在培养的细胞中,生理细胞应激诱导的线粒体自噬降解的线粒体不多。我们使用依赖于pH敏感荧光蛋白Keima的方法观察了HeLa细胞中的线粒体自噬。通过这种方法,我们发现羰基氰化物间氯苯酰腙处理几乎不诱导线粒体自噬,它被广泛用作PARK2/Parkin相关线粒体自噬的诱导剂,而在饥饿或缺氧条件下,线粒体自噬降解了少量但数量适中的线粒体。由饥饿或缺氧诱导的线粒体自噬被ATG7和ATG12或MAP1LC3B敲低所抑制,这对传统的巨自噬至关重要。此外,在Atg5敲除小鼠胚胎成纤维细胞中有效诱导线粒体自噬。然而,敲除RAB9A和RAB9B,这对替代性自噬至关重要,但不是常规的巨自噬,严重抑制线粒体自噬。最后,我们发现MAPKsMAPK1/ERK2和MAPK14/p38是线粒体自噬所必需的。基于这些发现,我们得出结论,哺乳动物细胞中的线粒体自噬主要通过另一种自噬途径发生,需要MAPK1和MAPK14信号通路。
In cultured cells, not many mitochondria are degraded by mitophagy induced by physiological cellular stress. We observed mitophagy in HeLa cells using a method that relies on the pH-sensitive fluorescent protein Keima. With this approach, we found that mitophagy was barely induced by carbonyl cyanide m-chlorophenyl hydrazone treatment, which is widely used as an inducer of PARK2/Parkin-related mitophagy, whereas a small but modest amount of mitochondria were degraded by mitophagy under conditions of starvation or hypoxia. Mitophagy induced by starvation or hypoxia was marginally suppressed by knockdown of ATG7 and ATG12, or MAP1LC3B, which are essential for conventional macroautophagy. In addition, mitophagy was efficiently induced in Atg5 knockout mouse embryonic fibroblasts. However, knockdown of RAB9A and RAB9B, which are essential for alternative autophagy, but not conventional macroautophagy, severely suppressed mitophagy. Finally, we found that the MAPKs MAPK1/ERK2 and MAPK14/p38 were required for mitophagy. Based on these findings, we conclude that mitophagy in mammalian cells predominantly occurs through an alternative autophagy pathway, requiring the MAPK1 and MAPK14 signaling pathways.