Previous studies indicated that CAPS (calcium-dependent activator protein for secretion) functions as an essential component for the Ca 2ϩ -dependent exocytosis of dense-core vesicles in neuroendocrine cells. However, recent mouse knock-out studies suggested an alternative role in the vesicular uptake or storage of catecholamines. To genetically assess the functional role of CAPS, we characterized the sole Caenorhabditis elegans CAPS ortholog UNC-31 (uncoordinated family member) and determined its role in dense-core vesiclemediated peptide secretion and in synaptic vesicle recycling. Novel assays for dense-core vesicle exocytosis were developed by expressing a prepro-atrial natriuretic factor-green fluorescent protein fusion protein in C. elegans. unc-31 mutants exhibited reduced peptide release in vivo and lacked evoked peptide release in cultured neurons. In contrast, cultured neurons from unc-31 mutants exhibited normal stimulated synaptic vesicle recycling measured by FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl) hexatrienyl)pyridinium dibromide] dye uptake. Conversely, UNC-13, which exhibits sequence homology to CAPS/UNC-31, was found to be essential for synaptic vesicle but not dense-core vesicle exocytosis. These findings indicate that CAPS/UNC-31 function is not restricted to catecholaminergic vesicles but is generally required for and specific to dense-core vesicle exocytosis. Our results suggest that CAPS/ UNC-31 and UNC-13 serve parallel and dedicated roles in dense-core vesicle and synaptic vesicle exocytosis, respectively, in the C. elegans nervous system.

Ca 2؉ -dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca 2؉ -triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P2 in acceptor liposomes and was independent of Ca 2؉ , but Ca 2؉ dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.vesicle exocytosis ͉ priming ͉ PIP2 ͉ contents mixing V esicle fusion in the secretory pathway employs SNARE proteins, members of a conserved family of membrane-associated proteins that contain approximately 60-aa heptad repeat motifs (1). A donor membrane SNARE protein associates in trans with acceptor membrane SNARE proteins to generate a 4-helix bundle (2, 3). Vesicle exocytosis in neuronal and endocrine cells utilizes VAMP2 (also known as synaptobrevin 2) on the vesicle (v-SNARE) and the target membrane SNAREs (t-SNAREs) syntaxin-1 and SNAP-25. The structure of a 4-helix bundle of neuronal SNAREs suggested how an assembled trans-SNARE complex could promote fusion through close membrane apposition (3). Evidence that neuronal SNARE complexes might directly catalyze membrane fusion was provided by lipid mixing studies with SNAREs reconstituted into proteoliposomes (4). The function of several key SNARE regulatory proteins, including synaptotagmin and Munc18-1, was successfully reconstituted in the lipid-mixing assay (5, 6).Dense-core vesicle exocytosis in cells proceeds through several stages before Ca 2ϩ -triggered fusion, consisting of docking/tethering and priming steps (7). Vesicles engage the plasma membrane in docking/tethering interactions that involve syntaxin-1 and SNAP-25 (8, 9). However, not all docked/tethered vesicles are competent for fusion, and priming reactions are needed to convert docked vesicles to a fusion-ready state (10). Priming involves the progressive assembly of trans-SNARE complexes, likely through the initial assembly of t-SNARE heterodimers with subsequent incorporation of VAMP2 by zippering N-terminal to C-terminal regions of the SNAREs (11-14). SNARE-binding proteins likely catalyze SNARE complex assembly during priming but little is known about how this is achieved.Molecular studies of factors that act in priming are needed to elucidate the pathway of SNARE co...

Neurotransmitters and peptide hormones are secreted by regulated vesicle exocytosis. CAPS (also known as CADPS) is a 145-kDa cytosolic and peripheral membrane protein required for vesicle docking and priming steps that precede Ca2+-triggered vesicle exocytosis. CAPS binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and SNARE proteins and is proposed to promote SNARE protein complex assembly for vesicle docking and priming. We characterized purified soluble CAPS as mainly monomer in equilibrium with small amounts of dimer. However, the active form of CAPS bound to PC12 cell membranes or to liposomes containing PI(4,5)P2 and Q-SNARE proteins was mainly dimer. CAPS dimer formation required its C2 domain based on mutation or deletion studies. Moreover, C2 domain mutations or deletions resulted in a loss of CAPS function in regulated vesicle exocytosis, indicating that dimerization is essential for CAPS function. Comparison of the CAPS C2 domain to a structurally defined Munc13-1 C2A domain dimer revealed conserved residues involved in CAPS dimerization. We conclude that CAPS functions as a C2 domain-mediated dimer in regulated vesicle exocytosis. The unique tandem C2-PH domain of CAPS may serve as a PI(4,5)P2-triggered switch for dimerization. CAPS dimerization may be coupled to oligomeric SNARE complex assembly for vesicle docking and priming.