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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence / genetics
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Animals
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Aspartic Acid / genetics
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Aspartic Acid / metabolism
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Calcium / metabolism
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Calcium Signaling / physiology*
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Calcium-Binding Proteins*
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Central Nervous System / metabolism
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Exocytosis / physiology*
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Membrane Glycoproteins / metabolism*
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Mutation / genetics
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / metabolism*
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Neurotransmitter Agents / metabolism
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PC12 Cells
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Presynaptic Terminals / metabolism*
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Protein Binding / physiology
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Protein Structure, Tertiary / genetics
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Rats
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SNARE Proteins
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Synaptic Membranes / metabolism*
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Synaptic Transmission / physiology
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Synaptic Vesicles / metabolism*
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Synaptosomal-Associated Protein 25
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Synaptotagmins
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Vesicular Transport Proteins*
Substances
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Calcium-Binding Proteins
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Membrane Glycoproteins
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Membrane Proteins
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Nerve Tissue Proteins
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Neurotransmitter Agents
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SNARE Proteins
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Snap25 protein, rat
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Synaptosomal-Associated Protein 25
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Vesicular Transport Proteins
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Synaptotagmins
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Aspartic Acid
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Calcium