JCB_201702099 2151..2166

Dense-core vesicle (DCV) exocytosis is essential for peptidergic and aminergic signaling in the nervous, endocrine, and immune systems. DCV biogenesis in the TGN, DCV transport across the cytoplasm, and DCV docking/priming and fusion at the plasma membrane use similar mechanisms in different secretory cell types, but many aspects remain incompletely understood. After budding from the TGN, immature DCVs undergo maturation by endosomal retrieval to become fully functional (Kögel and Gerdes, 2010). Mature DCVs are recruited to the plasma membrane where they are docked and primed by several protein factors that assemble vesicle and plasma membrane SNA RE proteins into trans-complexes (Rizo and Xu, 2015). The Ca2+-triggered fusion of DCVs with the plasma membrane is mediated by Ca2+ sensors acting on SNA RE complexes and the plasma membrane (Südhof and Rothman, 2009; Jahn and Fasshauer, 2012). Unlike synaptic vesicles that recycle locally, DCVs undergo compensatory endocytosis, which recycles DCV membrane proteins through retrograde trafficking to the TGN (Farquhar, 1983; Bauer et al., 2004). C2 domain–containing proteins play essential roles at multiple steps in anterograde DCV-mediated trafficking. C2 domains are comprised of eight antiparallel β-strands connected by surface loops (Sutton and Sprang, 1998). Some C2 domains bind phospholipids in a Ca2+-dependent manner through negatively charged surface loop residues that form Ca2+ binding sites with the membrane (Corbalan-Garcia and Gómez-Fernández, 2014). This enables C2 domain–containing proteins to mediate Ca2+-dependent membrane processes such as for Munc13-1/2 (C2B domain) function in vesicle priming or for synaptotagmin function in fusion triggering (Südhof, 2014). Some C2 domains do not bind Ca2+ but mediate protein–protein interactions, such as for Munc13-1/2 (C2A domain) and CAPS (calciumdependent activator protein for secretion 1) function in vesicle docking and priming (Lu et al., 2006; Petrie et al., 2016). Munc13 proteins are an important class of C2 domain– containing proteins, with Munc13-1, -2, and -3 regulating Ca2+-dependent vesicle priming in neurons and endocrine cells (Koch et al., 2000; Kwan et al., 2006; Man et al., 2015). Similarly, Munc13-4 is essential for the Ca2+-dependent priming of secretory granules in immune cells (Feldmann et al., 2003; Boswell et al., 2012; Johnson et al., 2016) but also functions in the Ca2+-dependent regulation of late endosomal fusion events Dense-core vesicle (DCV) exocytosis is a SNA RE (soluble N-ethylmaleimide–sensitive fusion attachment protein receptor)dependent anterograde trafficking pathway that requires multiple proteins for regulation. Several C2 domain– containing proteins are known to regulate Ca2+-dependent DCV exocytosis in neuroendocrine cells. In this study, we identified others by screening all (∼139) human C2 domain–containing proteins by RNA interference in neuroendocrine cells. 40 genes were identified, including several encoding proteins with known roles (CAPS [calcium-dependent activator protein for secretion 1], Munc13-2, RIM1, and SYT10) and many with unknown roles. One of the latter, BAI AP3, is a secretory cell–specific Munc13-4 paralog of unknown function. BAI AP3 knockdown caused accumulation of fusionincompetent DCVs in BON neuroendocrine cells and lysosomal degradation (crinophagy) of insulin-containing DCVs in INS-1 β cells. BAI AP3 localized to endosomes was required for Golgi trans-Golgi network 46 (TGN46) recycling, exhibited Ca2+-stimulated interactions with TGN SNA REs, and underwent Ca2+-stimulated TGN recruitment. Thus, unlike other Munc13 proteins, BAI AP3 functions indirectly in DCV exocytosis by affecting DCV maturation through its role in DCV protein recycling. Ca2+ rises that stimulate DCV exocytosis may stimulate BAI AP3-dependent retrograde trafficking to maintain DCV protein homeostasis and DCV function. BAI AP3, a C2 domain–containing Munc13 protein, controls the fate of dense-core vesicles in neuroendocrine cells