Sequential binding of cytosolic phox complex to phagosomes through regulated adaptor proteins: Evaluation using the novel monomeric kusabira-green system and live imaging of phagocytosis

We engineered a method for detecting intramolecular and intermolecular phox protein interactions in cells by fluorescence microscopy using fusion proteins of complementary fragments of a coral fluorescent reporter protein (monomeric Kusabira-Green). We confirmed the efficacy of the monomeric Kusabira-Green system by showing that the PX and PB1 domains of p40(phox) interact in intact cells, which we suggested maintains this protein in an inactive closed conformation. Using this system, we also explored intramolecular interactions within p47(phox) and showed that the PX domain interacts with the autoinhibited tandem Src homology 3 domains maintained in contact with the autoinhibitory region, along with residues 341-360. Furthermore, we demonstrated sequential interactions of p67(phox) , With phagosomes involving adaptor proteins, p47(phox) and p40(phox), during Fc gamma R-mediated phagocytosis. Although p67(phox) is not targeted to phagosomes by itself, p47(phox) functions as an adaptor for the ternary complex (p47(phox), -p67(phox)-p40(phox)) in early stages of phagocytosis before phagosome closure, while p40(phox) functions in later stages after phagosomal closure. Interestingly, a mutated open form of p40(phox), linked p47(phox), to closed phagosomes and prolonged p47(phox) and p67(phox) retention on phagosomes. These results indicate that binding of the ternary complex to phagosomes can be temporally regulated by switching between adaptor proteins that have PX domains with distinct lipid-binding specificities.