Synergistic membrane interactions of the two C2 domains of synaptotagmin.
Synaptotagmin, an integral membrane protein localized to secretory vesicles, has been implicated in the docking and fusion steps in calcium-regulated exocytosis. The large cytoplasmic domain contains two C2 motifs, each similar to the Ca2+ and phospholipid binding domain of protein kinase C. To study the membrane binding and aggregating properties of these C2 domains, three recombinant fragments of rat synaptotagmin I were expressed in Escherichia coli and purified. A recombinant protein containing both C2 domains ( p65 1-5) was found to bind to and aggregate bovine chromaffin granules in a calcium-dependent manner, with half-maximal binding and aggregation occurring at approximately p Ca2+ = 4. 2. However, recombinant proteins containing either the first ( p65 1-3) or second ( p65 3-5) C2 domain alone were not able to bind to the granules, indicating that both C2 domains are required for binding to chromaffin granules. p65 1-5 also bound to and aggregated liposomes made from chromaffin granule lipid extracts, as well as granules treated extensively with trypsin, suggesting that p65 1-5 binding to granules is mediated by the lipids in the granule membrane and not the granule membrane proteins. Although p65 1-3 and p65 3-5 did not bind to granules or lipids extracted from granules, both did bind to phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (10%-40%PS). Half-maximal binding of p65 1-3 to vesicles occurred at approximately p Ca2+ = 5.2, while p65 3-5 appeared to bind independently of calcium over the range of pCa2+ = 5.5-2.8. p65 1-5 exhibited binding to PS/PC vesicles with characteristics of both the smaller proteins, displaying some binding in EGTA and increased binding in calcium. Larger amounts of p65 1-5 bound to PS/PC vesicles than of either of the smaller fragments. These results suggest that the two C2 domains of synaptotagmin act synergistically to promote binding to biological membranes and to affect calcium sensitivity and membrane binding capacity.[1]References
- Synergistic membrane interactions of the two C2 domains of synaptotagmin. Damer, C.K., Creutz, C.E. J. Biol. Chem. (1994) [Pubmed]
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