Proteomic comparison of two fractions derived from the transsynaptic scaffold.
Proteomic comparison of two fractions derived from the transsynaptic scaffold.
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来自跨突触支架的两个级分的蛋白质组比较。
DOI：
10.1002/jnr.20614
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Colman,DavidR
中科院分区：
文献类型：
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作者：
Phillips,GregR;Florens,Laurence;Tanaka,Hidekazu;Khaing,ZinZ;Fidler,Lazar;Yates3rd,JohnR;Colman,DavidR
通过调节Triton X-100（TX-100）提取分离的跨突触支架的pH值，可以将源自突触前特化的部分（突触前颗粒部分; PPF）与突触后密度（PSD）分离。PPF的溶解对应于突触前特化的破坏。我们表明，PPF在pH 6.0下不溶于重复TX-100浸提，但在pH 8.0下可溶于洗涤剂。通过免疫定位，我们发现PPF的主要蛋白，网格蛋白和发动蛋白，集中在突触前区室。利用多维蛋白质鉴定技术，比较了PPF和PSD组分的蛋白质组成。我们共鉴定了341种蛋白质，其中50种在PPF中独特发现，231种在PSD组分中，60种在两种组分中。两个馏分的比较显示，肌动蛋白和相关蛋白的比例相对较低，囊泡或细胞内室蛋白的比例较高的PPF。我们得出结论，PPF由不连接到基于肌动蛋白的突触框架的突触前蛋白组成;其在pH 6中的不溶性和在pH 8缓冲洗涤剂中的溶解性表明网格蛋白可能是PPF中许多蛋白质的锚定支架。© 2005 Wiley利斯公司
A fraction derived from presynaptic specializations (presynaptic particle fraction; PPF) can be separated from postsynaptic densities (PSD) by adjusting the pH of Triton X‐100 (TX‐100) extraction of isolated transsynaptic scaffolds. Solubilization of the PPF corresponds to disruption of the presynaptic specialization. We show that the PPF is insoluble to repeated TX‐100 extraction at pH 6.0 but becomes soluble in detergent at pH 8.0. By immunolocalization, we find that the major proteins of the PPF, clathrin and dynamin, are concentrated in the presynaptic compartment. By using multidimensional protein identification technology, we compared the protein compositions of the PPF and the PSD fraction. We identified a total of 341 proteins, 50 of which were uniquely found in the PPF, 231 in the PSD fraction, and 60 in both fractions. Comparison of the two fractions revealed a relatively low proportion of actin and associated proteins and a high proportion of vesicle or intracellular compartment proteins in the PPF. We conclude that the PPF consists of presynaptic proteins not connected to the actin‐based synaptic framework; its insolubility in pH 6 and solubility in pH 8 buffered detergent suggests that clathrin might be an anchorage scaffold for many proteins in the PPF. © 2005 Wiley‐Liss, Inc.