The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon ‘breathing' during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone's stabilisation of the latter underpins the phenotype observed.

中文翻译：

---

分枝杆菌内酯通过将 Sec61 转位子捕获在 Ca2+ 可渗透状态来增强内质网的 Ca2+ 泄漏

溃疡分枝杆菌外毒素分枝杆菌内酯是通过 Sec61 复合物进行共翻译易位的抑制剂。先前已显示分枝杆菌内酯结合并改变主要转位子亚基 Sec61α 的结构，并改变其与核糖体新生链复合物的相互作用。除了在蛋白质易位到 ER 中的功能外，Sec61 还在细胞 Ca2+ 稳态中发挥关键作用，充当内质网 (ER) 和细胞质之间的泄漏通道。在这里，我们使用隔室特异性传感器分析了霉菌内酯对细胞溶质和内质网 Ca2+ 水平的影响。我们还使用分子对接分析来探索不同状态下易位子上霉菌内酯的潜在相互作用位点。这些结果表明，霉菌内酯通过 Sec61 转位子增强了 Ca2+ 离子的泄漏，导致 ER Ca2+ 缓慢但大量消耗。这种泄漏取决于霉菌内酯与 Sec61α 的结合，因为该蛋白质中的抗性突变完全消除了这种增加。分子对接支持在易位前存在一种与霉菌内酯结合的瞬时抑制状态，并表明霉菌内酯也可能在其空闲状态下结合 Sec61α。我们提出，在空闲状态和中间抑制状态之间的快速转换期间，翻译终止和/或易位子“呼吸”后延迟核糖体释放允许瞬时 Ca2+ 泄漏，并且后者的分支杆菌内酯稳定支持观察到的表型。分子对接支持在易位前存在一种与霉菌内酯结合的瞬时抑制状态，并表明霉菌内酯也可能在其空闲状态下结合 Sec61α。我们提出，在空闲状态和中间抑制状态之间的快速转换期间，翻译终止和/或易位子“呼吸”后延迟核糖体释放允许瞬时 Ca2+ 泄漏，并且后者的分支杆菌内酯稳定支持观察到的表型。分子对接支持在易位前存在一种与霉菌内酯结合的瞬时抑制状态，并表明霉菌内酯也可能在其空闲状态下结合 Sec61α。我们提出，在空闲状态和中间抑制状态之间的快速转换期间，翻译终止和/或易位子“呼吸”后延迟核糖体释放允许瞬时 Ca2+ 泄漏，并且后者的分支杆菌内酯稳定支持观察到的表型。