Subunit function in eukaryote cytochrome c oxidase. A mutation in the nuclear-coded subunit IV allows assembly but alters the function and stability of yeast cytochrome c oxidase.

Strains of the yeast Saccharomyces cerevisiae disrupted in YCOX4, the nuclear gene encoding cytochrome c oxidase subunit IV, do not assemble a functional or spectrally visible oxidase. We report the characterization of a yeast strain, RM1, expressing a mutated YCOX4 gene which is temperature sensitive for respiration at 37 degrees C, but incorporates cytochrome aa3 over all growth temperatures. The mutant enzyme is less stable than the wild type, with subunit IV readily proteolyzed without gross denaturation of the complex but with a concomitant loss of oxidase activity. When grown fermentatively at 37 degrees C, cytochrome c oxidase from the mutant strain had a turnover number of less than 3% of the normal complex, while Km values and subunit levels were comparable to normal. Thus alterations in subunit IV can perturb the enzyme structure and alter its catalytic rate, implying a role for this subunit in cytochrome c oxidase function as distinct from assembly.     

Interactions between peptidyl tRNA hydrolase homologs and the ribosomal release factor Mrf1 in S. pombe mitochondria

Mitochondrial translation synthesizes key subunits of the respiratory complexes. In Schizosaccharomyces pombe, strains lacking Mrf1, the mitochondrial stop codon recognition factor, are viable, suggesting that other factors can play a role in translation termination. S. pombe contains four predicted peptidyl tRNA hydrolases, two of which (Pth3 and Pth4), have a GGQ motif that is conserved in class I release factors. We show that high dosage of Pth4 can compensate for the absence of Mrf1 and loss of Pth4 exacerbates the lack of Mrf1. Also Pth4 is a component of the mitochondrial ribosome, suggesting that it could help recycling stalled ribosomes.     

rugose (rg), a Drosophila A kinase anchor protein,is required for retinal pattern formation and interacts genetically with multiple signaling pathways

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways.     

Intracellular Mitochondrial Triplasmy in a Patient with Two Heteroplasmic Base Changes

We report the clinical, biochemical, and genetic investigation of a patient with a severe mitochondrial encephalomyopathy. Genetic studies identified a novel, heteroplasmic tRNA mutation at nt 10010. This T-->C transition is located in the DHU loop of mitochondrial tRNA(Gly). In skeletal muscle, it was present at lower levels in cytochrome c oxidase (COX)-normal (87.2% +/- 11%) compared with COX-deficient fibers (97.3% +/- 2.6%); it was found in skin fibroblasts and blood cells, but at lower levels of heteroplasmy (15% +/- 6% and 17% +/- 10%, respectively). A second, heteroplasmic transition (A-->G), at nt 5656, showed a different distribution than the tRNA(Gly) mutation, with very low levels in skeletal muscle (< 3%) but higher levels in blood (22.7% +/- 3%) and skin fibroblasts (21% +/- 2%). These transitions were followed both in vivo, by repeat biopsy and blood sampling, and in vitro, by establishing primary cultures of myoblasts and skin fibroblasts. Repeat muscle biopsy showed a dramatic increase in COX-deficient fibers, but not of the tRNAGly mutation. Indeed, no significant change in heteroplasmy was measured for either substitution in muscle or blood. In vitro analysis gave very different results. The T10010C was not found in cultured myoblasts, even at early passage. In uncloned fibroblasts, the T10010C was stable (approximately 10%) for several passages but then gradually was lost. In contrast, the A5656G rose progressively from 27% to 91%. In cloned fibroblasts, different combinations of both base-pair changes and wild type could be identified, confirming the presence of clonal, intracellular triplasmy.     

Production of transmitochondrial cybrids containing naturally occurring pathogenic mtDNA variants

The human mitochondrial genome (mtDNA) encodes polypeptides that are critical for coupling oxidative phosphorylation. Our detailed understanding of the molecular processes that mediate mitochondrial gene expression and the structure–function relationships of the OXPHOS components could be greatly improved if we were able to transfect mitochondria and manipulate mtDNA in vivo. Increasing our knowledge of this process is not merely of fundamental importance, as mutations of the mitochondrial genome are known to cause a spectrum of clinical disorders and have been implicated in more common neurodegenerative disease and the ageing process. In organellar or in vitro reconstitution studies have identified many factors central to the mechanisms of mitochondrial gene expression, but being able to investigate the molecular aetiology of a limited number of cell lines from patients harbouring mutated mtDNA has been enormously beneficial. In the absence of a mechanism for manipulating mtDNA, a much larger pool of pathogenic mtDNA mutations would increase our knowledge of mitochondrial gene expression. Colonic crypts from ageing individuals harbour mutated mtDNA. Here we show that by generating cytoplasts from colonocytes, standard fusion techniques can be used to transfer mtDNA into rapidly dividing immortalized cells and, thereby, respiratory-deficient transmitochondrial cybrids can be isolated. A simple screen identified clones that carried putative pathogenic mutations in MTRNR1, MTRNR2, MTCOI and MTND2, MTND4 and MTND6. This method can therefore be exploited to produce a library of cell lines carrying pathogenic human mtDNA for further study.     

Translation termination in human mitochondrial ribosomes

Mitochondria are ubiquitous and essential organelles for all nucleated cells of higher eukaryotes. They contain their own genome [mtDNA (mitochondrial DNA)], and this autosomally replicating extranuclear DNA encodes a complement of genes whose products are required to couple oxidative phosphorylation. Sequencing of this human mtDNA more than 20?years ago revealed unusual features that included a modified codon usage. Specific deviations from the standard genetic code include recoding of the conventional UGA stop to tryptophan, and, strikingly, the apparent recoding of two arginine triplets (AGA and AGG) to termination signals. This latter reassignment was made because of the absence of cognate mtDNA-encoded tRNAs, and a lack of tRNAs imported from the cytosol. Each of these codons only occurs once and, in both cases, at the very end of an open reading frame. The presence of both AGA and AGG is rarely found in other mammals, and the molecular mechanism that has driven the change from encoding arginine to dictating a translational stop has posed a challenging conundrum. Mitochondria from the majority of other organisms studied use only UAA and UAG, leaving the intriguing question of why human organelles appear to have added the complication of a further two stop codons, AGA and AGG, or have they? In the present review, we report recent data to show that mammalian mitochondria can utilize a -1 frameshift such that only the standard UAA and UAG stop codons are required to terminate the synthesis of all 13 polypeptides.     

Inhibition of mitochondrial protein synthesis promotes autonomous regulation of mtDNA expression and generation of a new mitochondrial RNA species

Mammalian mitochondria are known to proliferate in response to several stimuli. Proliferation requires an increase in expression of genes encoding proteins involved in mitochondrial biogenesis, as well as in the replication and expression of mitochondrial DNA (mtDNA). In contrast, we report that inhibiting mitochondrial protein synthesis causes a modulation in mtDNA gene expression without the concomitant increase in proliferative markers. Further, inhibition results in the production of a previously unidentified light-strand mitochondrial RNA that spans the entire displacement loop, the function of which is currently unknown.