Abstract
Compound exocytosis is considered the most massive mode of exocytosis, during which the membranes of secretory granules (SGs) fuse with each other to form a channel through which the entire contents of their granules is released. The underlying mechanisms of compound exocytosis remain largely unresolved. Here we show that the small GTPase Rab5, a known regulator of endocytosis, is pivotal for compound exocytosis in mast cells. Silencing of Rab5 shifts receptor-triggered secretion from a compound to a full exocytosis mode, in which SGs individually fuse with the plasma membrane. Moreover, we show that Rab5 is essential for FcεRI-triggered association of the SNARE protein SNAP23 with the SGs. Direct evidence is provided for SNAP23 involvement in homotypic SG fusion that occurs in the activated cells. Finally, we show that this fusion event is prevented by inhibition of the IKKβ2 kinase, however, neither a phosphorylation-deficient nor a phosphomimetic mutant of SNAP23 can mediate homotypic SG fusion in triggered cells. Taken together our findings identify Rab5 as a heretofore-unrecognized regulator of compound exocytosis that is essential for SNAP23-mediated granule-granule fusion. Our results also implicate phosphorylation cycles in controlling SNAP23 SNARE function in homotypic SG fusion.
Highlights
Regulated exocytosis is a key mechanism for intercellular communication and contributes to host defenses against environmental challenges
Overexpression of neither the phosphorylation-deficient mutant (PD-SNAP23) nor the phosphomimetic mutant (PM-SNAP23) detectably affected IgE/Ag-stimulated exocytosis. These results suggest that both IKKβ2-mediated phosphorylation of SNAP23 and SNAP23 dephosphorylation are required for trigger-dependent SG fusion, we cannot exclude the possibility that the introduced mutations in PM-SNAP23 fail to fully recapitulate its genuine phosphorylated state
A role for SNAP23 has been implicated in compound exocytosis based on its redistribution from the plasma membrane to SGs, with kinetics paralleling that of compound exocytosis[13]
Summary
Regulated exocytosis is a key mechanism for intercellular communication and contributes to host defenses against environmental challenges. Knockdown of SNAP23 reduced FcεRI-stimulated secretion by 30%23,24, whereas redistribution from the plasma membrane to SGs occurred in permeabilized cells into which calcium and GTPγS had been introduced, conditions that parallel stimulated compound exocytosis[13]. These results do not identify the exact step that is regulated by this SNARE. The opposing effects exerted by SNAP23 on granule fusion with the plasma membrane in pancreatic exocrine and endocrine secretion[25], taken together with the well documented involvement of SNAP23 in multiple cellular processes, including the fusion of recycling endosomes with the plasma membrane[26], raises the possibilities that SNAP23 either may impact exocytosis indirectly, by affecting endocytic recycling which influences exocytosis[27,28], or may contribute to exocytosis directly, by enhancing or inhibiting SG fusion with the plasma membrane and/or mediating granule–granule fusion during compound exocytosis
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