Synaptotagmin function in dense core vesicle exocytosis studied in cracked PC12 cells.
Ca(2+)-triggered dense-core vesicle exocytosis in PC12 cells does not require vesicular synaptotagmins 1 and 2, but may use plasma membrane synaptotagmins 3 and 7 as Ca(2+) sensors. In support of this hypothesis, C(2) domains from the plasma membrane but not vesicular synaptotagmins inhibit PC12 cell exocytosis. Ca(2+) induces binding of both plasma membrane and vesicular synaptotagmins to phospholipids and SNAREs (soluble N-ethylmaleimide-sensitive attachment protein receptors), although with distinct apparent Ca(2+) affinities. Here we used gain-of-function C(2)-domain mutants of synaptotagmin 1 and loss-of-function C(2)-domain mutants of synaptotagmin 7 to examine how synaptotagmins function in dense-core vesicle exocytosis. Our data indicate that phospholipid- but not SNARE-binding by plasma membrane synaptotagmins is the primary determinant of Ca(2+)-triggered dense-core vesicle exocytosis. These results support a general lipid-based mechanism of action of synaptotagmins in exocytosis, with the specificity of various synaptotagmins for different types of fusion governed by their differential localizations and Ca(2+) affinities.
Shin
Ok-Ho
OH
Center for Basic Neuroscience, Department of Molecular Genetics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, Texas 75390, USA.
Rizo
Josep
J
Südhof
Thomas C
TC
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins; D002463:Cell Membrane Permeability / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005615:Freezing; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D050600:SNARE Proteins; D022142:Secretory Vesicles / Q000378:metabolism*; D013329:Structure-Activity Relationship; D050863:Synaptotagmin I; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D050600:SNARE Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; C494304:Syt7 protein, rat; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D002118:Calcium