Department of Biochemistry and Biophysics, MS 3474, Texas A&M University, College Station, TX 77843, USA.
Genetics. 2022 Jul 30;221(4). doi: 10.1093/genetics/iyac090.
Cytochrome c oxidase (CcO) is a multimeric copper-containing enzyme of the mitochondrial respiratory chain that powers cellular energy production. The two core subunits of cytochrome c oxidase, Cox1 and Cox2, harbor the catalytic CuB and CuA sites, respectively. Biogenesis of each copper site occurs separately and requires multiple proteins that constitute the mitochondrial copper delivery pathway. Currently, the identity of all the members of the pathway is not known, though several evolutionarily conserved twin CX9C motif-containing proteins have been implicated in this process. Here, we performed a targeted yeast suppressor screen that placed Coa4, a twin CX9C motif-containing protein, in the copper delivery pathway to the Cox1 subunit. Specifically, we show that overexpression of Cox11, a copper metallochaperone required for the formation of CuB site, can restore Cox1 abundance, cytochrome c oxidase assembly, and mitochondrial respiration in coa4Δ cells. This rescue is dependent on the copper-coordinating cysteines of Cox11. The abundance of Coa4 and Cox11 in mitochondria is reciprocally regulated, further linking Coa4 to the CuB site biogenesis. Additionally, we find that coa4Δ cells have reduced levels of copper and exogenous copper supplementation can partially ameliorate its respiratory-deficient phenotype, a finding that connects Coa4 to cellular copper homeostasis. Finally, we demonstrate that human COA4 can replace the function of yeast Coa4 indicating its evolutionarily conserved role. Our work provides genetic evidences for the role of Coa4 in the copper delivery pathway to the CuB site of cytochrome c oxidase.
细胞色素 c 氧化酶（CcO）是线粒体呼吸链中的一种多聚体铜含有酶，为细胞能量产生提供动力。细胞色素 c 氧化酶的两个核心亚基 Cox1 和 Cox2 分别含有催化 CuB 和 CuA 位点。每个铜位点的生物发生是分开进行的，需要多个构成线粒体铜递呈途径的蛋白质。目前，该途径的所有成员的身份尚不清楚，尽管几个进化上保守的双 CX9C 基序蛋白已被牵连到这个过程中。在这里，我们进行了靶向酵母抑制子筛选，将含有双 CX9C 基序的 Coa4 置于 Cox1 亚基的铜递呈途径中。具体来说，我们表明，过表达 Cox11，一种形成 CuB 位点所需的铜金属伴侣蛋白，可以恢复 coa4Δ 细胞中 Cox1 的丰度、细胞色素 c 氧化酶组装和线粒体呼吸作用。这种拯救依赖于 Cox11 的铜配位半胱氨酸。Coa4 和 Cox11 在线粒体中的丰度是相互调节的，这进一步将 Coa4 与 CuB 位点生物发生联系起来。此外，我们发现 coa4Δ 细胞的铜含量降低，外源性铜补充可以部分改善其呼吸缺陷表型，这一发现将 Coa4 与细胞内铜稳态联系起来。最后，我们证明了人 COA4 可以替代酵母 Coa4 的功能，表明其在进化上保守的作用。我们的工作为 Coa4 在细胞色素 c 氧化酶的 CuB 位点铜递呈途径中的作用提供了遗传证据。
