Towards an Atomistic Understanding of Mitochondrial Protein Biogenesis (Equipment Supplement)

项目总结 保持线粒体的完整性是正常的真核生理所必需的，并不令人惊讶的是， 线粒体功能障碍是疾病的病理标志，已被认为是主要的危险因素 治疗许多癌症和神经退行性疾病。线粒体功能的关键是它的双膜 提供适当的微环境以促进特定新陈代谢功能的架构，例如 氧化磷酸化-并允许在其他方面不兼容的过程在 手机。传统上，对线粒体的研究主要集中在生物能量学上，但最近的研究已经开始 阐明了线粒体蛋白质组和生物发生机制的错综复杂。 这一点尤其重要，因为99%的线粒体蛋白质组(人类约1500个蛋白质)是编码的 由核基因合成，并由胞质核糖体合成作为前体蛋白(前蛋白)。这些前蛋白 含有针对它们到线粒体的内源信号，它们随后在那里被转移 外膜，通过三种主要的蛋白质进口机械进行分类、分隔和适当折叠： 线粒体膜外膜(TOM)复合体的转位酶，内侧的线粒体转位酶 膜(TIM)-23复合体(TIM23)和TIM22复合体。这些蛋白质进口复合体是必需的 几乎所有线粒体蛋白的生物发生和失调对维持 正常的线粒体生理学。然而，结构信息的匮乏阻碍了分子的形成。 了解这些过程以及它们执行其关键功能的机制。 根据这个奖项，我将开发开创性的三维(3D)电子冷冻显微镜 (冷冻EM)技术开创了对这些至关重要的线粒体蛋白质输入复合体的研究， 提供对它们的功能及其在疾病状态中的作用的批判性见解。我会利用靶向生化 从天然来源中分离TOM、TIM23和TIM22复合体用于高分辨率低温电子显微镜的方法 学习。然后，我将开发新的EM样品准备、数据收集和数据处理策略，以产生 在活性前蛋白导入过程中，每台进口机器的一套高分辨率结构。那我会的 通过新的原子建模策略量化这些状态中的局部和全局动力学程度 作为定义构象地貌的一种手段。然后我将建立体外功能分析来测试密钥 在这些过程中的分子步骤。最后，我将开发相关的光学显微镜和高- 用于确定这些进口络合物结构的分辨率电子冷冻断层扫描(CryoET)方法 在它们的天然膜中。通过这些共同努力，我将回答与以下有关的基本问题 这些络合物的整体3D结构，并充分描述了这些 进口机器以进口和折叠线粒体前蛋白。重要的是，这些方法可以扩展 这超出了本奖项的直接范围，并将在整个CryoEM社区中普遍应用。

PROJECT SUMMARY Maintaining mitochondrial integrity is necessary for normal eukaryotic physiology and, not surprisingly, mitochondrial dysfunction is a pathological hallmark of diseases and has been implicated as a primary risk factor for many cancers and neurodegenerative disorders. Critical to mitochondrial function is its dual-membrane architecture which provides appropriate microenvironments that facilitate specific metabolic functions – such as oxidative phosphorylation – and allow for otherwise incompatible processes to occur simultaneously inside the cell. Traditionally, research on mitochondria have focused on bioenergetics, but recent studies have begun to shed light on the intricacies and complexities of the mitochondrial proteome and the biogenesis machineries. This is particularly important as >99% of the mitochondrial proteome (~1500 proteins in humans) are encoded by nuclear genes and synthesized by cytosolic ribosomes as precursor proteins (preproteins). These preproteins contain endogenous signals that target them to mitochondria, where they are subsequently translocated across the outer membrane, sorted, compartmentalized, and properly folded by three main protein import machineries: the translocase of the outer mitochondrial membrane (TOM) complex, the mitochondrial translocase of the inner membrane (TIM)-23 complex (TIM23), and the TIM22 complex. These protein import complexes are required for the biogenesis of nearly all mitochondrial proteins and dysregulation poses a significant challenge to maintaining normal mitochondrial physiology. However, a dearth of structural information has precluded a molecular understanding of these processes and the mechanisms by which they perform their critical functions. Under this award, I will develop groundbreaking three-dimensional (3D) electron cryomicroscopy (cryoEM) technologies to pioneer studies of these critically important mitochondrial protein import complexes, providing critical insights into their function and their roles in the disease state. I will utilize targeted biochemical approaches to isolate the TOM, TIM23, and TIM22 complexes from natural sources for high-resolution cryoEM studies. I will then develop novel EM sample preparation, data collection and data processing strategies to yield a suite of high-resolution structures of each of these import machines during active preprotein import. I will then quantify the degree of local and global dynamics within these states through novel atomic modeling strategies as a means to define the conformational landscape. I will then establish in vitro functional assays to test key molecular steps during these processes. Lastly, I will then develop correlated light microscopy and high- resolution electron cryotomography (cryoET) methodologies to determine structure of these import complexes in their native membranes. Through these combined efforts, I will answer fundamental questions pertaining to the overall 3D architecture of these complexes and fully describe the molecular motions necessary for these import machines to import and fold mitochondrial preproteins. Importantly, these methodologies can be extended beyond the immediate scope of this award and be applied ubiquitously across the cryoEM community.