Control of Mitochondrial Cox1 Synthesis by Nuclear Encoded Mss51 and Cox14

描述（由申请人提供）：铬c氧化酶合成所需的基因突变损害细胞能量生产。SURF1是细胞色素c氧化酶组装途径所需的一种蛋白，在Leigh综合征患者中发生突变。通过鉴定和表征酿酒酵母（Saccharomyces cerevisiae, SHY1）中的SURF1同源物，有助于SURF1的鉴定。酿酒酵母是兼性厌氧菌，是研究细胞色素c氧化酶组装的绝佳生物，因为破坏线粒体能量产生的基因改变并不致命。此外，细胞核和线粒体DNA可以通过基因转化被操纵并整合到细胞中。在本项目中，将利用酿酒酵母研究两种核编码蛋白Mss51和Cox14如何调节线粒体编码的Cox1翻译。酿酒酵母的MSS51突变可以抑制由shy1?引起的细胞色素c氧化酶缺陷，这一事实强调了这项研究的重要性。此外，已在人类和小鼠基因组中发现了Mss51的明显同源物，其功能仍有待确定。Cox14是另一种调节Cox1合成的核编码蛋白。这种调节被认为是通过控制可用于启动Cox1翻译的Mss51的数量而发生的。基于先前的工作，我们假设Mss51、Cox1和Cox14作为一系列依赖于Cox1合成和细胞色素c氧化酶组装需求的动态组装复合物存在，并且在缺乏Cox1编码区两侧的Cox1非翻译区时，Cox14对于Mss51-Cox1相互作用的稳定至关重要。在本项目中，Aim 1将使用蓝色天然凝胶电泳来鉴定启动Cox1翻译、稳定未组装的Cox1所需的Mss51、Cox1和Cox14蛋白复合物，并鉴定Cox1翻译被组装反馈抑制的菌株中存在的复合物。用35S标记Cox1合成和共免疫沉淀研究将在Aim 2中使用，以研究cox14？在COX1编码序列两侧没有COX1非翻译区的菌株。这项工作将定义Cox1合成所需的特定Mss51、Cox1和Cox14蛋白相互作用。确定这些调节Cox1合成的相互作用是研究这些复合物如何在Leigh综合征患者中改变的第一步。这反过来又可能导致更早的诊断或治疗患者的药物靶点。

DESCRIPTION (provided by applicant): Mutations in genes needed for chrome c oxidase synthesis compromise cellular energy production. SURF1 is one protein needed for the cytochrome c oxidase assembly pathway that is mutated in patients with Leigh syndrome. Identification of SURF1 was facilitated by identifying and characterizing a SURF1 homolog in Saccharomyces cerevisiae, SHY1. S. cerevisiae, being a facultative anaerobe, is an excellent organism to study cytochrome c oxidase assembly, since genetic alterations disrupting mitochondrial energy production are not lethal. Also, nuclear and mitochondrial DNA can be manipulated and incorporated into the cell through genetic transformation. In this project S. cerevisiae will be used to study how two nuclear encoded proteins, Mss51 and Cox14, regulate mitochondrially encoded Cox1 translation. The importance of this study is underscored by the fact that MSS51 mutations in S. cerevisiae can suppress the cytochrome c oxidase defect caused by a shy1?. Also, an apparent ortholog of Mss51 has been identified in the human and mouse genomes with its function remaining to be determined. Cox14 is another nuclear encoded protein that regulates Cox1 synthesis. This regulation is thought to occur by controlling the amount of Mss51 available for initiating Cox1 translation. Based on previous work it is hypothesized that Mss51, Cox1 and Cox14 exist as a series of dynamic assembly complexes that are dependent on the demand for Cox1 synthesis and cytochrome c oxidase assembly, and that Cox14 is essential for the stabilization of the Mss51-Cox1 interaction in the absence of the COX1 untranslated regions flanking the C0X1 coding region. In this project blue native gel electrophoresis will be used in Aim 1 to identify Mss51, Cox1 and Cox14 protein complexes needed for initiating Cox1 translation, stabilizing unassembled Cox1 and identify complexes present in strains where Cox1 translation is assembly feedback inhibited. Labeling Cox1 synthesis with 35S and co-immunoprecipitation studies will be used in Aim 2 to investigate the paradoxical inhibition of Cox1 synthesis in cox14? strains that do not have the COX1 untranslated regions flanking the COX1 coding sequence. This work will define specific Mss51, Cox1 and Cox14 protein interactions that are needed for Cox1 synthesis. Identifying these interactions that regulate Cox1 synthesis is a first step in studying how these complexes may be altered in Leigh syndrome patients. This in turn could potentially result in earlier diagnostics or drug targets to treat patients.