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 p40phox interact in intact 
cells, which we suggested maintains this protein in an inactive closed 
conformation. Using this system, we also explored intramolecular interactions 
within p47phox 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 p67phox with phagosomes involving adaptor proteins, p47phox and 
p40phox, during FcgammaR-mediated phagocytosis. Although p67phox is not targeted 
to phagosomes by itself, p47phox functions as an adaptor for the ternary complex 
(p47phox-p67phox-p40phox) in early stages of phagocytosis before phagosome 
closure, while p40phox functions in later stages after phagosomal closure. 
Interestingly, a mutated "open" form of p40phox linked p47phox to closed 
phagosomes and prolonged p47phox and p67phox 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.