Abstract
Dense-core vesicle (DCV) exocytosis is a SNARE (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, BAIAP3, is a secretory cell-specific Munc13-4 paralog of unknown function. BAIAP3 knockdown caused accumulation of fusion-incompetent DCVs in BON neuroendocrine cells and lysosomal degradation (crinophagy) of insulin-containing DCVs in INS-1 β cells. BAIAP3 localized to endosomes was required for Golgi trans-Golgi network 46 (TGN46) recycling, exhibited Ca2+-stimulated interactions with TGN SNAREs, and underwent Ca2+-stimulated TGN recruitment. Thus, unlike other Munc13 proteins, BAIAP3 functions indirectly in DCV exocytosis by affecting DCV maturation through its role in DCV protein recycling. Ca2+ rises that stimulate DCV exocytosis may stimulate BAIAP3-dependent retrograde trafficking to maintain DCV protein homeostasis and DCV function.
Highlights
Dense-core vesicle (DCV) exocytosis is essential for peptidergic and aminergic signaling in the nervous, endocrine, and immune systems
Z score, which measures the number of SDs from a sample siRNA to the nontargeting siRNA (Birmingham et al, 2009), was used to identify inhibitory siRNAs. siRNA pools targeting three genes required for regulated exocytosis (CADPS [CAPS], SNAP25, and STX1A [syntaxin 1A]) were used as controls. siRNAs targeting CADPS strongly inhibited the percent secretion of neuropeptide Y (NPY)-Venus (z score = −8.7), whereas targeting SNAP25 and STX1A resulted in milder inhibition (z score = −2.4 and −2.1, respectively)
We report the first systematic screen of C2 domain–containing proteins in the regulated secretory pathway of neuroendocrine cells
Summary
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 SNARE 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 SNARE 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)
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