A genome-wide copper-sensitized screen identifies novel regulators of mitochondrial cytochrome c oxidase activity.
A genome-wide copper-sensitized screen identifies novel regulators of mitochondrial cytochrome c oxidase activity.
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全基因组铜敏化筛选鉴定出线粒体细胞色素 c 氧化酶活性的新型调节因子。
DOI：
10.1016/j.jbc.2021.100485
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发表时间：
2021-01
期刊：
影响因子：
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通讯作者：
Gohil VM
中科院分区：
文献类型：
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作者：
Garza NM;Griffin AT;Zulkifli M;Qiu C;Kaplan CD;Gohil VM
铜对于细胞色素 C 氧化酶 (CcO) 的活性和稳定性至关重要，CcO 是线粒体呼吸链的末端酶。铜转运至 CcO 所需基因的功能缺失突变会导致致命的人类疾病。尽管铜在线粒体和有机体生理学中具有根本重要性，但缺乏对调节线粒体铜稳态的基因的系统鉴定。为了发现这些基因，我们使用在补充铜的培养基中生长的 DNA 条形码酵母缺失突变体库进行了全基因组筛选。我们的筛选恢复了许多已知参与细胞铜稳态的基因，以及以前与线粒体铜生物学无关的基因。这些新发现的基因包括接头蛋白 3 复合物 (AP-3) 的亚基以及细胞 pH 传感途径 Rim20 和 Rim21 的成分，已知这两者都会影响液泡功能。我们发现 AP-3 和 Rim 突变体表现出液泡酸度降低，进而扰乱线粒体铜稳态和 CcO 功能。这些突变体的 CcO 活性可以通过恢复液泡 pH 值或在生长培养基中添加额外的铜来恢复。与这些遗传数据一致，液泡质子泵的药理学抑制导致线粒体铜含量降低，并伴随 CcO 丰度和活性降低。综上所述，我们的研究发现了线粒体铜稳态的新型遗传调节因子，并提供了液泡 pH 通过铜稳态影响线粒体呼吸的机制。
Copper is essential for the activity and stability of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Loss-of-function mutations in genes required for copper transport to CcO result in fatal human disorders. Despite the fundamental importance of copper in mitochondrial and organismal physiology, systematic identification of genes that regulate mitochondrial copper homeostasis is lacking. To discover these genes, we performed a genome-wide screen using a library of DNA-barcoded yeast deletion mutants grown in copper-supplemented media. Our screen recovered a number of genes known to be involved in cellular copper homeostasis as well as genes previously not linked to mitochondrial copper biology. These newly identified genes include the subunits of the adaptor protein 3 complex (AP-3) and components of the cellular pH-sensing pathway Rim20 and Rim21, both of which are known to affect vacuolar function. We find that AP-3 and Rim mutants exhibit decreased vacuolar acidity, which in turn perturbs mitochondrial copper homeostasis and CcO function. CcO activity of these mutants could be rescued by either restoring vacuolar pH or supplementing growth media with additional copper. Consistent with these genetic data, pharmacological inhibition of the vacuolar proton pump leads to decreased mitochondrial copper content and a concomitant decrease in CcO abundance and activity. Taken together, our study uncovered novel genetic regulators of mitochondrial copper homeostasis and provided a mechanism by which vacuolar pH impacts mitochondrial respiration through copper homeostasis.