Recruitment of coat-protein-complex proteins on to phagosomal membranes isregulated 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 p articles are sorted from phagosomes into a heterogeneous population of vesi cles that fuse with endosomes. However, the mechanism and the protein machi nery involved in the formation of these phagosome-derived vesicles are larg ely unknown. It has been shown that vesicles coated with coat protein compl ex type I (COPI) have a role in both secretion and endocytosis. To address the possibility that COPI proteins might participate in the formation of ph agosome-derived vesicles we studied the recruitment of beta -COP to highly purified phagosomes. The binding of beta -COP to phagosomal membranes was r egulated by nucleotides and inhibited by brefeldin A (BFA). An ADP-ribosyla tion factor 1 (ARF1) mutant defective in GTP hydrolysis supported the bindi ng of beta -COP to phagosomes independently of added nucleotide. AlF, and G beta gamma subunits, agents known to modulate heterotrimeric G-protein act ivity, were tested in the beta -COP binding assay. A1F, increased beta -COP association, whereas binding was inhibited by the addition of G beta gamma subunits. Our results suggest that COP proteins are recruited to phagosoma l membranes by a mechanism that involves heterotrimeric GTP-binding protein s and a BFA-sensitive ARF. In addition, our findings indicate that COPI pro teins are involved in the recycling of components from phagosomes to the ce ll surface.