Glutathione (GSH) is a small-molecule thiol that is abundant in all eukaryotes and has key roles in oxidative metabolism¹. Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions². GSH is synthesized exclusively in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remains unknown. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, as a regulator of GSH transport into mitochondria. Loss of SLC25A39 reduces mitochondrial GSH import and abundance without affecting cellular GSH levels. Cells lacking both SLC25A39 and its paralogue SLC25A40 exhibit defects in the activity and stability of proteins containing iron–sulfur clusters. We find that mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enables mitochondrial GSH production and ameliorates the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH-import machinery.

谷胱甘肽 (GSH) 是一种小分子硫醇，在所有真核生物中含量丰富，在氧化代谢中具有关键作用¹ 。线粒体作为氧化反应的主要场所，必须维持足够的 GSH 水平才能发挥保护和生物合成功能² 。 GSH 仅在细胞质中合成，但参与线粒体 GSH 输入的分子机制仍然未知。在这里，利用细胞器蛋白质组学和代谢组学方法，我们确定了 SLC25A39（一种功能未知的线粒体膜载体）作为 GSH 转运到线粒体的调节剂。 SLC25A39 的缺失会减少线粒体 GSH 的输入和丰度，但不会影响细胞内的 GSH 水平。同时缺乏 SLC25A39 及其旁系同源物 SLC25A40 的细胞在含有铁硫簇的蛋白质的活性和稳定性方面表现出缺陷。我们发现线粒体 GSH 的输入对于体外细胞增殖和小鼠红细胞发育是必需的。工程化双功能细菌 GSH 生物合成酶 (GshF) 在线粒体中的异源表达能够实现线粒体 GSH 的产生，并改善因其消耗而引起的代谢和增殖缺陷。最后，GSH 的可用性对 SLC25A39 蛋白丰度产生负向调节，将氧化还原稳态与哺乳动物细胞中线粒体 GSH 的输入耦合起来。我们的工作将 SLC25A39 确定为线粒体 GSH 输入机制的重要且受调节的组成部分。