The DNAJA2 Substrate Release Mechanism Is Essential for Chaperone-mediated Folding
The DNAJA2 Substrate Release Mechanism Is Essential for Chaperone-mediated Folding
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DOI：
10.1074/jbc.m112.413278
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发表时间：
2012-12-07
影响因子：
4.8
通讯作者：
Young, Jason C.
中科院分区：
文献类型：
--
作者：
Baaklini, Imad;Wong, Michael J. H.;Young, Jason C.
DNAJA 1（DJA 1/Hdj 2）和DNAJA 2（DJA 2）是人Hsp 70/Hsc 70分子伴侣的主要J结构域伴侣。虽然它们与来自酵母和细菌的充分表征的I型共分子伴侣具有总体相似性，但它们在生物学上是不同的，并且它们的功能机制的特征很差。我们确定了DJA 2在荧光素酶折叠和抑制人类ether-a-go-go相关基因（HERG）运输中的特异性活性，这取决于其在细胞中的表达水平。DJA 2不同内部结构域的突变消除了这些影响。使用纯化的蛋白质，我们解决了机械缺陷。缺乏锌指基序之间的区域（DJA 2-Δ m2）的突变体能够与野生型类似地结合底物，但在其转移至Hsc 70期间不能释放底物。DJA 1中的等同突变也消除了其底物释放。一个DJA 2突变体（DJA-221），其C-末端二聚化区域被DJA 1取代，是无活性的，但保留了其释放底物的能力。释放机制需要J结构域和ATP水解Hsc 70，虽然核苷酸依赖性DJA 2和DJA 1之间的分歧。有限的蛋白水解表明两个野生型共分子伴侣和突变体之间的进一步构象差异。我们的研究结果表明，Hsc 70的折叠机制中的特定DJA结构域的重要作用。
DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Delta m2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70.