Fusion of Newly Formed Phagosomes with Endosomes in Intact Cells and in a Cell-free System

Phagosomes are membrane-bound vesicles, formed by the receptor-mediated internalization of particulate ligands, which exchange soluble and membrane proteins with other endocytic compartments as a part of their maturation process. This exchange of material is undoubtedly mediated by fusion of phagosomes with other membrane-bound compartments of the endocytic pathway. By using a particulate probe (fixed Staphylococcus aureus coated with mouse anti-dinitrophenol monoclonal antibody) localized in phagosomes and a soluble probe (dinitrophenol-derivitized beta-glucuronidase) internalized by receptor-mediated endocytosis, we have studied phagosome-endosome and phagosome-lysosome fusion in intact cells and in a cell-free system. Vesicle fusion was assessed by measuring beta-glucuronidase activity associated with S. aureus particles after lysis of the membranes. In intact macrophages, newly formed phagosomes fused with early endosomes and with lysosomes. Fusion with lysosomes was observed to commence after a short lag period of about 5 min. In broken-cell preparations, phagosomes were able to fuse with early endosomes. It was not possible to reconstitute phagosome-lysosome fusion in vitro. In vitro phagosome-endosome fusion required energy and cytosolic- and membrane-associated proteins. A nonhydrolyzable analog of GTP stimulated fusion at low cytosol concentrations and inhibited fusion at high cytosol concentrations. These observations indicate that the mechanisms mediating phagosome-endosome fusion are similar to those described for endosome-endosome fusion. Our results suggest that exchange of material with endosomes is an important step in the process of phagosome maturation.