Volume 272, Number 26, Issue of June 27, 1997 pp. 16147-16151
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Reconstitution of Phagosome-Lysosome Fusion in Streptolysin O-permeabilized Cells

(Received for publication, February 6, 1997, and in revised form, April 28, 1997)

Kouichi Funato , Walter Beron ^§ , Chun Z. Yang , Amitabha Mukhopadhyay ^** and Philip D. Stahl

From the  Animal and Cellular Systems Lab, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-01, Japan, ^§ Universidad Nacional de Cuyo, Facultad de Ciencias Medican, Instituto de Histologia y Embiologia, Casilla de Correo 56, 5500 Mendoza, Argentina, the ^** Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India, and the  Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

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 -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 GTPS 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.

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