac1(1−192) have distinct conformations, corresponding to the presence and absence of intrachimeric bonds, respectively.