de Heuvel E, Bell A W, Ramjaun A R, Wong K, Sossin W S, McPherson P S
Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.
J Biol Chem. 1997 Mar 28;272(13):8710-6. doi: 10.1074/jbc.272.13.8710.
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.
突触结合蛋白是一种在神经末梢中富集的肌醇5-磷酸酶，被认为在突触小泡的胞吞作用中发挥作用，部分是通过与发动蛋白的Src同源3结构域相互作用来实现的。我们利用杆状病毒介导表达后从Sf9细胞中纯化得到的突触结合蛋白，通过覆盖分析来鉴定大鼠脑中两种主要的突触结合蛋白结合蛋白。第一种，分子量为125 kDa，是发动蛋白。第二种，分子量为40 kDa，是检测到的主要突触结合蛋白结合蛋白，在脑中高度富集，集中在可溶性突触组分中，并与突触结合蛋白共免疫沉淀。40 kDa的蛋白不与缺乏富含脯氨酸的C末端的突触结合蛋白构建体结合，这表明它与突触结合蛋白的相互作用是通过Src同源3结构域介导的。通过三步程序从大鼠脑胞质溶胶中部分纯化了40 kDa的突触结合蛋白结合蛋白，该程序包括硫酸铵沉淀、蔗糖密度梯度离心和DEAE离子交换色谱。肽序列分析确定40 kDa的蛋白为SH3P4，它是一个含有Src同源3结构域的新型蛋白家族的成员。这些数据表明SH3P4在突触小泡胞吞作用中起重要作用。
