Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, Chlorhexidine and Alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found Alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that Alexidine severely perturbated the cristae structure. Notably, Alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of Alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.

线粒体从内共生细菌进化成为真核细胞的重要细胞器。线粒体膜独特的脂质组成和结构对于线粒体的正常功能至关重要。然而，有助于维持线粒体膜完整性的应激反应尚不清楚。缺乏洞察力的原因之一是缺乏专门损伤线粒体膜的有效工具。在这里，通过化合物筛选，我们发现两种双双胍化合物（氯己定和 Alexidine）通过改变 IMM 的完整性来修饰线粒体内膜 (IMM) 驻留蛋白酶 OMA1 的活性。这些化合物是众所周知的杀菌剂，其作用机制集中于它们对细菌膜的损伤诱导活性。我们发现 Alexidine 可能通过膜电位驱动的静电相互作用以及对阴离子磷脂的亲和力与 IMM 结合。电子显微镜分析显示，Alexidine 严重扰乱了嵴结构。值得注意的是，Alexidine 引发了其他典型线粒体应激源不会诱导的特定转录/蛋白质稳态特征，突显了 Alexidine 作为新型线粒体膜应激源的独特特性。我们的研究结果提供了一种化学生物学工具，可以描绘维持线粒体膜稳态所需的线粒体应激信号通路。