Abstract
The calcium-dependent activator protein for secretion (CAPS) is a novel neural/endocrine-specific cytosolic and peripheral membrane protein required for the Ca2+-regulated exocytosis of secretory vesicles. CAPS acts at a stage in exocytosis that follows ATP-dependent priming, which involves the essential synthesis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). In the present studies, CAPS is shown to bind liposomes that contain acidic phospholipids and binding was markedly enhanced by inclusion of PtdIns(4,5)P2 but not other phosphoinositides in the absence of Ca2+. PtdIns(4,5)P2, but not other phosphoinositides including PtdIns(3, 4)P2 and PtdIns(3,4,5)P3, altered the susceptibility of CAPS to proteolysis by trypsin and proteinase K, suggesting that phosphoinositide binding promoted a conformational change. Photoaffinity labeling studies with a photoactivatable benzoylcinnimidyl acyl chain derivative of PtdIns(4,5)P2 confirmed the phosphoinositide-binding properties of CAPS and suggested a hydrophobic aspect of the interaction. CAPS, as one of very few characterized proteins with a binding specificity for 4-, 5-phosphorylated inositides over 3-phosphorylated inositides, may function in regulated exocytosis as an effector of PtdIns(4,5)P2.
Highlights
The regulated secretion of neurotransmitters and peptide hormones from neural and endocrine cells is mediated by the fusion of secretory vesicles with the plasma membrane, but the molecular mechanisms that underlie the Ca2ϩ-dependent merger of phospholipid bilayers have not been fully elucidated
The exocytosis of large dense-core vesicles (LDCVs)1 in neuroendocrine cells can be reconstituted in broken cell (1, 2) or purified membrane preparations (3) where ATP hydrolysis is required for priming reactions that precede Ca2ϩ-dependent membrane fusion reactions (2)
Stereoselective Binding of Polyphosphoinositides to calcium-dependent activator protein for secretion (CAPS)— CAPS is a novel neural/endocrine-specific dimer of 145-kDa subunits that was identified by its activity in reconstituting Ca2ϩ-dependent secretion in permeable neuroendocrine cells (1, 27)
Summary
The regulated secretion of neurotransmitters and peptide hormones from neural and endocrine cells is mediated by the fusion of secretory vesicles with the plasma membrane, but the molecular mechanisms that underlie the Ca2ϩ-dependent merger of phospholipid bilayers have not been fully elucidated. The binding of CAPS to PtdChol liposomes that lacked or contained the acidic phospholipids PtdEt, PtdSer, or PtdIns was increased by the inclusion of Ca2ϩ in the binding mixtures. The phospholipid composition dependence of the effects of Ca2ϩ on CAPS binding were evident in studies where the liposome content of PtdIns(4,5)P2 was varied (Fig. 2C).
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