Identification of the major synaptojanin-binding proteins in brain
Identification of the major synaptojanin-binding proteins in brain
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DOI：
10.1074/jbc.272.13.8710
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发表时间：
1997-03-28
影响因子：
4.8
通讯作者：
McPherson, PS
中科院分区：
文献类型：
--
作者：
deHeuvel, E;Bell, AW;McPherson, PS
Synaptojanin是一种神经末梢富集的肌醇5-磷酸酶，被认为在突触囊泡内吞作用中起作用，部分通过与amphiphysin的Src同源3结构域相互作用。我们使用杆状病毒介导表达后从Sf9细胞中纯化的synaptojanin在覆盖实验中鉴定了大鼠脑中的两种主要的synaptojanin结合蛋白。第一个，125 kDa，是两栖素。第二种蛋白位于40 kDa，是检测到的主要的synaptojanin结合蛋白，在大脑中高度富集，浓缩在可溶的突触部分中，与synaptojanin共免疫沉淀。40kda蛋白不与缺乏富含脯氨酸的C末端的synaptojanin结构体结合，表明其与synaptojanin的相互作用是通过Src同源3结构域介导的。通过硫酸铵沉淀、蔗糖密度梯度离心和DEAE离子交换层析，从大鼠脑细胞质中部分纯化出40kda的synaptojanin结合蛋白。肽段序列分析鉴定该蛋白为SH3P4，是Src同源3结构域蛋白新家族的成员。这些数据表明SH3P4在突触囊泡内吞作用中起重要作用。
Synaptojanin is a nerve-terminal enriched inositol 5-phosphatase thought to function in synaptic vesicle endocytosis, in part through interactions with the Src homology 3 domain of amphiphysin. We have used synaptojanin purified from Sf9 cells after baculovirus mediated expression in overlay assays to identify two major synaptojanin-binding proteins in rat brain. The first, at 125 kDa, is amphiphysin. The second, at 40 kDa, is the major synaptojanin-binding protein detected, is highly enriched in brain, is concentrated in a soluble synaptic fraction, and co-immunoprecipitates with synaptojanin. The 40-kDa protein does not bind to a synaptojanin construct lacking the proline-rich C terminus, suggesting that its interaction with synaptojanin is mediated through an Src homology 3 domain. The 40-kDa synaptojanin-binding protein was partially purified from rat brain cytosol through a three-step procedure involving ammonium sulfate precipitation, sucrose density gradient centrifugation, and DEAE ion-exchange chromatography. Peptide sequence analysis identified the 40-kDa protein as SH3P4, a member of a novel family of Src homology 3 domain-containing proteins. These data suggest an important role for SH3P4 in synaptic vesicle endocytosis.