Barouch W, Prasad K, Greene L, Eisenberg E
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biochemistry. 1997 Apr 8;36(14):4303-8. doi: 10.1021/bi962727z.
We previously reported that a 100-kDa cofactor, recently identified as auxilin, is a DnaJ 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 microM. 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 AP180, 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.
我们之前报道过，一种最近被鉴定为辅助蛋白的100 kDa辅因子是一种DnaJ同源物，它是Hsc70解开网格蛋白笼所必需的。在本研究中，我们研究了辅助蛋白在pH 6时对Hsc70与纯网格蛋白笼相互作用的影响，此时不会发生去包被。在一个需要辅助蛋白的反应中，网格蛋白笼将Hsc70的ATP酶活性激活了100多倍，表观解离常数约为0.2 μM。最大ATP酶活性出现在辅助蛋白与网格蛋白三脚复合体的摩尔比为1:1时，与Hsc70浓度无关，这表明辅助蛋白主要与网格蛋白笼结合。Hsc70与网格蛋白笼的结合也需要辅助蛋白，但最大结合所需的辅助蛋白比最大ATP酶活性所需的少，这表明辅助蛋白可以催化诱导Hsc70的结合。这种结合也需要ATP；当ATP水解为ADP时，Hsc70以6分钟的半衰期从网格蛋白笼上解离。与辅助蛋白不同，组装蛋白AP-2和AP180不支持网格蛋白笼对Hsc70 ATP酶活性的激活，在pH 7时，存在辅助蛋白的可溶性网格蛋白三脚复合体也不会显著激活ATP酶活性。因此，辅助蛋白、网格蛋白笼和Hsc70-ATP之间的相互作用具有高度特异性，辅助蛋白首先与网格蛋白笼中的网格蛋白三脚复合体结合，然后Hsc70-ATP与辅助蛋白-网格蛋白复合体强烈结合；在ATP酶循环完成之前，辅助蛋白可以迁移到另一个网格蛋白三脚复合体。
