Defining the domains of Cia2 required for its essential function in vivo and in vitro.
Defining the domains of Cia2 required for its essential function in vivo and in vitro.
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DOI：
10.1039/c7mt00181a
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发表时间：
2017-11
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通讯作者：
Amanda Vo;Nicholas M Fleischman;Melissa D Marquez;Eric J Camire;Stephanie Esonwune;John D Grossman;K. Gay;Jessica A Cosman;Deborah L. Perlstein
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作者：
Amanda Vo;Nicholas M Fleischman;Melissa D Marquez;Eric J Camire;Stephanie Esonwune;John D Grossman;K. Gay;Jessica A Cosman;Deborah L. Perlstein
胞质铁硫簇组装 (CIA) 系统生物合成胞质和核蛋白的铁硫 (FeS) 簇辅助因子。酵母 Cia2 蛋白是靶向复合物的核心成分，该复合物在该途径的最后一步中识别脱辅基蛋白靶标。在此，我们确定 Cia2 包含五个保守基序，分布在本质上无序的 N 端结构域和未知功能 59 (DUF59) 的 C 端结构域之间。无序结构域对于体外结合靶向复合物 Met18 和 Cia1 以及 apo 靶标 Rad3 的其他亚基而言是可有可无的。虽然体内测定表明 C 端结构域足以支持活力，但一些表型测定表明 N 端结构域的删除会对 CIA 功能产生负面影响。我们还确定，Glu208 位于仅在真核 DUF59 蛋白中发现的保守基序内，对于体外 Cia1-Cia2 相互作用很重要。在体内，E208A-Cia2 会导致胞质铁硫簇蛋白 Leu1 的活性降低，但对羟基脲或甲磺酸甲酯的敏感性仅产生适度的影响。最后，我们证明 DUF59 结构域的两个高度保守的基序对于体外 Cia2 的任何相互作用都不是至关重要的，但 DUF59 结构域中 DPE 基序的突变会导致体内无功能的等位基因。我们观察到，Cia2 的五个高度保守基序中的四个对于靶向复合物形成和 apo 靶标结合是可有可无的，这表明 Cia2 不仅仅是蛋白质-蛋白质相互作用介体，而且在 CIA 最后的簇插入步骤中，它可能具有额外的、目前神秘的功能。
The cytosolic iron-sulfur cluster assembly (CIA) system biosynthesizes iron-sulfur (FeS) cluster cofactors for cytosolic and nuclear proteins. The yeast Cia2 protein is the central component of the targeting complex which identifies apo-protein targets in the final step of the pathway. Herein, we determine that Cia2 contains five conserved motifs distributed between an intrinsically disordered N-terminal domain and a C-terminal domain of unknown function 59 (DUF59). The disordered domain is dispensible for binding the other subunits of the targeting complex, Met18 and Cia1, and the apo-target Rad3 in vitro. While in vivo assays reveal that the C-terminal domain is sufficient to support viability, several phenotypic assays indicate that deletion of the N-terminal domain negatively impacts CIA function. We additionally establish that Glu208, located within a conserved motif found only in eukaryotic DUF59 proteins, is important for the Cia1-Cia2 interaction in vitro. In vivo, E208A-Cia2 results in a diminished activity of the cytosolic iron sulfur cluster protein, Leu1 but only modest effects on hydroxyurea or methylmethane sulfonate sensitivity. Finally, we demonstrate that neither of the two highly conserved motifs of the DUF59 domain are vital for any of Cia2's interactions in vitro yet mutation of the DPE motif in the DUF59 domain results in a nonfunctional allele in vivo. Our observation that four of the five highly conserved motifs of Cia2 are dispensable for targeting complex formation and apo-target binding suggests that Cia2 is not simply a protein-protein interaction mediator but it likely possesses an additional, currently cryptic, function during the final cluster insertion step of CIA.