Ca2+ and N-Ethylmaleimide-sensitive Factor Differentially Regulate Disassembly of SNARE Complexes on Early Endosomes

The endosome-associated protein Hrs inhibits the homotypic fusion of early endosomes. A helical region of Hrs containing a Q-SNARE motif mediates this effect as well as its endosomal membrane association via SNAP-25, an endosomal receptor for Hrs. Hrs inhibits formation of an early endosomal SNARE complex by displacing VAMP-2 from the complex, suggesting a mechanism by which Hrs inhibits early endosome fusion. We examined the regulation of endosomal SNARE complexes to probe how Hrs may function as a negative regulator. We show that although NSF dissociates the VAMP-2·SNAP-25·syntaxin 13 complex, it has no effect on the Hrs-containing complex. Whereas Ca 2+ dissociates the Hrs-containing complex but not the VAMP-2-containing SNARE complex. This is the first demonstration of differential regulation of R/Q-SNARE and all Q-SNARE-containing SNARE complexes. Ca 2+ also reverses the Hrs-induced inhibition of early endosome fusion in a tetanus toxin-sensitive manner and removes Hrs from early endosomal membranes. Moreover, Hrs inhibition of endosome fusion and its endosomal localization are sensitive to bafilomycin, implying a role for luminal Ca 2+ . Thus, Hrs may bind a SNARE protein on early endosomal membranes negatively regulating trans- SNARE pairing and endosomal fusion. The release of Ca 2+ from the endosome lumen dissociates Hrs, allowing a VAMP-2-containing complex to form enabling fusion.