Interactions of synapsin I with small synaptic vesicles: distinct sites in synapsin I bind to vesicle phospholipids and vesicle proteins.
Interactions of synapsin I with small synaptic vesicles: distinct sites in synapsin I bind to vesicle phospholipids and vesicle proteins.
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DOI：
10.1083/jcb.108.5.1863
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发表时间：
1989-05
期刊：
影响因子：
--
通讯作者：
Greengard P
中科院分区：
文献类型：
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作者：
Benfenati F;Bähler M;Jahn R;Greengard P
突触蛋白I是一种主要的神经元特异性磷酸化蛋白，特异定位于小突触囊泡的细胞质表面。本研究详细描述了突触素1与小突触囊泡的结合。当突触囊泡在磷脂酰胆碱中溶解和重组时，突触蛋白1的结合得以保留。在非变性条件下，突触囊泡的蛋白质和脂质成分分离后，突触蛋白1与这两种成分结合。使用疏水标记程序可以评估完整突触囊泡中磷脂和突触蛋白I之间的相互作用。疏水光标记后，半胱氨酸特异性切割突触蛋白1，表明突触蛋白1的头部结构域渗透到双分子层的疏水核心。纯化的nh2末端片段，通过半胱氨酸特异性切割从头部结构域获得，以高亲和力结合到突触囊泡上，证实了疏水光标记的结果。突触素I与突触囊泡的结合可以被整个分子或nh2末端和尾部片段的联合存在所抑制，但不能被过量的nh2末端或尾部片段单独抑制。纯化后的尾部片段与突触囊泡结合具有较高的亲和力，但与磷脂没有明显的相互作用。尾片段的结合由holosynapsin I完成；被磷酸化大大降低；并通过高离子强度条件或蛋白酶处理突触囊泡被消除。这些数据表明突触蛋白I与小突触囊泡之间存在两个相互作用位点：头部结构域与囊泡磷脂结合，尾部结构域与囊泡膜的蛋白质成分结合。后一种相互作用显然是突触蛋白1与小突触囊泡结合的盐依赖性和磷酸化依赖性的原因。
Synapsin I is a major neuron-specific phosphoprotein that is specifically localized to the cytoplasmic surface of small synaptic vesicles. In the present study, the binding of synapsin I to small synaptic vesicles was characterized in detail. The binding of synapsin I was preserved when synaptic vesicles were solubilized and reconstituted in phosphatidylcholine. After separation of the protein and lipid components of synaptic vesicles under nondenaturing conditions, synapsin I bound to both components. The use of hydrophobic labeling procedures allowed the assessment of interactions between phospholipids and synapsin I in intact synaptic vesicles. Hydrophobic photolabeling followed by cysteine-specific cleavage of synapsin I demonstrated that the head domain of synapsin I penetrates into the hydrophobic core of the bilayer. The purified NH2-terminal fragment, derived from the head domain by cysteine-specific cleavage, bound to synaptic vesicles with high affinity confirming the results obtained from hydrophobic photolabeling. Synapsin I binding to synaptic vesicles could be inhibited by the entire molecule or by the combined presence of the NH2-terminal and tail fragments, but not by an excess of either NH2-terminal or tail fragment alone. The purified tail fragment bound with relatively high affinity to synaptic vesicles, though it did not significantly interact with phospholipids. Binding of the tail fragment was competed by holosynapsin I; was greatly decreased by phosphorylation; and was abolished by high ionic strength conditions or protease treatment of synaptic vesicles. The data suggest the existence of two sites of interaction between synapsin I and small synaptic vesicles: binding of the head domain to vesicle phospholipids and of the tail domain to a protein component of the vesicle membrane. The latter interaction is apparently responsible for the salt and phosphorylation dependency of synapsin I binding to small synaptic vesicles.