Suppression of a defect in the 5'' untranslated leader of mitochondrial COX3 mRNA by a mutation affecting an mRNA-specific translational activator protein.

Translation of the Saccharomyces cerevisiae mitochondrial COX3 mRNA, encoding subunit III of cytochrome c oxidase, specifically requires the action of the nuclear gene products PET54, PET122, and PET494 at a site encoded in the 612-base 5' untranslated leader. To identify more precisely the site of action of the translational activators, we constructed two large deletions of the COX3 mRNA 5' untranslated leader. Both deletions blocked translation without affecting mRNA stability. However, one of the large deletions was able to revert to partial function by a small secondary deletion within the remaining 5' leader sequences. Translation of the resulting mutant (cox3-15) mRNA was still dependent on the nuclear-encoded specific activators but was cold sensitive. We selected revertants of this mitochondrial mutant at low temperature to identify genes encoding proteins that might interact with the COX3 mRNA 5' leader. One such revertant carried a missense mutation in the PET122 gene that was a strong and dominant suppressor of the cold-sensitive defect in the mRNA, indicating that the PET122 protein interacts functionally (possibly directly) with the COX3 mRNA 5' leader. The cox3-15 mutation was not suppressed by overproduction of the wild-type PET122 protein but was very weakly suppressed by overproduction of PET494 and slightly better suppressed by co-overproduction of PET494 and PET122.     

Analysis of the Saccharomyces cerevisiae mitochondrial COX3 mRNA 5' untranslated leader: translational activation and mRNA processing.

We used transformation of yeast mitochondria and homologous gene replacement to study features of the 613-base COX3 mRNA 5' untranslated leader (5'-UTL) required for translational activation by the protein products of the nuclear genes PET54, PET122, and PET494 in vivo. Elimination of the single AUG triplet in the 5'-UTL had no detectable effect on expression, indicating that activator proteins do not work by allowing ribosomes to bypass that AUG. Deletion of the entire 5'-UTL completely prevented translation, suggesting that the activator proteins do not function by antagonizing any other negative element in the 5'-UTL. Removal of the 15 terminal bases from the 5' end of the 5'-UTL did not block activator-dependent translation. The largest internal deletion that did not interfere with translation removed 125 bases from the upstream portion of the leader. However, two large deletions that blocked translation could be reverted to activator-dependent expression by secondary changes in the remaining 5'-UTL sequences, indicating that the original deletions had not removed the translational activator target but only deformed it. Taken together, the deletion mutations and revertants define a region of 151 bases (between positions -480 and -330 relative to the start codon) containing sequences that are sufficient for translational activation when modified slightly. Suppression of the respiratory phenotypes of two 5'-UTL mutations by overexpression of PET54, PET122, and PET494 indicated functional interactions between the leader and the three activator proteins. The mature COX3 mRNA is cleaved from a precursor immediately downstream of the preceding tRNAVal in a fashion resembling mRNA processing in vertebrate mitochondria. Our results indicate that the site of this cleavage in Saccharomyces cerevisiae is determined solely by the position of the tRNA.     

Site-Directed Mutagenesis of a Saccharomyces Cerevisiae Mitochondrial Translation Initiation Codon

We have used a generally applicable strategy for gene replacement in yeast mitochondria to mutate the translation initiation codon of the COX3 gene from AUG to AUA. The mutation, cox3-1, substantially reduced, but did not eliminate, translation of cytochrome c oxidase subunit III (coxIII). Strains bearing the mutation exhibited a leaky (partial) nonrespiratory growth phenotype and a reduced incorporation of radiolabeled amino acids into coxIII in vivo in the presence of cycloheximide. Hybridization experiments demonstrated that the mutation had little or no effect on levels of the COX3 mRNA. Residual translation of the cox3-1 mutant mRNA was dependent upon the three nuclearly coded mRNA-specific activators PET494, PET54 and PET122, known from previous studies to work through a site (or sites) upstream of the initiation codon to promote translation of the wild-type mRNA. Furthermore, respiratory growth of cox3-1 mutant strains was sensitive to decreased dosage of genes PET494 and PET122 in heterozygous mutant diploids, unlike the growth of strains carrying wild-type mtDNA. Some residual translation of the cox3-1 mRNA appeared to initiate at the mutant AUA codon, despite the fact that the 610-base 5'-mRNA leader contains numerous AUA triplets. We conclude that, while AUG is an important component of the COX3 translation initiation site, the site probably is also specified by other sequence or structural features.     

A Genetic Link between an mRNA-Specific Translational Activator and the Translation System in Yeast Mitochondria

Translation of the Saccharomyces cerevisiae mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the products of at least three nuclear genes, PET122, PET494 and PET54. pet122 mutations that remove 24-67 amino acid residues from the carboxyterminus of the gene product were found to be suppressed by unlinked nuclear mutations. These unlinked suppressors fail to suppress both a pet122 missense mutation and a complete pet122 deletion. One of the suppressor mutations causes a heat-sensitive nonrespiratory growth phenotype in an otherwise wild-type strain and reduces translation of all mitochondrial gene products in cells grown at high temperature. This suppressor maps to a newly identified gene on chromosome XV termed PET123. The sequence of a DNA fragment carrying PET123 contains one major open reading frame encoding a basic protein of 318 amino acids. Inactivation of the chromosomal copy of PET123 by interruption of this open reading frame causes cells to become rho- (sustain large deletions in their mtDNA). This phenotype is characteristic for null alleles of genes whose products are essential for general mitochondrial protein synthesis. Thus our data strongly suggest that the PET123 protein is a component of the mitochondrial translation apparatus that interacts directly with the coxIII-mRNA-specific translational activator PET122.     

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.     

Alteration of the Saccharomyces cerevisiae COX2 mRNA 5'-untranslated leader by mitochondrial gene replacement and functional interaction with the translational activator protein PET111.

The ability to replace wild-type mitochondrial DNA sequences in yeast with in vitro-generated mutations has been exploited to study the mechanism by which the nuclearly encoded PET111 protein specifically activates translation of the mitochondrially coded COX2 mRNA. We have generated three mutations in vitro that alter the COX2 mRNA 5'-untranslated leader (UTL) and introduced them into the mitochondrial genome, replacing the wild-type sequence. None of the mutations significantly affected the steady-state level of COX2 mRNA. Deletion of a single base at position -24 (relative to the translation initiation codon) in the 5'-UTL (cox2-11) reduced COX2 mRNA translation and respiratory growth, whereas insertion of four bases in place of the deleted base (cox2-12) and deletion of bases -30 to -2 (cox2-13) completely blocked both. Six spontaneous nuclear mutations were selected as suppressors of the single-base 5'-UTL deletion, cox2-11. One of these mapped to PET111 and was shown to be a missense mutation that changed residue 652 from Ala to Thr. This suppressor, PET111-20, failed to suppress the 29-base deletion, cox2-13, but very weakly suppressed the insertion mutation, cox2-12. PET111-20 also enhanced translation of a partially functional COX2 mRNA with a wild-type 5'-UTL but a mutant initiation codon. Although overexpression of the wild-type PET111 protein caused weak suppression of the single-base deletion, cox2-11, the PET111-20 suppressor mutation did not function simply by increasing the level of the protein. These results demonstrate an intimate functional interaction between the translational activator protein and the mRNA 5'-UTL and suggest that they may interact directly.     

Interactions among COX1, COX2, and COX3 mRNA-specific translational activator proteins on the inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae

The core of the cytochrome c oxidase complex is composed of its three largest subunits, Cox1p, Cox2p, and Cox3p, which are encoded in mitochondrial DNA of Saccharomyces cerevisiae and inserted into the inner membrane from the inside. Mitochondrial translation of the COX1, COX2, and COX3 mRNAs is activated mRNA specifically by the nuclearly coded proteins Pet309p, Pet111p, and the concerted action of Pet54p, Pet122p, and Pet494p, respectively. Because the translational activators recognize sites in the 5'-untranslated leaders of these mRNAs and because untranslated mRNA sequences contain information for targeting their protein products, the activators are likely to play a role in localizing translation. Herein, we report physical associations among the mRNA-specific translational activator proteins, located on the matrix side of the inner membrane. These interactions, detected by coimmune precipitation and by two-hybrid experiments, suggest that the translational activator proteins could be organized on the surface of the inner membrane such that synthesis of Cox1p, Cox2p, and Cox3p would be colocalized in a way that facilitates assembly of the core of the cytochrome c oxidase complex. In addition, we found interactions between Nam1p/Mtf2p and the translational activators, suggesting an organized delivery of mitochondrial mRNAs to the translation system.     

Highly diverged homologs of Saccharomyces cerevisiae mitochondrial mRNA-specific translational activators have orthologous functions in other budding yeasts

Translation of mitochondrially coded mRNAs in Saccharomyces cerevisiae depends on membrane-bound mRNA-specific activator proteins, whose targets lie in the mRNA 5'-untranslated leaders (5'-UTLs). In at least some cases, the activators function to localize translation of hydrophobic proteins on the inner membrane and are rate limiting for gene expression. We searched unsuccessfully in divergent budding yeasts for orthologs of the COX2- and COX3-specific translational activator genes, PET111, PET54, PET122, and PET494, by direct complementation. However, by screening for complementation of mutations in genes adjacent to the PET genes in S. cerevisiae, we obtained chromosomal segments containing highly diverged homologs of PET111 and PET122 from Saccharomyces kluyveri and of PET111 from Kluyveromyces lactis. All three of these genes failed to function in S. cerevisiae. We also found that the 5'-UTLs of the COX2 and COX3 mRNAs of S. kluyveri and K. lactis have little similarity to each other or to those of S. cerevisiae. To determine whether the PET111 and PET122 homologs carry out orthologous functions, we deleted them from the S. kluyveri genome and deleted PET111 from the K. lactis genome. The pet111 mutations in both species prevented COX2 translation, and the S. kluyveri pet122 mutation prevented COX3 translation. Thus, while the sequences of these translational activator proteins and their 5'-UTL targets are highly diverged, their mRNA-specific functions are orthologous.     

At least two nuclear gene products are specifically required for translation of a single yeast mitochondrial mRNA.

Mitochondrial translation of the oxi2 mRNA, encoding yeast cytochrome c oxidase subunit III (coxIII), has previously been shown to specifically require the mitochondrially located protein product of the nuclear gene PET494. We show here that this specific translational activation involves at least one other newly identified gene termed PET54. Mutations in PET54 cause an absence of the coxIII protein despite the presence of normal levels of its mRNA. pet494 mutations are known to be suppressible by mitochondrial gene rearrangements that replace the normal 5'-untranslated leader of the oxi2 mRNA with the leaders of other mitochondrial mRNAs. In this study we show that pet54, pet494 double mutants are suppressed by the same mitochondrial gene rearrangements, showing that the PET54 product is specifically required, in addition to the PET494 protein, for translation of the oxi2 mRNA. Since, as we show here, PET54 is not an activator of PET494 gene expression, our results suggest that the products of both of these genes may act together to stimulate coxIII translation.     

Functional Interactions among Two Yeast Mitochondrial Ribosomal Proteins and an mRNA-Specific Translational Activator

Expression of the Saccharomyces cerevisiae mitochondrial gene coding cytochrome c oxidase subunit III is specifically activated at the level of translation by at least three nuclear genes, PET122, PET494 and PET54. We have shown previously that carboxy-terminal deletions of PET122 are allele-specifically suppressed by mutations in an unlinked nuclear gene, termed PET123, that encodes a small subunit ribosomal protein. Here we describe additional pet122 suppressors generated by mutations in a second gene which we show to be the previously identified nuclear gene MRP1. Like PET123, MRP1 encodes a component of the small subunit of mitochondrial ribosomes. Our mrp1 mutations are allele-specific suppressors of carboxyl-terminal truncations of the PET122 protein and do not bypass the requirement for residual function of PET122. None of our mrp1 mutations has an intrinsic phenotype in an otherwise wild-type background. However, some of the mrp1 mutations cause a non-conditional respiratory-defective phenotype in combination with certain pet123 alleles. This synthetic defective phenotype suggests that the ribosomal proteins PET123 and MRP1 interact functionally with each other. The fact that they can both mutate to suppress certain alleles of the mRNA-specific translational activator PET122 strongly suggests that the PET122 protein promotes translation of the coxIII mRNA via an interaction with the small subunit of mitochondrial ribosomes.     

Suppression of carboxy-terminal truncations of the yeast mitochondrial mRNA-specific translational activator PET122 by mutations in two new genes, MRP17 and PET127

Summary The PET122 protein is one of three Saccharomyces cerevisiae nuclear gene products required specifically to activate translation of the mitochondrially coded COX3 mRNA. We have previously observed that mutations which remove the carboxy-terminal region of PET122 block translation of the COX3 mRNA but can be suppressed by unlinked nuclear mutations in several genes, two of which have been shown to code for proteins of the small subunit of mitochondrial ribosomes. Here we describe and map two more new genes identified as allele-specific suppressors that compensate for carboxy-terminal truncation of PET122. One of these genes, MRP17, is essential for the expression of all mitochondrial genes and encodes a protein of M_r 17343. The MRP17 protein is a component of the small ribosomal subunit in mitochondria, as demonstrated by the fact that a missense mutation, mrp17-1, predicted to cause a charge change indeed alters the charge of a mitochondrial ribosomal protein of the expected size. In addition, mrp17-1, in combination with some mutations affecting another mitochondrial ribosomal protein, caused a synthetic defective phenotype. These findings are consistent with a model in which PET122 functionally interacts with the ribosomal small subunit. The second new suppressor gene described here, PET127, encodes a protein too large (M_r 95900) to be a ribosomal protein and appears to operate by a different mechanism. PET127 is not absolutely required for mitochondrial gene expression and allele-specific suppression of pet122 mutations results from the loss of PET127 function: a pet127 deletion exhibited the same recessive suppressor activity as the original suppressor mutation. These findings suggest the possibility that PET127 could be a novel component of the mitochondrial translation system with a role in promoting accuracy of translational initiation.     

Genetic evidence that different functional domains of the PET54 gene product facilitate expression of the mitochondrial genes COX1 and COX3 in Saccharomyces cerevisiae.

Expression of the yeast mitochondrial genes COX1 and COX3, which encode subunits I and III of cytochrome oxidase, respectively, is controlled by a common nuclear-encoded trans-acting factor. This protein, encoded by the PET54 gene, controls expression of COX1 at the level of RNA splicing and COX3 at the level of mRNA translation. While the steps of COX1 and COX3 gene expression affected by the PET54 gene product are different, it is possible that the PET54 protein is monofunctional and affects expression of each gene by a single mechanism, such as modulation of RNA secondary structure. The goal of this study was to address whether the PET54 protein is monofunctional or multifunctional with respect to its role in COX1 and COX3 gene expression. Ten insertion mutations, which each resulted in the in-frame addition of four amino acids within the PET54 polypeptide, were generated, and the resulting mutants were characterized for respiration phenotype and mitochondrial gene expression. Five of the ten mutants were respiration deficient. Two of these five mutants were defective in expression of COX3 but not in expression of COX1, while two other mutants had the opposite phenotype (primarily defective in expression of COX1). The fifth mutant was equally defective in expression of both genes. These results demonstrate that the two functions of PET54 are genetically separable and support the idea that the PET54 protein is multifunctional.     

Alteration of a novel dispensable mitochondrial ribosomal small-subunit protein, Rsm28p, allows translation of defective COX2 mRNAs

Mutations affecting the RNA sequence of the first 10 codons of the Saccharomyces cerevisiae mitochondrial gene COX2 strongly reduce translation of the mRNA, which encodes the precursor of cytochrome c oxidase subunit II. A dominant chromosomal mutation that suppresses these defects is an internal in-frame deletion of 67 codons from the gene YDR494w. Wild-type YDR494w encodes a 361-residue polypeptide with no similarity to proteins of known function. The epitope-tagged product of this gene, now named RSM28, is both peripherally associated with the inner surface of the inner mitochondrial membrane and soluble in the matrix. Epitope-tagged Rsm28p from Triton X-100-solubilized mitochondria sedimented with the small subunit of mitochondrial ribosomes in a sucrose gradient containing 500 mM NH4Cl. Complete deletion of RSM28 caused only a modest decrease in growth on nonfermentable carbon sources in otherwise wild-type strains and enhanced the respiratory defect of the suppressible cox2 mutations. The rsm28 null mutation also reduced translation of an ARG8m reporter sequence inserted at the COX1, COX2, and COX3 mitochondrial loci. We tested the ability of RSM28-1 to suppress a variety of cox2 and cox3 mutations and found that initiation codon mutations in both genes were suppressed. We conclude that Rsm28p is a dispensable small-subunit mitochondrial ribosomal protein previously undetected in systematic investigations of these ribosomes, with a positive role in translation of several mitochondrial mRNAs.     

Pet111 Acts in the 5''-Leader of the Saccharomyces Cerevisiae Mitochondrial Cox2 mRNA to Promote Its Translation

PET111 is a yeast nuclear gene specifically required for the expression of the mitochondrial gene COX2, encoding cytochrome c oxidase subunit II (coxII). Previous studies have shown that PET111 activates translation of the COX2 mRNA. To map the site of PET111 action we have constructed, in vitro, genes coding for chimeric mRNAs, introduced them into mitochondria by transformation and studied their expression. Translation of a chimeric mRNA with the 612-base 5'-untranslated leader of the COX3 mRNA fused precisely to the structural gene for the coxII-precursor protein is independent of PET111, but does require a COX3 mRNA-specific translational activator known to work on the COX3 5'-leader. This result demonstrates that PET111 is not required for any posttranslational step. Translation of a chimeric mRNA with the 54-base 5'-leader of the COX2 mRNA fused precisely to the structural gene for cytochrome c oxidase subunit III was dependent on PET111 activity. These results demonstrate that PET111 acts specifically at a site in the short COX2 5'-leader to activate translation of downstream coding sequences.