The post-transcriptional role of Mss51p in mitochondrial gene expression is of great interest since MSS51 mutations suppress the respiratory defect caused by shy1 mutations. SHY1 is a Saccharomyces cerevisiae homolog of human SURF1, which when mutated causes a cytochrome oxidase assembly defect. We found that MSS51 is required for expression of the mitochondrial reporter gene ARG8 m when it is inserted at the COX1 locus, but not when it is at COX2 or COX3. Unlike the COX1 mRNA-speci®c translational activator PET309, MSS51 has at least two targets in COX1 mRNA. MSS51 acts in the untranslated regions of the COX1 mRNA, since it was required to synthesize Arg8p when ARG8 m completely replaced the COX1 codons. MSS51 also acts on a target speci®ed by the COX1 coding region, since it was required to translate either COX1 or COX1:: ARG8 m coding sequences from an ectopic COX2 locus. Mss51p was found to interact physically with newly synthesized Cox1p, suggesting that it could coordinate Cox1p synthesis with insertion into the inner membrane or cytochrome oxidase assembly.

Functional interactions of the translational activator Mss51 with both the mitochondrially encoded COX1 mRNA 5-untranslated region and with newly synthesized unassembled Cox1 protein suggest that it has a key role in coupling Cox1 synthesis with assembly of cytochrome c oxidase. Mss51 is present at levels that are near rate limiting for expression of a reporter gene inserted at COX1 in mitochondrial DNA, and a substantial fraction of Mss51 is associated with Cox1 protein in assembly intermediates. Thus, sequestration of Mss51 in assembly intermediates could limit Cox1 synthesis in wild type, and account for the reduced Cox1 synthesis caused by most yeast mutations that block assembly. Mss51 does not stably interact with newly synthesized Cox1 in a mutant lacking Cox14, suggesting that the failure of nuclear cox14 mutants to decrease Cox1 synthesis, despite their inability to assemble cytochrome c oxidase, is due to a failure to sequester Mss51. The physical interaction between Mss51 and Cox14 is dependent upon Cox1 synthesis, indicating dynamic assembly of early cytochrome c oxidase intermediates nucleated by Cox1. Regulation of COX1 mRNA translation by Mss51 seems to be an example of a homeostatic mechanism in which a positive effector of gene expression interacts with the product it regulates in a posttranslational assembly process. INTRODUCTIONThe largest subunit of mitochondrial cytochrome c oxidase, Cox1, is encoded in the mitochondrial DNA (mtDNA) of all eukaryotic species that have been examined (Gray et al., 2004), and it is synthesized by their organellar genetic systems. Cox1 is highly hydrophobic, spanning the inner mitochondrial membrane 12 times, and it is complexed with several metal ions and two heme A moieties that participate directly in electron transport (Tsukihara et al., 1996). It is assembled into the core of cytochrome c oxidase, largely surrounded by subunits encoded by nuclear genes. The processes by which Cox1 is assembled with the other subunits and cofactors into an active enzyme are highly complex, requiring at least 30 genes in Saccharomyces cerevisiae (Herrmann and Funes, 2005;Khalimonchuk and Rodel, 2005;Cobine et al., 2006;Fontanesi et al., 2006;Barrientos et al., 2009). The assembly pathway is not understood in detail. In mammals, analysis of mutant and drug-treated cell lines indicates that Cox1 is a component of the earliest assembly intermediates (Nijtmans et al., 1998;Williams et al., 2004), and similar analysis in yeast is consistent with this idea (Horan et al., 2005).An important function of this assembly process may be to prevent incompletely assembled components of cytochrome c oxidase from generating damaging reactive oxygen species, before they are contained by the holoenzyme. Indeed, mutations in several yeast genes required for cytochrome c oxidase assembly cause hypersensitivity to hydrogen peroxide (Pungartnik et al., 1999;Williams et al., 2005;Banting and Glerum, 2006), and a key component of the reactive prooxidant species is Cox1 . One feature of the assembly p...

No abstract

The mitochondrial genomes of Chlamydomonad algae lack the cox2 gene that encodes the essential subunit COX II of cytochrome c oxidase. COX II is normally a single polypeptide encoded by a single mitochondrial gene. In this work we cloned two nuclear genes encoding COX II from both Chlamydomonas reinhardtii and Polytomella sp. The cox2a gene encodes a protein, COX IIA, corresponding to the N-terminal portion of subunit II of cytochrome c oxidase, and the cox2b gene encodes COX IIB, corresponding to the C-terminal region. The cox2a and cox2b genes are located in the nucleus and are independently transcribed into mRNAs that are translated into separate polypeptides. These two proteins assemble with other cytochrome c oxidase subunits in the inner mitochondrial membrane to form the mature multi-subunit complex. We propose that during the evolution of the Chlorophyte algae, the cox2 gene was divided into two mitochondrial genes that were subsequently transferred to the nucleus. This event was evolutionarily distinct from the transfer of an intact cox2 gene to the nucleus in some members the Leguminosae plant family.

Synthesis of the largest cytochrome c oxidase (CcO) subunit, Cox1, on yeast mitochondrial ribosomes is coupled to assembly of CcO. The translational activator Mss51 is sequestered in early assembly intermediate complexes by an interaction with Cox14 that depends on the presence of newly synthesized Cox1. If CcO assembly is prevented, the level of Mss51 available for translational activation is reduced. We deleted the C-terminal 11 or 15 residues of Cox1 by site-directed mutagenesis of mtDNA. Although these deletions did not prevent respiratory growth of yeast, they eliminated the assembly-feedback control of Cox1 synthesis. Furthermore, these deletions reduced the strength of the Mss51-Cox14 interaction as detected by co-immunoprecipitation, confirming the importance of the Cox1 C-terminal residues for Mss51 sequestration. We surveyed a panel of mutations that block CcO assembly for the strength of their effect on Cox1 synthesis, both by pulse labeling and expression of the ARG8 m reporter fused to COX1. Deletion of the nuclear gene encoding Cox6, one of the first subunits to be added to assembling CcO, caused the most severe reduction in Cox1 synthesis. Deletion of the C-terminal 15 amino acids of Cox1 increased Cox1 synthesis in the presence of each of these mutations, except pet54. Our data suggest a novel activity of Pet54 required for normal synthesis of Cox1 that is independent of the Cox1 C-terminal end.