Interaction of assembly protein AP-2 and its isolated subunits with clathrin.
Interaction of assembly protein AP-2 and its isolated subunits with clathrin.
复制标题
组装蛋白 AP-2 及其分离亚基与网格蛋白的相互作用。
DOI：
10.1021/bi00236a036
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Keen,JH
中科院分区：
文献类型：
--
作者：
Prasad,K;Keen,JH
1991年1月28日收到的修订手稿摘要：网格蛋白组装蛋白复合物AP-2是一种多聚亚基复合物，由两个100-115-kDa亚基组成，称为a和/3以及50-和16-kDa亚基。亚基已被解离和分离的离子交换色谱在7.5 M尿素。获得了高度富集α或β亚基的级分。a级分与网格蛋白相互作用，如通过其与预组装网格蛋白笼结合的能力所证明的。它还与解离的网格蛋白三聚体反应的条件下，有利于组装的外套结构，但不产生离散的网格蛋白多边形晶格。富集的β组分（含有少量α）与网格蛋白反应，产生完整的涂层，并将大约等量的α和β亚基掺入聚合物质中;过量的游离β亚基不起反应。AP-2复合物也完全解离在一个高度变性的溶剂，6 M Gdn-HCl，和100-115，50，和16 kDa的组成亚基通过凝胶过滤分离。在与网格蛋白的共组装测定中，网格蛋白聚合活性仅与100-kDa亚基组分相关，其中100 kDa的α和β亚基化学计量掺入聚合的外壳中，并且不需要50-或16-kDa亚基。这些观察结果表明复合物的组装活性与α和0亚基有关，并表明这两个亚基通过与网格蛋白的独立相互作用，是表达完整的晶格组装活性所必需的。
Revised Manuscript Received January 28, 1991 abstract: The clathrin assembly protein complex AP-2 is a multimeric subunitcomplex consisting of two 100-115-kDa subunits known as a and/3 and 50-and 16-kDa subunits. The subunits have been dissociated and separated by ion-exchange chromatography in 7.5 M urea. Fractions highly enriched in either the a or (3 subunit were obtained. The a fraction interacted with clathrin as evidenced by its ability to bind to preassembled clathrin cages. It also reacted with dissociated clathrin trimers under conditions that favor assembly of coat structures, but did not yield discrete clathrin polygonal lattices. The enriched $ fraction (containing small amounts of a) reacted with clathrin to yieldintact coats with the incorporation of approximately equivalent amounts of a and (8 subunits into the polymerized species; excess free/3 subunit was unreactive. The AP-2 complex was also completely dissociated in a highly denaturing solvent, 6 M Gdn-HCl, and the constituent subunits of 100-115, 50, and 16 kDa were separated by gel filtration. In a coassembly assay with clathrin, the clathrin polymerizing activity was exclusively associated with the 100-kDa subunitfraction with stoichiometric incorporation of both a and/3 subunits of 100 kDa into the polymerized coats, and with no requirement for 50-or 16-kDa subunits. These observations demonstrate that theassembly activity of the complex is associated with the a and 0 subunits and suggest that both subunits, through independent interactions with clathrin, are required for expression of complete lattice assembly activity.