A Fluorescently Tagged C-Terminal Fragment of p47phoxDetects NADPH Oxidase Dynamics during Phagocytosis

Abstract

The assembly of cytosolic p47^phoxand p67^phoxwith flavocytochrome b₅₅₈at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47^phoxand p67^phoxare linked via a high-affinity, tail-to-tail interaction involving a proline-rich region (PRR) and a C-terminal SH3 domain (SH3b), respectively, in their C-termini. This interaction mediates p67^phoxtranslocation in neutrophils, but is not required for oxidase activity in model systems. Here we examined phagocytosis-induced NADPH oxidase assembly, showing the sequential recruitment of YFP-tagged p67^phoxto the phagosomal cup, and, after phagosome internalization, a probe for PI(3)P followed by a YFP-tagged fragment derived from the p47^phoxPRR. This fragment was recruited in a flavocytochrome b₅₅₈-dependent, p67^phox-specific, and PI(3)P-independent manner. These findings indicate that p47PRR fragment probes the status of the p67^phoxSH3b domain and suggest that the p47^phox/p67^phoxtail-to-tail interaction is disrupted after oxidase assembly such that the p67^phox-SH3b domain becomes accessible. Superoxide generation was sustained within phagosomes, indicating that this change does not correlate with loss of enzyme activity. This study defines a sequence of events during phagocytosis-induced NADPH oxidase assembly and provides experimental evidence that intermolecular interactions within this complex are dynamic and modulated after assembly on phagosomes.