Mitophagy plays an important role in the maintenance of mitochondrial homeostasis and can be categorized into two types: ubiquitin-mediated and receptor-mediated pathways. During receptor-mediated mitophagy, mitophagy receptors facilitate mitophagy by tethering the isolation membrane to mitochondria. Although at least five outer mitochondrial membrane proteins have been identified as mitophagy receptors, their individual contribution and interrelationship remain unclear. Here, we show that HeLa cells lacking BNIP3 and NIX, two of the five receptors, exhibit a complete loss of mitophagy in various conditions. Conversely, cells deficient in the other three receptors show normal mitophagy. Using BNIP3/NIX double knockout (DKO) cells as a model, we reveal that mitophagy deficiency elevates mitochondrial reactive oxygen species (mtROS), which leads to activation of the Nrf2 antioxidant pathway. Notably, BNIP3/NIX DKO cells are highly sensitive to ferroptosis when Nrf2-driven antioxidant enzymes are compromised. Moreover, the sensitivity of BNIP3/NIX DKO cells is fully rescued upon the introduction of wild-type BNIP3 and NIX, but not the mutant forms incapable of facilitating mitophagy. Consequently, our results demonstrate that BNIP3 and NIX-mediated mitophagy plays a role in regulating mtROS levels and protects cells from ferroptosis.

线粒体自噬在维持线粒体稳态中发挥着重要作用，可分为两种类型：泛素介导的途径和受体介导的途径。在受体介导的线粒体自噬过程中，线粒体自噬受体通过将隔离膜束缚到线粒体来促进线粒体自噬。尽管至少有五种线粒体外膜蛋白已被鉴定为线粒体自噬受体，但它们各自的贡献和相互关系仍不清楚。在这里，我们发现缺乏 BNIP3 和 NIX（五种受体中的两种）的 HeLa 细胞在各种条件下表现出线粒体自噬的完全丧失。相反，缺乏其他三种受体的细胞表现出正常的线粒体自噬。使用 BNIP3/NIX 双敲除 (DKO) 细胞作为模型，我们发现线粒体自噬缺陷会升高线粒体活性氧 (mtROS)，从而导致 Nrf2 抗氧化途径激活。值得注意的是，当 Nrf2 驱动的抗氧化酶受到损害时，BNIP3/NIX DKO 细胞对铁死亡高度敏感。此外，在引入野生型BNIP3和NIX后，BNIP3/NIX DKO细胞的敏感性得到了完全恢复，但不能促进线粒体自噬的突变形式却无法恢复。因此，我们的结果表明 BNIP3 和 NIX 介导的线粒体自噬在调节 mtROS 水平并保护细胞免于铁死亡方面发挥作用。