Reconstitution of phagosome-lysosome fusion in streptolysinO-permeabilized cells

Funato, Kouichi ; Beron, Walter ; Yang, Z. Chun ; Mukhopadhyay, Amitabha ; Stahl, D. Philip (1997) Reconstitution of phagosome-lysosome fusion in streptolysinO-permeabilized cells Journal of Biological Chemistry, 272 (26). pp. 16147-16151. ISSN 0021-9258

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We have reconstituted fusion between phagosomes and lysosomes in streptolysin O-permeabilized J774-E macrophages. Fusion was assessed by measuring the delivery of avidin-conjugated horseradish peroxidase pre-internalized into lysosomes to phagosomes containing biotinylated β -glucuronidase-conjugated paramagnetic beads (1-2 μm). Fusion was dependent on energy and exogenously supplied cytosol. Phagosome-lysosome fusion was greatly inhibited when microtubules were depolymerized by nocodazole treatment, suggesting that fusion occurs via microtubule-dependent transport. Furthermore, fusion was inhibited by GTPγ S and Rab GDP dissociation inhibitor. These results suggest that rab proteins 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 that NSF is required for fusion between phagosomes and lysosomes.

Item Type:Article
Source:Copyright of this article belongs to The American Society for Biochemistry and Molecular Biology.
ID Code:70210
Deposited On:21 Nov 2011 11:00
Last Modified:21 Nov 2011 11:00

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