Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis
K L Lynch1, R R L Gerona, E C Larsen
1Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
Molecular Biology of the Cell
|October 5, 2007
Summary
Calcium binding to synaptotagmin (SYT) is crucial for vesicle exocytosis. Specific SYT-SNARE interactions, not just membrane binding, are essential for calcium-triggered fusion.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Synaptotagmins (SYTs) are key calcium (Ca2+) sensors regulating vesicle exocytosis.
- The precise mechanism linking Ca2+ influx to membrane fusion remains unclear.
- SYTs interact with SNARE proteins, crucial for membrane fusion, but the functional significance is debated.
Purpose of the Study:
- To investigate the role of specific Ca2+-dependent interactions between synaptotagmin-1 and SNAREs in vesicle exocytosis.
- To determine if Ca2+-dependent membrane binding alone is sufficient for triggering fusion.
Main Methods:
- Zero-length cross-linking to identify Ca2+-dependent binding sites between synaptotagmin-1 and SNAP25.
- Site-directed mutagenesis of identified binding sites within the C2A and C2B domains of synaptotagmin-1.
- Assays for Ca2+-dependent SNARE binding, liposome fusion, and vesicle exocytosis in synaptotagmin-deficient cells.
Main Results:
- Specific sites mediating Ca2+-dependent SNAP25 binding on synaptotagmin-1 were identified.
- Mutations at these sites abolished Ca2+-dependent SNARE binding but not Ca2+-dependent membrane binding.
- Mutant synaptotagmin-1 failed to restore Ca2+-regulated liposome fusion and vesicle exocytosis in deficient cells.
Conclusions:
- Ca2+-dependent binding of synaptotagmin to SNAREs is essential for triggering vesicle exocytosis.
- Ca2+-dependent membrane binding by itself is insufficient to induce membrane fusion.
- A structural model suggests simultaneous Ca2+-dependent SNARE and membrane binding.
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