Ca2+-dependent synaptotagmin binding to SNAP-25 is essential for Ca2+-triggered exocytosis.
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.
Zhang
Xiaodong
X
Department of Biochemistry, University of Wisconsin, Madison 53706, USA.
Kim-Miller
Mindy J
MJ
Fukuda
Mitsunori
M
Kowalchyk
Judith A
JA
Martin
Thomas F J
TF
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002135:Calcium-Binding Proteins; D002490:Central Nervous System / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D016716:PC12 Cells; D017729:Presynaptic Terminals / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D050600:SNARE Proteins; D013570:Synaptic Membranes / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology; D013572:Synaptic Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D001224:Aspartic Acid; D002118:Calcium