Pet127p, a membrane-associated protein involved in stability and processing of Saccharomyces cerevisiae mitochondrial RNAs.

Nuclear mutations that inactivate the Saccharomyces cerevisiae gene PET127 dramatically increased the levels of mutant COX3 and COX2 mitochondrial mRNAs that were destabilized by mutations in their 5' untranslated leaders. The stabilizing effect of pet127 delta mutations occurred both in the presence and in the absence of translation. In addition, pet127 delta mutations had pleiotropic effects on the stability and 5' end processing of some wild-type mRNAs and the 15S rRNA but produced only a leaky nonrespiratory phenotype at 37 degrees C. Overexpression of PET127 completely blocked respiratory growth and caused cells to lose wild-type mitochondrial DNA, suggesting that too much Pet127p prevents mitochondrial gene expression. Epitope-tagged Pet127p was specifically located in mitochondria and associated with membranes. These findings suggest that Pet127p plays a role in RNA surveillance and/or RNA processing and that these functions may be membrane bound in yeast mitochondria.     

Overexpression of the COX2 translational activator, Pet111p, prevents translation of COX1 mRNA and cytochrome c oxidase assembly in mitochondria of Saccharomyces cerevisiae

Dramatically elevated levels of the COX2 mitochondrial mRNA-specific translational activator protein Pet111p interfere with respiratory growth and cytochrome c oxidase accumulation. The respiratory phenotype appears to be caused primarily by inhibition of the COX1 mitochondrial mRNA translation, a finding confirmed by lack of cox1Delta::ARG8(m) reporter mRNA translation. Interference with Cox1p synthesis depends to a limited extent upon increased translation of the COX2 mRNA, but is largely independent of it. Respiratory growth is partially restored by a chimeric COX1 mRNA bearing the untranslated regions of the COX2 mRNA, and by overproduction of the COX1 mRNA-specific activators, Pet309p and Mss51p. These results suggest that excess Pet111p interacts unproductively with factors required for normal COX1 mRNA translation. Certain missense mutations in PET111 alleviate the interference with COX1 mRNA translation but do not completely restore normal respiratory growth in strains overproducing Pet111p, suggesting that elevated Pet111p also perturbs assembly of newly synthesized subunits into active cytochrome c oxidase. Thus, this severe imbalance in translational activator levels appears to cause multiple problems in mitochondrial gene expression, reflecting the dual role of balanced translational activators in cooperatively regulating both the levels and locations of organellar translation.     

The pentatricopeptide repeats present in Pet309 are necessary for translation but not for stability of the mitochondrial COX1 mRNA in yeast

Pet309 is a protein essential for respiratory growth. It is involved in translation of the yeast mitochondrial COX1 gene, which encodes subunit I of the cytochrome c oxidase. Pet309 is also involved in stabilization of the COX1 mRNA. Mutations in a similar human protein, Lrp130, are associated with Leigh syndrome, where cytochrome c oxidase activity is affected. The sequence of Pet309 reveals the presence of at least seven pentatricopeptide repeats (PPRs) located in tandem in the central portion of the protein. Proteins containing PPR motifs are present in mitochondria and chloroplasts and are in general involved in RNA metabolism. Despite the increasing number of proteins from this family found to play essential roles in mitochondria and chloroplasts, little is understood about the mechanism of action of the PPR domains present in these proteins. In a series of in vivo analyses we constructed a pet309 mutant lacking the PPR motifs. Although the stability of the COX1 mRNA was not affected, synthesis of Cox1 was abolished. The deletion of one PPR motif at a time showed that all the PPR motifs are required for COX1 mRNA translation and respiratory growth. Mutations of basic residues in PPR3 caused reduced respiratory growth. According to a molecular model, these residues are facing a central cavity that could be involved in mRNA-binding activity, forming a possible path for this molecule on Pet309. Our results show that the RNA metabolism function of Pet309 is found in at least two separate domains of the protein.     

The yeast nuclear gene MRF1 encodes a mitochondrial peptide chain release factor and cures several mitochondrial RNA splicing defects.

We report the molecular cloning, sequencing and genetic characterization of the first gene encoding an organellar polypeptide chain release factor, the MRF1 gene of the yeast Saccharomyces cerevisiae. The MRF1 gene was cloned by genetic complementation of a respiratory deficient mutant disturbed in the expression of the mitochondrial genes encoding cytochrome c oxidase subunit 1 and 2, COX1 and COX2. For COX1 this defect has been attributed to an impaired processing of several introns. Sequence analysis of the MRF1 gene revealed that it encodes a protein highly similar to prokaryotic peptide chain release factors, especially RF-1. Disruption of the gene results in a high instability of the mitochondrial genome, a hallmark for a strict lesion in mitochondrial protein synthesis. The respiratory negative phenotype of mrf1 mutants lacking all known mitochondrial introns and the reduced synthesis of mitochondrial translation products encoded by unsplit genes confirm a primary defect in mitochondrial protein synthesis. Over-expression of the MRF1 gene in a mitochondrial nonsense suppressor strain reduces suppression in a dosage-dependent manner, shedding new light on the role of the '530 region' of 16S-like ribosomal RNA in translational fidelity.     

Properties of an abundant RNA-binding protein in yeast mitochondria

We have previously identified a protein with Mr approximately 40,000 (p40) that binds with high specificity and affinity to the 5'-untranslated leaders of mitochondrial mRNAs in yeast. Here we show that this protein is abundant, comprising about 0.4% of total mitochondrial protein. p40 is present in a cytoplasmic (rho degree) petite mutant that lacks mitochondrial protein synthesis and is therefore nuclear encoded. p40 can be detected by immunological techniques in cell lysates of several different pet mutants, specifically disturbed in the translation of individual mitochondrial mRNAs. It is thus not one of the translation factors defined by any of these mutations. In the case of a pet111 mutant, which is specifically blocked in the translation of COX2 mRNA, extracts still display COX2 mRNA binding activity, indicating that p40 complex formation in vitro is not dependent on the presence of PET111.