The mitochondrial genetic systems of animals and fungi synthesize a small number of hydrophobic proteins encoded in mitochondrial DNA (mtDNA) that are essential components of the oxidative phosphorylation machinery. This proposal aims broadly to delineate mechanisms that govern expression of mitochondrial genes at the level of translation, and that couple the synthesis of mitochondrial gene products to their insertion in the inner membrane and assembly into enzyme complexes. Production of cytochrome oxidase in mitochondria of Saccharomyces cerevisiae is the focus of this work. Genetic tools available in yeast, including reporter genes inserted into the mtDNA of living cells, allow in vivo experimentation that is impossible in animals.
One aim of this proposal is to understand how ongoing assembly of cytochrome oxidase promotes the synthesis in mitochondria of its largest subunit, Cox1. A component of this feedback system, the nuclearly encoded protein Mss51, has dual roles in controlling Cox1 synthesis. It acts on the COX1 mRNA leader and also interacts with newly synthesized Cox1 itself at the surface of the inner membrane. Using a reporter gene inserted at the COX1 locus in mtDNA, the functions of Mss51 will be dissected genetically.
The second aim will be to explore the functions of Oxa1 and Cox18 in membrane insertion and assembly of Cox2. Oxa1 and Cox18 are distantly related integral membrane proteins, functionally conserved between humans and yeast. The mechanism by which recessive nuclear mutations allow overproduced Oxa1 to compensate for the absence of Cox18 will be explored. A non-Mendelian element causing the same phenotype as the nuclear mutations will be characterized to test the possibility that it is a novel prion-like structure.
The third aim will be to study genetic changes that allow phenotypic expression of a mitochondrially located gene specifying the pyrimidine biosynthetic enzyme Ura3. Ura3 is normally located in the cytoplasm and cannot function in the mitochondrial matrix. Ura3 has been fused to the exported C-tail domain of Cox2. Characterization of mutations that allow it to confer a Ura+ phenotype should reveal functions that affect protein export from the matrix, inner membrane integrity, and/or the environment in the mitochondrial intermembrane space. Defects in mitochondrial gene expression cause a number of inherited neurological diseases, and mitochondrial mutations accumulate as we all age. The fact that mice with elevated mtDNA mutation rates die young after what appears to be rapid aging, underscores the importance of accurate mitochondrial gene expression for a long and healthy life. The further study of mitochondrial gene expression in the model organism yeast will continue to contribute to our understanding of similar processes in humans.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics B Study Section (MGB)
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Bender, Michael T
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Cornell University
Schools of Earth Sciences/Natur
United States
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Elliott, Leah E; Saracco, Scott A; Fox, Thomas D (2012) Multiple roles of the Cox20 chaperone in assembly of Saccharomyces cerevisiae cytochrome c oxidase. Genetics 190:559-67
Fox, Thomas D (2012) Mitochondrial protein synthesis, import, and assembly. Genetics 192:1203-34
Kuhl, Inge; Fox, Thomas D; Bonnefoy, Nathalie (2012) Schizosaccharomyces pombe homologs of the Saccharomyces cerevisiae mitochondrial proteins Cbp6 and Mss51 function at a post-translational step of respiratory complex biogenesis. Mitochondrion 12:381-90
Mick, David U; Fox, Thomas D; Rehling, Peter (2011) Inventory control: cytochrome c oxidase assembly regulates mitochondrial translation. Nat Rev Mol Cell Biol 12:14-20
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