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J. Cell Biol.,
Volume 139, Number 3, November 3, 1997 665-673
Department of Pathology, Division of Cell Biology and Immunology, University of Utah Health Science Center, Salt Lake City,
Utah 84132
Lysosomes are dynamic structures capable of
fusing with endosomes as well as other lysosomes. We
examined the biochemical requirements for homotypic
lysosome fusion in vitro using lysosomes obtained from
rabbit alveolar macrophages or the cultured macrophage-like cell line, J774E. The in vitro assay measures
the formation of a biotinylated HRP-avidin conjugate,
in which biotinylated HRP and avidin were accumulated in lysosomes by receptor-mediated endocytosis.
We determined that lysosome fusion in vitro was time- and temperature-dependent and required ATP and an
N-ethylmaleimide (NEM)-sensitive factor from cytosol.
The NEM-sensitive factor was NSF as purified recombinant NSF could completely replace cytosol in the fusion assay whereas a dominant-negative mutant NSF
inhibited fusion. Fusion in vitro was extensive; up to
30% of purified macrophage lysosomes were capable of
self-fusion. Addition of GTP
s to the in vitro assay inhibited fusion in a concentration-dependent manner. Purified GDP-dissociation inhibitor inhibited homotypic lysosome fusion suggesting the involvement of
rabs. Fusion was also inhibited by the heterotrimeric G
protein activator mastoparan, but not by its inactive analogue Mas-17. Pertussis toxin, a G
i activator, inhibited in vitro lysosome fusion whereas cholera toxin, a
Gs activator did not inhibit the fusion reaction. Addition of agents that either promoted or disrupted microtubule function had little effect on either the extent or
rate of lysosome fusion. The high value of homotypic
fusion was supported by in vivo experiments examining
lysosome fusion in heterokaryons formed between cells containing fluorescently labeled lysosomes. In both
macrophages and J774E cells, almost complete mixing
of the lysosome labels was observed within 1-3 h of UV
sendai-mediated cell fusion. These studies provide a
model system for identifying the components required
for lysosome fusion.
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