RECONSTITUTION OF PHAGOSOME-LYSOSOME FUSION IN STREPTOLYSIN O-PERMEABILIZED CELLS

We have reconstituted fusion between phagosomes and lysosomes in strep tolysin O-permeabilized J774-E macrophages. Fusion was assessed by mea suring the delivery of avidin-conjugated horseradish peroxidase pre-in ternalized into lysosomes to phagosomes containing biotinylated beta-g lucuronidase-conjugated paramagnetic beads (1-2 mu m). Fusion was depe ndent on energy and exogenously supplied cytosol. Phagosome-lysosome f usion was greatly inhibited when microtubules were depolymerized by no codazole treatment, suggesting that fusion occurs via microtubule-depe ndent transport. Furthermore, fusion was inhibited by GTP gamma S and Rab GDP dissociation inhibitor. These results suggest that rab protein s are involved in the regulation of fusion. Lastly, anti-NEM-sensitive factor (NSF) antibodies inhibited fusion, and addition of recombinant NSF wild type partially restored the fusogenic activity, indicating t hat NSF is required for fusion between phagosomes and lysosomes.