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2.
Proc Natl Acad Sci U S A ; 106(41): 17308-13, 2009 Oct 13.
Article in English | MEDLINE | ID: mdl-19805029

ABSTRACT

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)P(2) 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.


Subject(s)
Qa-SNARE Proteins/metabolism , SNARE Proteins/metabolism , Synaptosomal-Associated Protein 25/metabolism , Animals , Cell Membrane/metabolism , Cell Membrane/physiology , Exocytosis/physiology , Homeostasis , Lecithins/metabolism , Liposomes/metabolism , Membrane Fusion/physiology , PC12 Cells/physiology , Phosphatidylserines/metabolism , Rats , Synaptotagmins/metabolism , Syntaxin 1/metabolism , Vesicle-Associated Membrane Protein 2/metabolism
3.
J Cell Biol ; 182(2): 355-66, 2008 Jul 28.
Article in English | MEDLINE | ID: mdl-18644890

ABSTRACT

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. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.


Subject(s)
Cell Membrane/metabolism , Exocytosis/physiology , Membrane Fusion/physiology , Phosphatidylinositol 4,5-Diphosphate/metabolism , SNARE Proteins/metabolism , Transport Vesicles/metabolism , Animals , Cell Membrane/drug effects , Cell Membrane/ultrastructure , Dose-Response Relationship, Drug , Exocytosis/drug effects , Liposomes/metabolism , Membrane Fusion/drug effects , Membrane Microdomains/drug effects , Membrane Microdomains/metabolism , Membrane Microdomains/ultrastructure , PC12 Cells , Phosphatidylinositol 4,5-Diphosphate/pharmacology , Rats , Syntaxin 1/drug effects , Syntaxin 1/metabolism , Transport Vesicles/drug effects , Transport Vesicles/ultrastructure
4.
Nat Neurosci ; 8(4): 421-5, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15778713

ABSTRACT

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. Here we characterize direct inhibitory actions of G protein betagamma (Gbetagamma) on Ca(2+)-triggered vesicle exocytosis in permeable PC12 cells. Gbetagamma inhibition was rapid (<1 s) and was attenuated by cleavage of synaptosome-associated protein of 25 kD (SNAP25). Gbetagamma bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, and binding was reduced to SNARE complexes containing cleaved SNAP25 or by Ca(2+)-dependent synaptotagmin binding. Here we show inhibitory coupling between GPCRs and vesicle exocytosis mediated directly by Gbetagamma interactions with the Ca(2+)-dependent fusion machinery.


Subject(s)
Exocytosis/physiology , GTP-Binding Protein beta Subunits/physiology , GTP-Binding Protein gamma Subunits/physiology , Secretory Vesicles/metabolism , Vesicular Transport Proteins/metabolism , Animals , Blotting, Western/methods , Calcium/pharmacology , Calcium-Binding Proteins/metabolism , Cattle , Dose-Response Relationship, Drug , Drug Interactions , Exocytosis/drug effects , Eye Proteins/pharmacology , GTP-Binding Protein Regulators , GTP-Binding Protein beta Subunits/pharmacology , GTP-Binding Protein gamma Subunits/pharmacology , Membrane Glycoproteins/metabolism , Membrane Proteins/pharmacology , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/pharmacology , Neural Inhibition/drug effects , Norepinephrine/metabolism , PC12 Cells , Phosphoproteins/pharmacology , Protein Binding/drug effects , Rats , Reaction Time/drug effects , Recombinant Fusion Proteins/metabolism , SNARE Proteins , Secretory Vesicles/drug effects , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins , Synaptosomal-Associated Protein 25 , Synaptotagmins , Time Factors
5.
Neuron ; 43(4): 551-62, 2004 Aug 19.
Article in English | MEDLINE | ID: mdl-15312653

ABSTRACT

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. CAPS-1 activity depended upon prior ATP-dependent priming during which PIP2 synthesis occurs. CAPS-1 activity and binding to the plasma membrane depended upon PIP2. Ca2+ was ineffective in triggering vesicle fusion in the absence of CAPS-1 but instead promoted desensitization to CAPS-1 resulting from decreased plasma membrane PIP2. We conclude that CAPS-1 functions following ATP-dependent priming as a PIP2 binding protein to enhance Ca2+-dependent DCV exocytosis. Essential prefusion steps in dense-core vesicle exocytosis involve sequential ATP-dependent synthesis of PIP2 and the subsequent PIP2-dependent action of CAPS-1. Regulation of PIP2 levels and CAPS-1 activity would control the secretion of neuropeptides and monoaminergic transmitters.


Subject(s)
Calcium-Binding Proteins/physiology , Exocytosis/physiology , Phosphatidylinositol 4,5-Diphosphate/metabolism , Secretory Vesicles/metabolism , Animals , Calcium/metabolism , Calcium/physiology , Calcium-Binding Proteins/biosynthesis , Calcium-Binding Proteins/genetics , PC12 Cells , Phosphatidylinositol 4,5-Diphosphate/antagonists & inhibitors , Protein Binding/physiology , Rats , Rats, Inbred Strains , Vesicular Transport Proteins
6.
Neuron ; 34(4): 599-611, 2002 May 16.
Article in English | MEDLINE | ID: mdl-12062043

ABSTRACT

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. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion.


Subject(s)
Calcium Signaling/physiology , Calcium-Binding Proteins , Exocytosis/physiology , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Presynaptic Terminals/metabolism , Synaptic Membranes/metabolism , Synaptic Vesicles/metabolism , Vesicular Transport Proteins , Amino Acid Sequence/genetics , Animals , Aspartic Acid/genetics , Aspartic Acid/metabolism , Calcium/metabolism , Central Nervous System/metabolism , Membrane Proteins/genetics , Mutation/genetics , Nerve Tissue Proteins/genetics , Neurotransmitter Agents/metabolism , PC12 Cells , Protein Binding/physiology , Protein Structure, Tertiary/genetics , Rats , SNARE Proteins , Synaptic Transmission/physiology , Synaptosomal-Associated Protein 25 , Synaptotagmins
7.
J Biol Chem ; 277(24): 22025-34, 2002 Jun 14.
Article in English | MEDLINE | ID: mdl-11927595

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.


Subject(s)
Calcium/chemistry , Cell Membrane/metabolism , Exocytosis , Algorithms , Amino Acid Sequence , Animals , COS Cells , Dose-Response Relationship, Drug , Green Fluorescent Proteins , Immunohistochemistry , Lipid Metabolism , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , PC12 Cells , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/metabolism , Transfection
8.
J Biol Chem ; 277(7): 4601-4, 2002 Feb 15.
Article in English | MEDLINE | ID: mdl-11751925

ABSTRACT

Synaptotagmin (Syt) I-deficient phaeochromocytoma (PC12) cell lines show normal Ca(2+)-dependent norepinephrine (NE) release (Shoji-Kasai, Y., Yoshida, A., Sato, K., Hoshino, T., Ogura, A., Kondo, S., Fujimoto, Y., Kuwahara, R., Kato, R., and Takahashi, M. (1992) Science 256, 1821-1823). To identify an alternative Ca(2+) sensor, we searched for other Syt isoforms in Syt I-deficient PC12 cells and identified Syt IX, an isoform closely related to Syt I, as an abundantly expressed dense-core vesicle protein. Here we show that Syt IX is required for the Ca(2+)-dependent release of NE from PC12 cells. Antibodies directed against the C2A domain of either Syt IX or Syt I inhibited Ca(2+)-dependent NE release in permeable PC12 cells indicating that both Syt proteins function in dense-core vesicle exocytosis. Our results support the idea that Syt family proteins that co-reside on secretory vesicles may function cooperatively and redundantly as potential Ca(2+) sensors for exocytosis.


Subject(s)
Calcium-Binding Proteins , Calcium/metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Animals , COS Cells , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Exocytosis , PC12 Cells , Phylogeny , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , Rats , Recombinant Proteins/metabolism , Synaptotagmins , Time Factors , Tissue Distribution
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