The Role of Cardiolipin in Assembly and Function of the Mitochondrial Respirasome

线粒体单个呼吸复合体与高阶超复合体处于动态平衡状态 构成呼吸系统的组织。线粒体的生理和病理变化 心磷脂(CL)池直接影响和调节这种平衡。衰老、神经退行性疾病、心脏 衰竭、缺血/再灌流、癌症和Barth综合征与CL池异常相关。 缺乏CL的酿酒酵母突变株表现出与CL降低的哺乳动物细胞相似的表型 水平使酵母成为一个很好的模型系统。我们第一个报道了酵母的形成 四聚体呼吸超复合体(SC III2IV2)和细胞色素c(Cytc)在复合体III之间的通道 SC内的(CIII，Bc1复合体)和IV(CIV，Cyt c氧化酶)直接依赖于CL。使用我们最近的 获得了用于单粒子电子冷冻显微镜的K2 Summit直接电子探测器，我们有显著的 改进了EM图像的质量。我们得到了比四聚体Sc更高分辨率的3D密度图 我们之前曾报道过。我们现在能够以前所未有的速度获得SC的3D密度图- 纳米分辨率。我们提出了一种高分辨率结构确定的创新组合， 酵母细胞的功能分析、遗传操作和新的脂类依赖重建研究 建立依赖CL的呼吸干细胞形成和功能的分子基础。目标1：a)获得 四聚体SC的亚纳米分辨率3D密度图，以确定脂质的精确尺寸- 填充的间隙和CIII和CIV之间的界面；B)确定结合的Cytc在四聚体中的位置(S) C)解析III2IV1三聚体的结构 了解与四聚体的结构差异如何导致CL非依赖性的形成和Cyt c的缺乏 D)进行缺少偶联CIII亚单位Qcr6的SC四聚体的结构研究 通过脂质分析(在目标2下)确定Qcr6p是否保持CIII和CIII之间的脂质填充间隙 Civ，这使得SC的形成对CL水平很敏感。目标2：a)将结构分析与定量分析结合起来 B)确定支持四聚体SC的CL的特征 采用结构类似物的创新的体外SC-重建系统的形成和功能 C)模拟导致CL池改变的病理条件，以了解CL水平和CL 氧化作用影响SC的结构和功能。目的3：A)确定CIII和CIV表面暴露的CL结合部位 使用可光激活的CL类似物潜在地负责SC的形成；B)使用分子动力学 模拟以预测表面暴露的CL结合位点；c)在化学上使用定点突变 位于CIII-CIV界面上的修饰和预测的CL结合位点，以验证它们参与了 SC队形。这一创新和集成的方法将建立化学发光的分子机制。 依赖SC的形成和功能。在未来，类似的研究将扩展到哺乳动物呼吸系统。

Individual respiratory complexes of mitochondria are in dynamic equilibrium with higher order supercomplex organizations that compose the respirasome. Physiological and pathological perturbations in the mitochondrial cardiolipin (CL) pool directly affect and regulate this equilibrium. Aging, neurodegenerative diseases, heart failure, ischemia/reperfusion, cancer and Barth Syndrome are associated with abnormal CL pools. Saccharomyces cerevisiae mutants lacking CL display similar phenotypes to mammalian cells with reduced CL levels making yeast an excellent model system. We were the first to report that formation of the yeast tetrameric respiratory supercomplex (SC III2IV2) and cytochrome c (cyt c) channeling between complexes III (CIII, bc1 complex) and IV (CIV, cyt c oxidase) within the SC are directly dependent on CL. Using our recently acquired K2 Summit direct electron detector for single particle electron cryo-microscopy, we have significantly improved the quality of EM images. We achieved a higher resolution 3D density map of the tetrameric SC than we previously reported. We are now in a position to attain 3D density maps of SCs at an unprecedented sub- nanometer resolution. We propose an innovative combination of high resolution structural determinations, functional assays, genetic manipulation of yeast cells and novel lipid-dependent reconstitution studies to establish the molecular basis for CL-dependent formation and function of respiratory SCs. Aim 1: A) obtain a sub-nanometer resolution 3D density map for the tetrameric SC to establish the precise dimensions of the lipid- filled gaps and the interface between CIII and CIV; B) determine the location(s) of bound cyt c in the tetrameric SC to decipher how SC formation makes cyt c channeling possible; C) resolve the structure of the III2IV1 trimer to understand how structural differences with the tetramer results in CL-independent formation and lack of cyt c channeling in the trimer; D) perform structural studies of the SC tetramer lacking subunit Qcr6 of CIII coupled with lipid analysis (under Aim 2) to determine whether Qcr6p maintains the lipid-filled gap between CIII and CIV, which makes SC formation sensitive to CL levels. Aim 2: A) integrate structural with quantitative analysis of CL and other lipids present in the above SCs; B) determine the features of CL that support tetrameric SC formation and function using an innovative in vitro SC-reconstitution system employing structural analogs of CL; C) mimic pathological conditions resulting in CL pool alterations to understand how CL levels and CL oxidation affect SC structure and function. Aim 3: A) determine CIII and CIV surface exposed CL-binding sites potentially responsible for SC formation using a photoactivatible CL analog; B) use Molecular Dynamic Simulations to predict surface exposed CL-binding sites; C) employ site-directed mutagenesis at chemically modified and predicted CL-binding sites that lie at the CIII-CIV interface for verification of their involvement in SC formation. This innovative and integrated approach will establish the molecular mechanism of CL- dependent SC formation and function. In the future similar studies will be extended mammalian respirasomes.