Abstract
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. The first of these, a centrally located pleckstrin homology domain, exhibited three properties: charge-based binding to acidic phospholipids, binding to plasma membrane but not DCV membrane, and stereoselective binding to phosphatidylinositol 4,5-bisphosphate. Mutagenesis studies revealed that the former two properties but not the latter were essential for CAPS function. The central pleckstrin homology domain may mediate transient CAPS interactions with the plasma membrane during Ca2+-triggered exocytosis. The second membrane association domain comprising distal C-terminal sequences mediated CAPS targeting to and association with neuroendocrine DCVs. The CAPS C-terminal domain was also essential for optimal activity in regulated exocytosis. The presence of two membrane association domains with distinct binding specificities may enable CAPS to bind both target membranes to facilitate DCV-plasma membrane fusion.
Highlights
Because Ca2ϩ-dependent activator protein for secretion (CAPS) functions in membrane fusion and is associated with dense-core vesicles (DCVs) and plasma membranes as a peripherally bound protein (14), we determined the properties of the pleckstrin homology (PH) domain and whether it is essential for CAPS function in exocytosis
That mediate CAPS binding to plasma membranes and DCV membranes. Because both membrane association domains were essential for CAPS function in Ca2ϩ-dependent exocytosis, these findings suggest that CAPS may bridge donor and acceptor membranes at a critical point during DCV exocytosis
Almost all of the PH domains that have been studied to date bind phosphoinositides or inositol phosphates, the role, if any, of phosphoinositide binding in protein function is unknown for the majority of these proteins (19, 27, 33)
Summary
The cytosolic proteins required for MgATP-dependent priming consist of phosphatidylinositol transfer protein and PtdIns(4)P 5-kinase (4, 5), which mediate PtdIns(4,5)P2 synthesis that is essential for Ca2ϩ-triggered vesicle fusion (3). CAPS binds membranes after MgATP-dependent priming in the absence of Ca2ϩ and is required during Ca2ϩ-triggered fusion,[2] indicating that CAPS functions in parallel with the membrane fusion machinery consisting of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (8). Studies in permeable synaptosomes indicated that CAPS was selectively required for Ca2ϩ-dependent DCV but not synaptic vesicle exocytosis (13). The properties of the CAPS PH domain that were essential for function were its charge-based interactions with acidic phospholipids and its ability to associate with specific cellular membranes (plasma membrane but not DCV membrane). Membrane Association Domains of CAPS ities may enable CAPS to bind both target membranes to facilitate DCV-plasma membrane fusion
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