Membrane Association Domains in Ca2+-dependent Activator Protein for Secretion Mediate Plasma Membrane and Dense-core Vesicle Binding Required for Ca2+-dependent Exocytosis (original) (raw)
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Reconstitution of calcium-mediated exocytosis of dense-core vesicles
Science advances, 2017
Regulated exocytosis is a process by which neurotransmitters, hormones, and secretory proteins are released from the cell in response to elevated levels of calcium. In cells, secretory vesicles are targeted to the plasma membrane, where they dock, undergo priming, and then fuse with the plasma membrane in response to calcium. The specific roles of essential proteins and how calcium regulates progression through these sequential steps are currently incompletely resolved. We have used purified neuroendocrine dense-core vesicles and artificial membranes to reconstruct in vitro the serial events that mimic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent membrane docking and fusion during exocytosis. Calcium recruits these vesicles to the target membrane aided by the protein CAPS (calcium-dependent activator protein for secretion), whereas synaptotagmin catalyzes calcium-dependent fusion; both processes are dependent on phosphatidylinositol 4,5-bis...
Docked Secretory Vesicles Undergo Ca2+-activated Exocytosis in a Cell-free System
Journal of Biological Chemistry, 1997
The Ca 2؉-activated fusion of secretory vesicles with the plasma membrane responsible for regulated neurotransmitter and hormone secretion has previously been studied in permeable neuroendocrine cells, where requirements for ATP and cytosolic proteins were identified. As reported here, Ca 2؉-activated fusion mechanisms are also preserved following cell homogenization. The release of norepinephrine (NE) and other vesicle constituents from a PC12 cell membrane fraction was activated by micromolar Ca 2؉ (EC 50 ϳ 3 M) and exhibited a dependence upon MgATP and cytosol. Ca 2؉-dependent NE release was inhibited by botulinum neurotoxins and by CAPS (Ca 2؉-dependent activator protein for secretion) antibody implying that syntaxin, synaptobrevin, SNAP-25 (synaptosomal-associated protein of 25 kDa), and CAPS are required for regulated exocytosis in this system. The exocytosis-competent membrane fraction consisted of rapidly sedimenting dense core vesicles associated with plasma membrane fragments. Free vesicles did not release NE either in the absence or presence of plasma membranes, indicating that only docked vesicles were competent for exocytosis under the reconstitution conditions used. A cell-free system for Ca 2؉-activated fusion will facilitate studies on the roles of essential proteins such as syntaxin, synaptobrevin, SNAP-25, and CAPS that act at post-docking steps in the regulated exocytotic pathway.
CAPS Acts at a Prefusion Step in Dense-Core Vesicle Exocytosis as a PIP2 Binding Protein
Neuron, 2004
is currently known as CAPS-1 because of the character-University of Wisconsin ization of a second highly homologous and more ubiqui-Madison, Wisconsin 53706 tously expressed CAPS-2 protein in higher vertebrates (Speidel et al., 2003; Cisternas et al., 2003). Null mutants in the single CAPS gene in C. elegans (Avery et al., 1993; Summary Ann et al., 1997) and D. melanogaster (Renden et al., 2001) exhibit loss of secretion of a subset of transmit-CAPS-1 is required for Ca 2؉-triggered fusion of denseters. Mammalian CAPS-1 is cytosolic as well as memcore vesicles with the plasma membrane, but its site brane-associated in neurons and localizes to the plasma of action and mechanism are unknown. We analyzed membrane and to DCVs but not SVs (Berwin et al., 1998; the kinetics of Ca 2؉-triggered exocytosis reconstituted Grishanin et al., 2002). Neutralizing CAPS-1 antibodies in permeable PC12 cells. CAPS-1 increased the initial disrupt DCV but not SV exocytosis (Berwin et al., 1998; rate of Ca 2؉-triggered vesicle exocytosis by acting at a Tandon et al., 1998; Elhamdani et al., 1999; Rupnik et rate-limiting, Ca 2؉-dependent prefusion step. CAPS-1 al., 2000; Olsen et al., 2003), indicating that CAPS-1 activity depended upon prior ATP-dependent priming functions selectively in DCV exocytosis. during which PIP 2 synthesis occurs. CAPS-1 activity In neuroendocrine PC12 cells, CAPS-1 acts at a late and binding to the plasma membrane depended upon step in the DCV exocytic pathway after vesicle docking PIP 2. Ca 2؉ was ineffective in triggering vesicle fusion (Walent et al., 1992; Hay and Martin, 1992; Ann et al., in the absence of CAPS-1 but instead promoted desen-1997; Martin and Kowalchyk, 1997). CAPS-1 exhibits sitization to CAPS-1 resulting from decreased plasma specific PIP 2 binding in vitro, but the role of this property membrane PIP 2. We conclude that CAPS-1 functions in CAPS-1 function has not been determined (Loyet et following ATP-dependent priming as a PIP 2 binding al., 1998; Grishanin et al., 2002). In addition, two memprotein to enhance Ca 2؉-dependent DCV exocytosis. brane-association domains in CAPS-1, a central pleck-Essential prefusion steps in dense-core vesicle exostrin homology (PH) domain and a C-terminal region, cytosis involve sequential ATP-dependent synthesis mediate plasma membrane and DCV associations, reof PIP 2 and the subsequent PIP 2-dependent action of spectively, which suggests that CAPS-1 could bridge CAPS-1. Regulation of PIP 2 levels and CAPS-1 activity donor and acceptor membranes during DCV fusion would control the secretion of neuropeptides and (Grishanin et al., 2002). Studies of single exocytic events monoaminergic transmitters. in CAPS-1 antibody-injected chromaffin cells indicated that CAPS-1 also regulates fusion pore dilation (Elham-Introduction dani et al., 1999). The mechanism by which CAPS-1 functions and the precise point at which it acts relative The secretion of neurotransmitters and neuropeptides to Ca 2ϩ-triggered DCV fusion remain to be clarified. is mediated by the Ca 2ϩ-triggered fusion of secretory Ca 2ϩ-triggered DCV exocytosis in PC12 cells exhibits vesicles with the plasma membrane. Two classes of relatively long latencies and slow rates compared to secretory vesicles have been identified, small clear synadrenal chromaffin cells (Ninomiya et al., 1997; Elhamaptic vesicles (SVs) and dense-core vesicles (DCVs) (Dedani et al., 2000; Wang et al., 2001). In the current work Camilli and Jahn, 1990; Kelly, 1993), the contents, oriwith PC12 cells, we found that recombinant CAPS-1 gins, and functions of which are distinct. Exocytosis of markedly increases the rate of a relatively slow (03ف the two vesicle types exhibits distinct regulation and is s) Ca 2ϩ-dependent step in DCV exocytosis that occurs elicited by different patterns of stimulation (Jan and Jan, after vesicle docking and ATP-dependent priming but 1982; Matteoli et al., 1988; DeCamilli and Jahn, 1990; prior to Ca 2ϩ-triggered fusion. Ca 2ϩ , acting in the ab-Verhage et al., 1991). Whereas rapid evoked SV exosence of CAPS-1, was found to promote desensitization cytosis is essential for fast synaptic transmission, DCV to CAPS-1, and studies of desensitization indicated that exocytosis is slower and mediates the release of transit resulted from decreases in plasma membrane PIP 2 mitters that modulate pre-and postsynaptic events. levels. Moreover, PIP 2 was found to be essential for Mechanisms for SV and DCV exocytosis utilize a number of common components such as SNARE proteins, but CAPS-1 activity and binding to PC12 cell plasma memthere are constituents specific to either pathway that branes. The results indicate that CAPS-1 acts as a PIP 2 may confer kinetic and regulatory properties distinct for binding protein that facilitates DCV exocytosis. Essential each pathway (Rettig and Neher, 2002; Martin, 2003). prefusion steps in DCV exocytosis involve sequential ATP-dependent synthesis of PIP 2 and the subsequent PIP 2-dependent action of CAPS-1.
A Family of Ca2+-Dependent Activator Proteins for Secretion
Journal of Biological Chemistry, 2003
Ca 2؉-dependent activator protein for secretion (CAPS) 1 is an essential cytosolic component of the protein machinery involved in large dense-core vesicle (LDCV) exocytosis and in the secretion of a subset of neurotransmitters. In the present study, we report the identification, cloning, and comparative characterization of a second mammalian CAPS isoform, CAPS2. The structure of CAPS2 and its function in LDCV exocytosis from PC12 cells are very similar to those of CAPS1. Both isoforms are strongly expressed in neuroendocrine cells and in the brain. In subcellular fractions of the brain, both CAPS isoforms are enriched in synaptic cytosol fractions and also present on vesicular fractions. In contrast to CAPS1, which is expressed almost exclusively in brain and neuroendocrine tissues, CAPS2 is also expressed in lung, liver, and testis. Within the brain, CAPS2 expression seems to be restricted to certain brain regions and cell populations, whereas CAPS1 expression is strong in all neurons. During development, CAPS2 expression is constant between embryonic day 10 and postnatal day 60, whereas CAPS1 expression is very low before birth and increases after postnatal day 0 to reach a plateau at postnatal day 21. Light microscopic data indicate that both CAPS isoforms are specifically enriched in synaptic terminals. Ultrastructural analyses show that CAPS1 is specifically localized to glutamatergic nerve terminals. We conclude that at the functional level, CAPS2 is largely redundant with CAPS1. Differences in the spatial and temporal expression patterns of the two CAPS isoforms most likely reflect as yet unidentified subtle functional differences required in particular cell types or during a particular developmental period. The abundance of CAPS proteins in synaptic terminals indicates that they may also be important for neuronal functions that are not exclusively related to LDCV exocytosis.
Journal of Biological Chemistry, 2003
Ca 2؉-dependent activator protein for secretion (CAPS) 1 is an essential cytosolic component of the protein machinery involved in large dense-core vesicle (LDCV) exocytosis and in the secretion of a subset of neurotransmitters. In the present study, we report the identification, cloning, and comparative characterization of a second mammalian CAPS isoform, CAPS2. The structure of CAPS2 and its function in LDCV exocytosis from PC12 cells are very similar to those of CAPS1. Both isoforms are strongly expressed in neuroendocrine cells and in the brain. In subcellular fractions of the brain, both CAPS isoforms are enriched in synaptic cytosol fractions and also present on vesicular fractions. In contrast to CAPS1, which is expressed almost exclusively in brain and neuroendocrine tissues, CAPS2 is also expressed in lung, liver, and testis. Within the brain, CAPS2 expression seems to be restricted to certain brain regions and cell populations, whereas CAPS1 expression is strong in all neurons. During development, CAPS2 expression is constant between embryonic day 10 and postnatal day 60, whereas CAPS1 expression is very low before birth and increases after postnatal day 0 to reach a plateau at postnatal day 21. Light microscopic data indicate that both CAPS isoforms are specifically enriched in synaptic terminals. Ultrastructural analyses show that CAPS1 is specifically localized to glutamatergic nerve terminals. We conclude that at the functional level, CAPS2 is largely redundant with CAPS1. Differences in the spatial and temporal expression patterns of the two CAPS isoforms most likely reflect as yet unidentified subtle functional differences required in particular cell types or during a particular developmental period. The abundance of CAPS proteins in synaptic terminals indicates that they may also be important for neuronal functions that are not exclusively related to LDCV exocytosis.
Phosphatidylserine Regulation of Ca2+-triggered Exocytosis and Fusion Pores in PC12 Cells
Molecular Biology of the Cell, 2009
The synaptic vesicle protein synaptotagmin I (Syt I) binds phosphatidylserine (PS) in a Ca 2+ -dependent manner. This interaction is thought to play a role in exocytosis, but its precise functions remain unclear. To determine potential roles for Syt I-PS binding we varied the PS content in PC12 cells and liposomes, and studied the effects on the kinetics of exocytosis and Syt I binding in parallel. Raising PS produced a steeply nonlinear, saturating increase in Ca 2+ -triggered fusion, and a graded slowing of the rate of fusion pore dilation. Ca 2+ -Syt I bound liposomes more tightly as PS content was raised, with a steep increase in binding at low PS, and a further gradual increase at higher PS. These two phases in the PS dependence of Ca 2+ -dependent Syt I binding to lipid may correspond to the two distinct and opposing kinetic effects of PS on exocytosis. PS influences exocytosis in two ways, enhancing an early step leading to fusion pore opening, and slowing a later step when fusion pores dilate. The possible relevance of these results to Ca 2+ -triggered Syt I binding is discussed along with other possible roles of PS. Amatore, C., Arbault, S., Bonifas, I., Bouret, Y., Erard, M., Ewing, A.G., and Sombers, L.A. (2005). Correlation between vesicle quantal size and fusion pore release in chromaffin cell exocytosis. Biophys J 88, 4411-4420. Augustine, G.J. (2001). How does calcium trigger neurotransmitter release? Curr Opin Neurobiol 11, 320-326. Bai, J., Wang, C.T., Richards, D.A., Jackson, M.B., and Chapman, E.R. (2004). Fusion pore dynamics are regulated by synaptotagmin*t-SNARE interactions. Neuron 41, 929-942. Bai, J., Wang, P., and Chapman, E.R. (2002). C2A activates a cryptic Ca 2+ -triggered membrane penetration activity within the C2B domain of synaptotagmin I.
Journal of Biological Chemistry, 2010
CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca 2؉-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can coreside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.
Journal of Biological Chemistry, 1998
The calcium-dependent activator protein for secretion (CAPS) is a novel neural/endocrine-specific cytosolic and peripheral membrane protein required for the Ca 2؉ -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)P 2 ). 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)P 2 but not other phosphoinositides in the absence of Ca 2؉ . PtdIns(4,5)P 2 , but not other phosphoinositides including PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 , 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)P 2 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-,5phosphorylated inositides over 3-phosphorylated inositides, may function in regulated exocytosis as an effector of PtdIns(4,5)P 2 .
A novel Ca2+-dependent step in exocytosis subsequent to vesicle fusion
FEBS Letters, 1995
Exocytosis begins with formation of a small fusion pore which then expands allowing rapid release of granular contents. We studied the influence of cytoplasmic free Ca z+ (ICaZ+[0 on the conductance of the initial pore and on the dynamics of subsequent expansion in horse eosinophils using the patch clamp technique. The mean initial conductance is ~200 pS independent of [CaZ+]i . This value is close to that previously found in beige mouse mast cells. The pore subsequently expands by 18 nS/s at ICa2+li < 10 nM, by 40 nSIs at [Ca2+]i = 1.5//M and by 90 nSIs at ]Ca2+]i = 10/zM. These results show that the structure of the initial fusion pore is independent of cytoplasmic Ca 2÷. However, the pore expansion is a Ca2+-dependent process modulating secretion at a step later than vesicle-plasma membrane fusion.
The Ca2+-dependent Activator Protein for Secretion CAPS: Do I Dock or do I Prime?
Molecular Neurobiology, 2009
The "Ca 2+ -dependent activator protein for secretion" (CAPS) is a protein which reconstitutes regulated secretion in permeabilized neuroendocrine cells. It is generally accepted that CAPS plays an important role in the release of the contents of dense core vesicles in the nervous system as well as in a variety of other secretory tissues. At which step in the exocytotic process CAPS functions as well as its role in the fusion of synaptic vesicles is still under dispute. A recent growth spurt in the CAPS field has been fueled by genetic approaches in Caenorhabditis elegans and Drosophila as well as the application of knockout and knockdown approaches in mouse cells and in cell lines, respectively. We have attempted to review the body of work that established CAPS as an important regulator of secretion and to describe new information that has furthered our understanding of how CAPS may function. We discuss the conclusions, point out areas where controversy remains, and suggest directions for future experiments.