Munc13-4 functions as a Ca(2+) sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles

Munc13-4 is a Ca(2+)-dependent SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca(2+)-evoked secretion in various secretory cells. Studies in mast cell–like RBL-2H3 cells provide direct evidence that Munc13–4 with its two Ca(2+)-binding C2 domains functions as a Ca(2+) sensor for SG exocytosis. Unexpectedly, Ca(2+) stimulation also generated large (>2.4 μm in diameter) Munc13-4(+)/Rab7(+)/Rab11(+) endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4(+)/Rab7(+) SGs, followed by a merge with Rab11(+) endosomes, and depended on Ca(2+) binding to Munc13-4. Munc13-4 promoted the Ca(2+)-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca(2+) sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells.