ERdj3, a luminal ER DnaJ homologue, binds directly to unfolded proteins in the mammalian ER: identification of critical residues.
ERdj3, a luminal ER DnaJ homologue, binds directly to unfolded proteins in the mammalian ER: identification of critical residues.
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DOI：
10.1021/bi8015923
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发表时间：
2009-01-13
期刊：
影响因子：
2.9
通讯作者：
Hendershot, Linda M.
中科院分区：
文献类型：
--
作者：
Jin, Yi;Zhuang, Min;Hendershot, Linda M.
ERdj3 被鉴定为可溶性腔内 DnaJ 家族成员，可与哺乳动物细胞内质网中未组装的免疫球蛋白重链 (HC) 以及 BiP 分子伴侣复合物结合。在这里，我们证明了 ERdj3 直接与未折叠的底物结合。二级结构预测表明，ERdj3 的底物结合结构域很可能与酵母胞质 DnaJ 家族成员 Ydj1 非常相似，Ydj1 先前与由结构域 I、II 和 III 组成的 C 末端片段结合的肽结晶。结构域 I 中的保守残基（形成 Ydj1 中的肽结合位点）的突变影响了 ERdj3 在哺乳动物细胞和体外结合研究中的底物结合能力。有点出乎意料的是，我们发现结构域 II 在 ERdj3 同源物中高度保守，但与 Ydj1 的结构域 II 非常不同，对于底物结合也至关重要。此外，我们证明ERdj3在细胞中形成多聚体，并发现保守的羧基末端残基苯丙氨酸326在自组装中发挥着关键作用。体外结合测定表明，该残基突变为丙氨酸会降低 ERdj3 的底物结合能力，表明多聚化对于底物结合很重要。这些研究共同表明，Ydj1 结构在另一个家族成员中是保守的，并揭示了在这组 DnaJ 蛋白中，结构域 II（在密切相关的 II 型家族成员中不存在）也在底物结合中发挥着重要作用。
ERdj3 was identified as a soluble, lumenal DnaJ family member that binds to unassembled immunoglobulin heavy chains (HC) along with the BiP chaperone complex in the endoplasmic reticulum of mammalian cells. Here we demonstrated that ERdj3 binds directly to unfolded substrates. Secondary structure predictions suggested that the substrate binding domain of ERdj3 was likely to closely resemble Ydj1, a yeast cytosolic DnaJ family member, which was previously crystallized with a peptide bound to the Cterminal fragment composed of domains I, II, and III. Mutation of conserved residues in domain I, which formed the peptide binding site in Ydj1, affected ERdj3’s substrate binding ability in mammalian cells and in vitro binding studies. Somewhat unexpectedly, we found that domain II, which is highly conserved among ERdj3 homologues, but very different from domain II of Ydj1, was also critical for substrate binding. In addition, we demonstrated that ERdj3 forms multimers in cells and found that the conserved carboxyterminal residue phenylalanine 326 played a critical role in self-assembly. In vitro binding assays revealed that mutation of this residue to alanine diminished ERdj3’s substrate binding ability, arguing that multimerization is important for substrate binding. Together these studies demonstrate that the Ydj1 structure is conserved in another family member and reveal that among this group of DnaJ proteins domain II, which is not resent in the closely related type II family members, also plays an essential role in ubstrate binding.
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