Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA.
J Biol Chem. 2021 Jan-Jun;296:100485. doi: 10.1016/j.jbc.2021.100485. Epub 2021 Mar 1.
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.
铜对于细胞色素 c 氧化酶（CcO）的活性和稳定性是必需的，CcO 是线粒体呼吸链的末端酶。导致铜向 CcO 运输的功能丧失的基因突变会导致致命的人类疾病。尽管铜在线粒体和机体生理学中具有重要意义，但调节线粒体铜稳态的基因的系统鉴定仍然缺乏。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文库进行了全基因组筛选。我们的筛选回收了许多已知参与细胞内铜稳态的基因，以及以前与线粒体铜生物学没有联系的基因。这些新鉴定的基因包括衔接蛋白 3 复合物（AP-3）的亚基和细胞 pH 感应途径 Rim20 和 Rim21 的成分，两者都已知会影响液泡功能。我们发现，AP-3 和 Rim 突变体表现出液泡酸度降低，这反过来又扰乱了线粒体铜稳态和 CcO 功能。这些突变体的 CcO 活性可以通过恢复液泡 pH 或在生长培养基中补充额外的铜来挽救。这些遗传数据一致表明，液泡质子泵的药理学抑制会导致线粒体铜含量减少，同时 CcO 丰度和活性也会降低。总之，我们的研究揭示了线粒体铜稳态的新的遗传调节因子，并提供了一种机制，即通过铜稳态，液泡 pH 会影响线粒体呼吸。
