Damer C K, Creutz C E
Program in Neuroscience, University of Virginia, Charlottesville 22908.
J Biol Chem. 1994 Dec 9;269(49):31115-23.
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.
突触结合蛋白是一种定位于分泌小泡的整合膜蛋白，与钙调节的胞吐作用中的对接和融合步骤有关。其大的胞质结构域包含两个C2基序，每个基序都类似于蛋白激酶C的Ca2+和磷脂结合结构域。为了研究这些C2结构域的膜结合和聚集特性，在大肠杆菌中表达并纯化了大鼠突触结合蛋白I的三个重组片段。发现含有两个C2结构域的重组蛋白（p65 1-5）以钙依赖的方式结合并聚集牛嗜铬颗粒，在约p Ca2+ = 4.2时出现半数最大结合和聚集。然而，仅含有第一个（p65 1-3）或第二个（p65 3-5）C2结构域的重组蛋白无法结合颗粒，这表明两个C2结构域对于结合嗜铬颗粒都是必需的。p65 1-5还结合并聚集了由嗜铬颗粒脂质提取物制成的脂质体，以及用胰蛋白酶广泛处理的颗粒，这表明p65 1-5与颗粒的结合是由颗粒膜中的脂质介导的，而不是颗粒膜蛋白。尽管p65 1-3和p65 3-5不与颗粒或从颗粒中提取的脂质结合，但它们都与磷脂酰丝氨酸（PS）/磷脂酰胆碱（PC）脂质体（10%-40%PS）结合。p65 1-3与脂质体的半数最大结合发生在约p Ca2+ = 5.2时，而p65 3-5在pCa2+ = 5.5-2.8范围内似乎与钙无关地结合。p65 1-5与PS/PC脂质体的结合表现出两种较小蛋白质的特征，在EGTA中显示出一些结合，在钙存在下结合增加。与任何一个较小片段相比，更多的p65 1-5与PS/PC脂质体结合。这些结果表明，突触结合蛋白的两个C2结构域协同作用，促进与生物膜的结合，并影响钙敏感性和膜结合能力。
