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(Received for publication, June 30, 1995, and in revised form, March 20, 1996)
From the Survival or destruction of a pathogen following
phagocytosis depends, in part, on fusion events between the phagosome
and the endosomal or lysosomal compartments. Here we use an in
vitro assay to show that phagosome-endosome fusion is regulated
by the small GTPase rab5 and that fusion events are influenced by an
internalized live organism, Listeria monocytogenes (LM). We
compare the in vitro fusion of phagosomes containing
heat-killed organisms (dead LM) with that of phagosomes containing a
live nonhemolytic mutant (live LMhly
Volume 271, Number 23,
Issue of June 7, 1996
pp. 13834-13843
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,
,¶
,
Department of Cell Biology and Physiology,
Washington University School of Medicine, St. Louis, Missouri
63110, ¶ Instituto de Histologia y Embriologia, CONICET, Facultad
de Ciencias Medicas, Universidad Nacional de Cuyo, Mendoza,
Argentina, and the '' Department of Biochemistry, Molecular Biology
and Cell Biology, Northwestern University, Evanston, Illinois
60208
). Unlike the
wild-type organism, LMhly remains trapped inside the
phagosome. Phagosome-endosome fusion was reconstituted using
biotinylated organisms and endosomes containing horseradish
peroxidase conjugated with avidin. With both live
LMhly and dead LM preparations, in vitro
phagosome-endosome fusion was time-, temperature-, and
cytosol-dependent. Live LMhly phagosomes
exhibited a faster rate of fusion. Fusion in both preparations was
regulated by rab5 and possibly by other GTPases. Anti-rab5 antibodies
and immunodepletion of cytosolic rab5 inhibited fusion. Addition of
glutatione S-transferase-rab5 in the GTP form stimulated
phagosome-endosome fusion, whereas addition of a dominant negative
mutant of rab5 blocked fusion. Purified live LMhly
phagosomal membranes were enriched in rab5 as revealed by Western
blotting, compared with dead LM phagosomes. Fusion of endosomes with
dead LM containing phagosomes required ATP and was inhibited by ATP
depletion and by N-ethylmaleimide (NEM) and
anti-NEM-sensitive factor (NSF) antibodies. Unexpectedly,
phagosome-endosome fusion with live LMhly-containing
phagosomes was not inhibited by ATP depletion nor by NEM or anti-NSF
antibodies. Western blot analysis revealed that live
LMhly-containing phagosomes were enriched for
membrane-bound NSF, while dead LM containing phagosomes contained low
or undetectable quantities. Washing live LMhly-containing
phagosomes with 0.5 M KCl removed NSF associated with the
membranes and rendered them NEM, ATP, anti-NSF antibody sensitive for
fusion. We conclude that rab5 regulates phagosome-endosome fusion and
that live microorganisms can up-regulate this process by recruiting
rab5 to the membrane.
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