CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions.

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.

Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion.

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited.

G protein betagamma directly regulates SNARE protein fusion machinery for secretory granule exocytosis.

The activation of G protein-coupled receptors (GPCRs) can result in an inhibition of Ca(2+)-dependent hormone and neurotransmitter secretion. This has been attributed in part to G protein inhibition of Ca(2+) influx. However, a frequently dominant inhibitory effect, of unknown mechanism, also occurs distal to Ca(2+) entry.

CAPS acts at a prefusion step in dense-core vesicle exocytosis as a PIP2 binding protein.

CAPS-1 is required for Ca2+-triggered fusion of dense-core vesicles with the plasma membrane, but its site of action and mechanism are unknown. We analyzed the kinetics of Ca2+-triggered exocytosis reconstituted in permeable PC12 cells. CAPS-1 increased the initial rate of Ca2+-triggered vesicle exocytosis by acting at a rate-limiting, Ca2+-dependent prefusion step.

Ca2+-dependent synaptotagmin binding to SNAP-25 is essential for Ca2+-triggered exocytosis.

Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca2+ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca2+ regulation of secretion. We found that synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca2+-dependent synaptotagmin binding.

Membrane association domains in Ca2+-dependent activator protein for secretion mediate plasma membrane and dense-core vesicle binding required for Ca2+-dependent exocytosis.

Ca2+-dependent activator protein for secretion (CAPS) is a cytosolic protein essential for the Ca2+-dependent fusion of dense-core vesicles (DCVs) with the plasma membrane and the regulated secretion of a subset of neurotransmitters. The mechanism by which CAPS functions in exocytosis and the means by which it associates with target membranes are unknown. We identified two domains in CAPS with distinct membrane-binding properties that were each essential for CAPS activity in regulated exocytosis.

Synaptotagmin IX regulates Ca2+-dependent secretion in PC12 cells.

Synaptotagmin (Syt) I-deficient phaeochromocytoma (PC12) cell lines show normal Ca(2+)-dependent norepinephrine (NE) release (Shoji-Kasai, Y., Yoshida, A., Sato, K.