The mitochondrial genetic systems of eukaryotes have evolved mechanisms for synthesis of small numbers of hydrophobic proteins encoded in mtDNA, insertion of those proteins into the inner membrane, and assembly of those proteins with imported nuclearly encoded subunits into active respiratory complexes. While some aspects of mitochondrial gene expression are highly divergent among different eukaryotic lineages, others are conserved. The overall goal of this proposal is to exploit the model organism Saccharomyces cerevisiae to elucidate mechanisms, conserved in humans, that control expression and assembly of mitochondrially coded proteins, focusing on the assembly of respiratory complex IV, cytochrome c oxidase. The first two specific aims seek to elucidate conserved mechanisms that regulate the synthesis and assembly of mitochondrially coded Cox1, a core catalytic subunit the enzyme. First, we will determine the function in mitochondrial gene expression of Ygr021w, a protein highly homologous to human TACO1. TACO1 is the recently described COX1 mRNA-specific translational activator whose absence causes a late-onset cytochrome oxidase deficiency disease. We will determine the cause of defective expression of the mitochondrial ARG8m reporter from the yeast COX1 locus produced by ygr021w deletion, as well as determine the physical and functional interactions of this nuclearly encoded protein. Success here will shed new light on the relatively unexplored mechanisms of translational control in human mitochondrial gene expression.
Our second aim will be to study several functions of Mss51, a COX1 mRNA-specific translational activator that couples yeast Cox1 synthesis with cytochrome oxidase assembly, and has an apparent human/mouse homolog. We hypothesize, based on previous evidence and the E. coli SecM precedent, that one function of Mss51 is to release an intrinsic Cox1 translation-elongation arrest by binding to nascent Cox1. We will test this idea using cells with mtDNA that has ARG8m translationally fused downstream of COX1, such that Arg+ growth is dependent upon this Mss51 activity. Selection of mutations in these that allow Arg+ growth of mss51 cells, will identify components required for the intrinsic arrest (possibly, for example, affecting the ribosomal exit tunnel or a Cox1 arrest sequence). Additional studies will examine other activities of Mss51 in Cox1 synthesis and early assembly steps.
Our third aim will be to identify mechanisms by which the highly conserved paralogous inner membrane translocases Oxa1 and Cox18, which have distinct functions in topogenesis of mitochondrially synthesized Cox2, promote Cox2 assembly after translocation of its hydrophilic domains to the IMS. We will test the idea that Cox18, and mutant forms of Oxa1, facilitate Cox2 C-tail interaction with the conserved IMS chaperone Cox20 and/or novel factors.
A large number of human inherited disorders are due to defects in mitochondrial gene expression or the assembly of respiratory complexes, which taken together are responsible for a significant fraction of the overall incidence of inherited disease. Thus, better understanding of mechanisms underlying the assembly of the mitochondrial oxidative phosphorylation system is a significant health related priority for basic research, and could lead to novel therapies. This proposal seeks to exploit the model organism budding yeast to elucidate key steps, that are conserved in humans, in the assembly of the respiratory complex cytochrome c oxidase.
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