Prohibitins (PHBs) are ubiquitously expressed proteins in the mitochondrial inner membrane (MIM) that provide membrane scaffolds for both mitochondrial proteins and phospholipids. Eukaryotic PHB complexes contain two highly homologous PHB subunits, PHB1 and PHB2, which are involved in various cellular processes, including metabolic control through the regulation of mitochondrial dynamics and integrity. Their mechanistic actions at the molecular level, however, particularly those of PHB1, remain poorly understood. To gain insight into the mechanistic actions of PHB1, we established an overexpression system for the full-length recombinant protein using silkworm larvae and characterized its biophysical properties in vitro. Using recombinant PHB1 proteoliposomes reconstituted into MIM-mimicking phospholipids, we found that PHB1 forms an oligomer via its carboxy-terminal coiled-coil region. A proline substitution into the PHB1 coiled-coil collapsed its well-ordered oligomeric state, and its destabilization correlated with mitochondrial morphologic defects. Negative-staining electron microscopy revealed that homotypic PHB1-PHB1 interactions via the coiled-coil also induced liposome tethering with remodeling of the lipid membrane structure. We clarified that PHB1 promotes membrane fusion mediated by optic atrophy 1 (OPA1), a key regulator of MIM fusion. Additionally, the presence of PHB1 reduces the dependency of lipids and OPA1 for completing the fusion process. Our in vitro study provides structural insight into how the mitochondrial scaffold plays a crucial role in regulating mitochondrial dynamics. Modulating the structure and/or function of PHB1 may offer new therapeutic potential, not only for mitochondrial dysfunction but also for other cell-related disorders.

中文翻译：

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Prohibitin 1 栓系脂质膜并调节 OPA1 介导的膜融合。


禁止蛋白 （PHB） 是线粒体内膜 （MIM） 中普遍表达的蛋白质，可为线粒体蛋白和磷脂提供膜支架。真核 PHB 复合物包含两个高度同源的 PHB 亚基 PHB1 和 PHB2，它们参与各种细胞过程，包括通过调节线粒体动力学和完整性进行代谢控制。然而，它们在分子水平上的机制作用，尤其是 PHB1 的机制作用，仍然知之甚少。为了深入了解 PHB1 的机制作用，我们利用家蚕幼虫建立了全长重组蛋白的过表达系统，并在体外表征了其生物物理特性。使用重组 PHB1 蛋白脂质体重构成 MIM 模拟磷脂，我们发现 PHB1 通过其羧基末端卷曲螺旋区域形成寡聚体。PHB1 卷曲螺旋中的脯氨酸取代破坏了其有序的寡聚状态，并且其不稳定与线粒体形态缺陷相关。负染色电子显微镜显示，通过卷曲螺旋的同型 PHB1-PHB1 相互作用也诱导脂质体栓系，重塑脂质膜结构。我们阐明了 PHB1 促进视神经萎缩 1 （OPA1） 介导的膜融合，视萎缩 1 （OPA1） 是 MIM 融合的关键调节因子。此外，PHB1 的存在降低了脂质和 OPA1 对完成融合过程的依赖性。我们的体外研究为线粒体支架如何在调节线粒体动力学中发挥关键作用提供了结构见解。调节 PHB1 的结构和/或功能可能提供新的治疗潜力，不仅适用于线粒体功能障碍，也适用于其他细胞相关疾病。