Fis1 regulation of mitochondrial fission

描述（由申请人提供）：本提案的长期目标是确定线粒体分裂的分子机制及其在细胞凋亡中的作用。线粒体分裂缺陷会造成严重后果，甚至死亡。然而，我们对裂变如何起作用以及如何与其他细胞过程协调知之甚少。遗传和细胞研究，主要是在酿酒酵母菌，由其他研究人员导致了线粒体裂变模型的发展。在这个模型中，Fis1蛋白通过介导与动力蛋白相关的GTPase Dnm1和衔接蛋白Mdv1在线粒体外膜收缩部位的组装来调节裂变。虽然来自定性模型的数据具有启发性，但该模型并没有解释这一重要过程是如何调节的。通过将细胞生物学、生物化学和生物物理数据与裂变机制的低分辨率和高分辨率结构相结合，目前的建议旨在建立一个线粒体裂变的综合模型。第一个具体目标是通过实验定义支持这些相互作用的结构域，这些相互作用的化学计量学和亲和性及其对Dnm1活性的影响，来定义溶液中对裂变很重要的蛋白质-蛋白质相互作用。所提出的裂变机制的低分辨率图像将通过电子显微镜获得。Fis1、二元Fis1/Mdv1配合物和三元Fis1/Mdv1/Dnm1配合物的高分辨率结构将通过核磁共振波谱和x射线晶体学进行研究。第二个具体目标是鉴定酵母Fis1残基对同二聚化、Mdv1结合和Dnm1结合很重要。我们将识别影响寡聚化的Fis1突变体，并测试这些突变体的活性变化，以确定它们在裂变机制组装中的重要性。第三个具体目的是通过合成脂质和分离线粒体的膜实验，确定Fis1是否影响Dnm1和Mdv1在膜上的组装。这些实验还应允许确定组装的顺序。这三种方法的结果数据将被整合成一幅线粒体裂变如何完成的完整图景。分析也承诺相当普遍的洞察基础的动力蛋白为基础的膜动力学，以及蛋白质-蛋白质和蛋白质-脂质相互作用。线粒体分裂机制的人类同源物存在，据报道在调节细胞凋亡中起重要作用，而细胞凋亡与许多重要疾病有关。因此，线粒体分裂的详细信息可能有助于设计抑制或诱导细胞凋亡的策略。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to identify the molecular mechanism of mitochondrial fission and its role in apoptosis. Defects in mitochondrial fission have severe consequences and even death. Yet little is known about how fission works and is coordinated with other cellular processes. Genetic and cellular studies, primarily in Saccharomyces cerevisiae, by other investigators have led to the development of a model for mitochondrial fission. In this model, the protein Fis1 regulates fission by mediating the assembly of a dynamin- related GTPase, Dnm1, and an adaptor protein, Mdv1, at the sites of constriction on the mitochondrial outer membrane. Although the data from the qualitative model are suggestive, the model does not explain how this important process is regulated. By integrating cell biological, biochemical, and biophysical data with low- and high-resolution structures of the fission machinery, the current proposal aims to develop a comprehensive model for mitochondrial fission. The first specific aim is to define the protein-protein interactions in solution that are important to fission through experiments that will define domains that support these interactions, the stoichiometries and affinities of these interactions and their consequences on Dnm1 activity. Low-resolution images of the proposed fission machinery will be obtained by electron microscopy. High-resolution structures of Fis1, the binary Fis1/Mdv1 complex, and the ternary Fis1/Mdv1/Dnm1 complex will be pursued by NMR spectroscopy and x-ray crystallography. The second specific aim is to identify yeast Fis1 residues important for homodimerization, Mdv1 binding, and Dnm1 binding. We will identify mutants of Fis1 that affect oligomerization and test these mutants for altered activities to define their importance in assembly of the fission machinery. The third specific aim is to determine whether Fis1 affects the assembly of Dnm1 and Mdv1 on the membrane through experiments with membranes derived from synthetic lipids and isolated mitochondria. These experiments should also allow determination of the order of assembly. The resulting data from all three approaches will be integrated into a complete picture of how mitochondrial fission is accomplished. The analyses also promise considerable general insight into the basis of dynamin-based membrane dynamics, as well as protein-protein and protein-lipid interactions. Human homologues of the mitochondrial fission machinery exist and are reported to be important in regulating apoptosis, which is linked to many important diseases. Therefore, detailed information on mitochondrial fission might be helpful in designing strategies to inhibit or induce apoptosis. PUBLIC HEALTH RELEVANCE: Mitochondria perform many essential functions that are thought to require frequent mitochondrial fission and fusion events, which are accomplished by distinct protein machineries. A point mutant in the mitochondrial fission protein, Dnm1, caused infant death. Additionally, mitochondrial fission increases during apoptosis, a process whose misregulation contributes to many human diseases. The work proposed will illuminate mechanistic details of these processes and represents an important step towards the discovery of new therapeutic strategies for human diseases.