The tandem C2 domains of synaptotagmin contain redundant Ca2+ binding sites that cooperate to engage t-SNAREs and trigger exocytosis.
Real-time voltammetry measurements from cracked PC12 cells were used to analyze the role of synaptotagmin-SNARE interactions during Ca2+-triggered exocytosis. The isolated C2A domain of synaptotagmin I neither binds SNAREs nor inhibits norepinephrine secretion. In contrast, two C2 domains in tandem (either C2A-C2B or C2A-C2A) bind strongly to SNAREs, displace native synaptotagmin from SNARE complexes, and rapidly inhibit exocytosis. The tandem C2 domains of synaptotagmin cooperate via a novel mechanism in which the disruptive effects of Ca2+ ligand mutations in one C2 domain can be partially alleviated by the presence of an adjacent C2 domain. Complete disruption of Ca2+-triggered membrane and target membrane SNARE interactions required simultaneous neutralization of Ca2+ ligands in both C2 domains of the protein. We conclude that synaptotagmin-SNARE interactions regulate membrane fusion and that cooperation between synaptotagmin's C2 domains is crucial to its function.
Earles
C A
CA
Department of Physiology, University of Wisconsin, Madison, WI 53706, USA.
Bai
J
J
Wang
P
P
Chapman
E R
ER
D000818:Animals; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002135:Calcium-Binding Proteins; D004563:Electrochemistry; D005089:Exocytosis / Q000187:drug effects*; D008024:Ligands; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D009638:Norepinephrine / Q000378:metabolism; D016716:PC12 Cells; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats; D050600:SNARE Proteins; D050863:Synaptotagmin I; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
D002135:Calcium-Binding Proteins; D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D002118:Calcium; D009638:Norepinephrine