Syntaxin 7 and VAMP-7 are soluble N-ethylmaleimide-sensitive factor attachment protein receptors required for late endosome-lysosome and homotypic lysosome fusion in alveolar macrophages

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 popula tion was capable of homotypic fusion. All vesicle populations were also cap able 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. Lys osomes fuse with 12-min endosomes but not with earlier endosomes. Using hom ogenous populations of endosomes, we have identified Syntaxin 7 as a solubl e N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) req uired for late endosome-lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inh ibited homotypic late endosome and lysosome fusion as well as heterotypic l ate endosome-lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on ho motypic early endosome fusion. Previous work suggested that human VAMP-7 (v esicle-associated membrane protein-7) was a SNARE required for late endosom e-lysosome fusion. A bacterially expressed human VAMP-7 lacking the transme mbrane domain inhibited both late endosome-lysosome fusion and homotypic ly sosome fusion in vitro. These studies indicate that: 1) fusion along the en docytic 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.