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 beta-COP to highly purified phagosomes. The binding of beta-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 beta-COP to phagosomes independently of added nucleotide. AlF(4) and Gbetagamma subunits, agents known to modulate heterotrimeric G-protein activity, were tested in the beta-COP binding assay. AlF(4) increased beta-COP association, whereas binding was inhibited by the addition of Gbetagamma 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.
Berón
W
W
Instituto de Histología y Embriología-CONICET, Universidad Nacional de Cuyo, Casilla de Correo 56, (5500) Mendoza, Argentina. wberon@fmed2.uncu.edu.ar
Mayorga
L S
LS
Colombo
M I
MI
Stahl
P D
PD
D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology*; D002460:Cell Line; D020756:Coatomer Protein / Q000378:metabolism; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008854:Microscopy, Electron; D010588:Phagosomes / Q000378:metabolism* / Q000648:ultrastructure
D020756:Coatomer Protein; D020126:Brefeldin A; D020727:ADP-Ribosylation Factors