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.
