Auxilin-induced interaction of the molecular chaperone Hsc70 with clathrin baskets
Auxilin-induced interaction of the molecular chaperone Hsc70 with clathrin baskets
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DOI：
10.1021/bi962727z
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发表时间：
1997-04-08
期刊：
影响因子：
2.9
通讯作者：
Eisenberg, E
中科院分区：
文献类型：
--
作者：
Barouch, W;Prasad, K;Eisenberg, E
我们之前曾报道过一个100 kDa的辅因子，最近被鉴定为Axin，它是Hsc70解开网状蛋白篮子所需的DNAT同源物。在目前的研究中，我们研究了在pH为6的条件下，在没有脱膜的情况下，生长素对Hsc70与纯络合蛋白篮子相互作用的影响。在一个需要生长素的反应中，篮子激活了Hsc70 ATPase活性100多倍，表观解离常数约为0.2mM。最大ATPase活性出现在与Hsc70浓度无关的摩尔比为1：1的分子筛上，这表明Axin主要与笼状蛋白篮络合。Hsc70与篮子的结合也需要生长素，但最大结合所需的生长素比最大ATPase活性所需的更少，这表明生长素可以催化诱导Hsc70的结合。结合也需要三磷酸腺苷；当三磷酸腺苷被水解成ADP时，Hsc70从篮子中解离，半衰期为6分钟。与生长素相反，组装蛋白AP-2和AP(180)不支持分子筛对Hsc70 ATPase活性的激活，也不支持在pH为7的可溶性分子筛存在的情况下激活ATPase活性。因此，生长素、网织物篮和Hsc70-ATP之间的相互作用是高度特异的，首先，生长素与篮子中的一个网织物三棱柱结合，然后Hsc70-ATP与生长素-网壳蛋白复合体强烈结合；然后，在ATPase循环完成之前，生长素可以迁移到另一个网织物三棱柱上。
We previously reported that a 100-kDa cofactor, recently identified as auxilin, is a DnaT homolog which is required for Hsc70 to uncoat clathrin baskets. In the present study we investigated the effect of auxilin on the interaction of Hsc70 with pure clathrin baskets at pH 6, where no uncoating occurs. In a reaction which required auxilin, the baskets activated the Hsc70 ATPase activity more than 100-fold with an apparent dissociation constant of about 0.2 mu M. Maximal ATPase activity occurred at a 1 to 1 molar ratio of auxilin to clathrin triskelion independent of the Hsc70 concentration suggesting that auxilin is primarily complexed with the clathrin baskets. The binding of Hsc70 to baskets also required auxilin, but less auxilin was needed for maximum binding than for maximum ATPase activity showing that auxilin can catalytically induce binding of Hsc70. The binding also required ATP; Hsc70 dissociated from baskets with a 6 min half-life when ATP was hydrolyzed to ADP. In contrast to auxilin, the assembly proteins, AP-2 and AP(180), did not support activation of the Hsc70 ATPase activity by clathrin baskets nor did Soluble clathrin triskelions at pH 7 significantly activate the ATPase activity with auxilin present. Therefore, the interaction of auxilin, clathrin baskets, and Hsc70-ATP is highly specific with auxilin first binding to a clathrin triskelion in the baskets and then Hsc70-ATP strongly binding to the auxilin-clathrin complex; the auxilin can then migrate to another clathrin triskelion before the ATPase cycle is complete.