Damer C K, Creutz C E
Neuroscience Graduate Program, University of Virginia, Charlottesville 22908, USA.
J Neurochem. 1996 Oct;67(4):1661-8. doi: 10.1046/j.1471-4159.1996.67041661.x.
Synaptotagmin I, an integral membrane protein of secretory vesicles, appears to have an essential role in calcium-triggered hormone and neurotransmitter release. The large cytoplasmic domain of synaptotagmin I has two C2 domains that are thought to mediate calcium and phospholipid binding. A recombinant protein (p65 1-5) comprised of the cytoplasmic domain was previously shown to aggregate purified chromaffin granules and artificial phospholipid vesicles in a calcium-dependent manner. p65 1-5 may be able to aggregate membrane vesicles by a self-association reaction. This hypothesis led us to investigate the ability of synaptotagmin I protein fragments to multimerize in vitro. We found that p65 1-5, in the absence of membranes, was able to self-associate to form large aggregates in a calcium-dependent manner as shown by light-scattering assays and electron microscopy. In addition, a recombinant protein comprised of only the second half of the cytoplasmic domain, including the second C2 domain, was also able to self-associate and aggregate phospholipid vesicles in a calcium-dependent manner. A recombinant protein comprised of only the first C2 domain was not able to self-associate or aggregate vesicles. These results suggest that synaptotagmin I is able to bind calcium in the absence of membranes and that the second half of the cytoplasmic domain is able to bind calcium and mediate its multimerization in a calcium-dependent manner. The ability of synaptotagmin I protein fragments to multimerize in a calcium-dependent manner in vitro suggests that multimerization may have an important function in vivo.
突触结合蛋白I是分泌囊泡的一种整合膜蛋白，似乎在钙触发的激素和神经递质释放中起关键作用。突触结合蛋白I的大细胞质结构域有两个C2结构域，被认为介导钙和磷脂结合。先前已证明，由细胞质结构域组成的重组蛋白（p65 1-5）能以钙依赖的方式聚集纯化的嗜铬粒蛋白颗粒和人工磷脂囊泡。p65 1-5可能能够通过自缔合反应聚集膜囊泡。这一假设促使我们研究突触结合蛋白I蛋白片段在体外多聚化的能力。我们发现，如光散射分析和电子显微镜所示，在没有膜的情况下，p65 1-5能够以钙依赖的方式自缔合形成大聚集体。此外，仅由细胞质结构域后半部分（包括第二个C2结构域）组成的重组蛋白也能够以钙依赖的方式自缔合并聚集磷脂囊泡。仅由第一个C2结构域组成的重组蛋白不能自缔合或聚集囊泡。这些结果表明，突触结合蛋白I在没有膜的情况下能够结合钙，并且细胞质结构域的后半部分能够结合钙并以钙依赖的方式介导其多聚化。突触结合蛋白I蛋白片段在体外以钙依赖的方式多聚化的能力表明，多聚化在体内可能具有重要功能。
