The R-SNARE endobrevin/VAMP-8 mediates homotypic fusion of early endosomesand late endosomes

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown i n which intracellular fusion step it operates. Using subcellular fractionat ion and quantitative immunogold electron microscopy, we found that endobrev in/VAMP-8 is present on all membranes known to communicate with early endos omes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of the trans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potent ly inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevi n/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of ea rly and late endosomes.