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首页> 外文期刊>Biochemistry >ERdj3, a Luminal ER DnaJ Homologue, Binds Directly to Unfolded Proteins in the Mammalian ER: Identification of Critical Residues
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ERdj3, a Luminal ER DnaJ Homologue, Binds Directly to Unfolded Proteins in the Mammalian ER: Identification of Critical Residues

机译：ERdj3，一种发光的ER DnaJ同源物，直接与哺乳动物ER中的未折叠蛋白结合：鉴定关键残基

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摘要

ERdj3 was identified as a soluble, lumenal DnaJ family member that binds to unassembled immunoglobulin heavy chains 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 C-terminal 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 carboxy-terminal 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 present in the closely related type II family members, also plays an essential role in substrate binding.
机译：ERdj3被鉴定为可溶的腔内DnaJ家族成员，与哺乳动物细胞内质网中的未组装免疫球蛋白重链以及BiP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的底物结合域可能与酵母胞质DnaJ家族成员Ydj1非常相似，后者先前已与结合至由域I，II和III构成的C端片段的肽结晶。结构域I中保守的残基突变（在Ydj1中形成肽结合位点）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。出乎意料的是，我们发现在ERdj3同源物中高度保守的域II与Ydj1的域II非常不同，它对底物结合也很关键。此外，我们证明了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在自组装中起关键作用。体外结合试验表明，该残基突变为丙氨酸会减弱ERdj3的底物结合能力，理由是多聚化对于底物结合很重要。总之，这些研究表明Ydj1结构在另一个家族成员中是保守的，并揭示了在这组DnaJ蛋白质结构域II中（在紧密相关的II型家族成员中不存在）在底物结合中也起着重要作用。

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    《Biochemistry》 |2009年第1期|p.41-49|共9页
  • 作者

    Yi Jin Min Zhuang and Linda M. Hendershot;

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    Departments of Genetics and Tumor Cell Biology and of Structural Biology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, Tennessee 38105, and Integrated Program in Biological Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, Tennessee 38163;

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