Recruitment of coat-protein-complex proteins on to phagosomal membranes is regulated by a brefeldin A-sensitive ADP-ribosylation factor

Particle internalization in macrophages is followed by a complex maturation process. We have previously observed that proteins bound to phagocytosed particles are sorted from phagosomes into a heterogeneous population of vesicles that fuse with endosomes. However, the mechanism and the protein machinery involved in the formation of these phagosome-derived vesicles are largely unknown. It has been shown that vesicles coated with coat protein complex type I (COPI) have a role in both secretion and endocytosis. To address the possibility that COPI proteins might participate in the formation of phagosome-derived vesicles we studied the recruitment of β-COP to highly purified phagosomes. The binding of β-COP to phagosomal membranes was regulated by nucleotides and inhibited by brefeldin A (BFA). An ADP-ribosylation factor 1 (ARF1) mutant defective in GTP hydrolysis supported the binding of β-COP to phagosomes independently of added nucleotide. AlF4 and Gβγ subunits, agents known to modulate heterotrimeric G-protein activity, were tested in the β-COP binding assay. AlF4 increased β-COP association, whereas binding was inhibited by the addition of Gβγ subunits. Our results suggest that COP proteins are recruited to phagosomal membranes by a mechanism that involves heterotrimeric GTP-binding proteins and a BFA-sensitive ARF. In addition, our findings indicate that COPI proteins are involved in the recycling of components from phagosomes to the cell surface.