CLATHRIN-COATED VESICLES - ISOLATION, DISSOCIATION AND FACTOR-DEPENDENT REASSOCIATION OF CLATHRIN BASKETS
CLATHRIN-COATED VESICLES - ISOLATION, DISSOCIATION AND FACTOR-DEPENDENT REASSOCIATION OF CLATHRIN BASKETS
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DOI：
10.1016/0092-8674(79)90007-2
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发表时间：
1979-01-01
期刊：
影响因子：
64.5
通讯作者：
PASTAN, IH
中科院分区：
文献类型：
--
作者：
KEEN, JH;WILLINGHAM, MC;PASTAN, IH
网格蛋白包被囊泡上的蛋白质外壳的性质和负责网格蛋白篮的结构和改造的相互作用进行了研究。包被的囊泡从牛脑中分离使用快速，1天的修改的Pearse方法。囊泡主要由网格蛋白（175，000）和少量110，000和55，000 MW多肽组成。网格蛋白被释放在一个溶解的形式从这些囊泡通过处理与0.5 M的三（羟甲基）甲基氯化铵（Tris-Cl）或其他质子化胺在中性pH值。它没有释放与硫醇，硫醇试剂，Triton X-100或NaCl处理，这表明特定的氨基羧酸盐连接是必要的篮子结构的维护。在EM下，Tris-Cl中的增溶蛋白以丝状聚集体的形式存在，并且没有观察到篮状结构。在Tris-Cl中对提取物进行凝胶过滤，从主要由110，000 MW多肽（II）组成的组分中分离出含网格蛋白的组分（I）。来自未分级Tris-Cl提取物的篮状结构不能通过用囊泡分离缓冲液（一种中等离子强度的溶液（<$MA/ 2 = 0.11）。无论是单独的解决部分（I或II）产生篮透析，但重建是成功的，当I和II合并和透析。在II，这似乎是一个篮子组装因子的活动，是热不稳定的，不能被牛血清白蛋白或脑钙依赖性调节蛋白取代。如果使用低离子强度（<$/ 2 = 0.01）作为透析液，单独的网格蛋白级分（I）能够重整篮。网格蛋白外壳显然是一种不稳定的结构，可以通过非变性处理溶解，并且篮子可以从提取的材料中重新形成。
The nature of the protein coat on clathrin-coated vesicles and the interactions responsible for the structure and reformation of clathrin baskets were investigated. Coated vesicles were isolated from bovine brain using a rapid, 1-day modification of the Pearse method. The vesicles are composed predominantly of clathrin (175,000) with smaller amounts of 110,000 and 55,000 MW polypeptides. Clathrin was released in a solubilized form from these vesicles by treatment with 0.5 M Tris(hydroxymethyl)methyl ammonium chloride (Tris-Cl) or other protonated amines at neutral pH. It was not released on treatment with thiols, thiol reagents, Triton X-100 or NaCl, suggesting that specific amino-carboxylate salt linkages are necessary for maintenance of the basket structure. Under EM the solubilized proteins in Tris-Cl are present in the form of filamentous aggregates and no basket structures are observed. Gel filtration of the extract in Tris-Cl resolves a clathrin-containing fraction (I) from one consisting predominantly of the 110,000 MW polypeptide (II). Basket structures from the unfractionated Tris-Cl extract could not be reconstituted by dialyzing it against the vesicle isolation buffer, a solution of moderate ionic strength (.GAMMA./2 = 0.11). Neither of the resolved fractions (I or II) alone yielded baskets on dialysis, but reconstitution was successful when both I and II were combined and dialyzed. The activity in II, which appears to be a basket-assembly factor, was heat-labile and could not be replaced by bovine serum albumin or brain Ca-dependent modulator protein. If a solution of low ionic strength (.GAMMA./2 = 0.01) containing Ca was used as the dialysate, the clathrin fraction (I) alone was capable of reforming baskets. The clathrin coat is apparently a labile structure that can be solubilized by nondenaturing treatments and baskets can be reformed from the extracted material.