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 FcγR-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.