Mitochondria critically rely on protein import and its tight regulation. Here, we found that the complex I assembly factor NDUFAF8 follows a two-step import pathway linking IMS and matrix import systems. A weak targeting sequence drives TIM23-dependent NDUFAF8 matrix import, and en route, allows exposure to the IMS disulfide relay, which oxidizes NDUFAF8. Import is closely surveyed by proteases: YME1L prevents accumulation of excess NDUFAF8 in the IMS, while CLPP degrades reduced NDUFAF8 in the matrix. Therefore, NDUFAF8 can only fulfil its function in complex I biogenesis if both oxidation in the IMS and subsequent matrix import work efficiently. We propose that the two-step import pathway for NDUFAF8 allows integration of the activity of matrix complex I biogenesis pathways with the activity of the mitochondrial disulfide relay system in the IMS. Such coordination might not be limited to NDUFAF8 as we identified further proteins that can follow such a two-step import pathway.

中文翻译：

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两步线粒体输入途径将二硫键中继与基质复合物 I 生物发生结合起来

线粒体严重依赖蛋白质输入及其严格调节。在这里，我们发现复合物 I 组装因子 NDUFAF8 遵循连接 IMS 和基质导入系统的两步导入途径。弱靶向序列驱动 TIM23 依赖性 NDUFAF8 矩阵导入，并在途中允许暴露于 IMS 二硫化物中继，从而氧化 NDUFAF8。蛋白酶对导入进行密切监测：YME1L 防止 IMS 中过量 NDUFAF8 的积累，而 CLPP 则降解基质中减少的 NDUFAF8。因此，只有在 IMS 中的氧化和随后的基质导入有效发挥作用的情况下，NDUFAF8 才能在复合物 I 生物发生中发挥其功能。我们提出NDUFAF8的两步导入途径允许将基质复合物I生物发生途径的活性与IMS中线粒体二硫键中继系统的活性整合。这种协调可能不仅限于 NDUFAF8，因为我们还发现了可以遵循这种两步导入途径的更多蛋白质。