SNAP-25 and synaptotagmin 1 function in Ca2+-dependent reversible docking of granules to the plasma membrane.
In neuroendocrine cells, Ca2+ triggers fusion of granules with the plasma membrane and functions at earlier steps by increasing the size of the readily releasable pool of vesicles. The effect of Ca2+ at early steps of secretion may be due to the recruitment at the plasma membrane of granules localized in the cytoplasm. To study the mechanism of granule docking, a new in vitro assay is designed using membrane fractions from mouse pituitary AtT-20 cells. By using this assay, it is found that granule docking to the plasma membrane is controlled by Ca2+ concentrations in the micromolar range, is reversible and requires intact SNAP-25, but not VAMP-2. In the docking assay, addition of Ca2+ induces the formation of a SNAP-25-Synaptotagmin 1 complex. The cytosolic domain C2AB of Synaptotagmin 1 and anti-Synaptotagmin 1 antibodies block granule docking. These results show that Ca2+ modulates dynamic docking of granules to the plasma membrane and that this process is due to a Ca2+-dependent interaction between SNAP-25 and Synaptotagmin 1.
Chieregatti
Evelina
E
Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA.
Witkin
Joan W
JW
Baldini
Giulia
G
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism*; D053379:Chromogranin A; D002864:Chromogranins / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology; D008561:Membrane Fusion / Q000502:physiology; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D016253:Microscopy, Immunoelectron; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D053379:Chromogranin A; D002864:Chromogranins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; C494311:Syt1 protein, mouse; D050857:Synaptotagmins; D002118:Calcium