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Vol. 11, Issue 7, 2327-2333, July 2000
*Division of Cell Biology and Immunology, Department of Pathology,
University of Utah School of Medicine, Salt Lake City, Utah 84132; and
Endocytosis in alveolar macrophages can be reversibly inhibited,
permitting the isolation of endocytic vesicles at defined stages of
maturation. Using an in vitro fusion assay, we determined that each
isolated endosome population was capable of homotypic fusion. All
vesicle populations were also capable of heterotypic fusion in a
temporally specific manner; early endosomes, isolated 4 min after
internalization, could fuse with endosomes isolated 8 min after
internalization but not with 12-min endosomes or lysosomes. Lysosomes
fuse with 12-min endosomes but not with earlier endosomes. Using
homogenous populations of endosomes, we have identified Syntaxin 7 as a
soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome-lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late
endosome and lysosome fusion as well as heterotypic late endosome-lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no
affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was
a SNARE required for late endosome-lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both
late endosome-lysosome fusion and homotypic lysosome fusion in vitro.
These studies indicate that: 1) fusion along the endocytic pathway is a
highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and
human VAMP-7, are involved in fusion of vesicles in the late endocytic
pathway in alveolar macrophages.
Department of Molecular Neurobiology, Kennedy Krieger
Institute and The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
Corresponding author. E-mail
address: kaplan{at}bioscience.biology.utah.edu.
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