Protein synthesis in mitochondria from yeast strains carrying nam and mim suppressor genes

Yeast mitochondria isolated from two different wild type strains (gal+ and gal-), whether grown on galactose or glucose, synthesize all mitochondrial polypeptides with similar efficiencies and in proportions approximating those detected in vivo. Mitochondria isolated from mit- mutants synthesize in vitro a mutant pattern of mitochondrial proteins, indistinguishable from the in vivo products. The mutant pattern is restored to the wild type one in mitochondria isolated from pseudorevertant strains carrying an additional nuclear (nam3-1 and R705) or mitochondrial (mim3-1) informational suppressor gene. Suppression is expressed in isolated mitochondria without the obligatory presence of cytosol at the level of both respiratory control and specific polypeptide synthesis. Translation in isolated mitochondria is sensitive to paromomycin. The antibiotic differentiates between translation in mitochondria from wild type strains and that in nam-type gene carrying strains. This strongly suggests that nam-type mutations affect the mitoribosome, enhancing ambiguity of translation, thus allowing for the pseudoreversion of mit- phenotypes.     

Nuclear suppressors of the mitochondrial mutation oxi1-V25 in Saccharomyces cerevisiae. Genetic analysis of the suppressors: absence of complementation between non-allelic mutants

Ten informational nuclear suppressors of the oxi1- mitochondrial mutation of Saccharomyces cerevisiae are recessive. They are linked to each other, but their allelism is uncertain. Some of them unfavourably affect functions of standard (mit+) mitochondrial genomes. One suppressor severely impairs or entirely prevents mitochondrial functions of the spore clones carrying it. The spectrum of mit- mutations on which these suppressors act is similar to that exhibited by nam3-1. In double heterozygotes namx/NAM3+, NAM+x/nam3-1 the oxi1- (and box3-) mutation is suppressed, yet one of our suppressors (R705) and nam3-1 show independent segregation in tetrads. This indicates that there may be absence of complementation between non-allelic suppressors.     

Long range control circuits within mitochondria and between nucleus and mitochondria

Summary In the preceding paper of this series (Dujardin et al. 1980a) we described general methods of selecting and genetically characterizing suppressor mutations that restore the respiratory capacity of mit ^- mitochondrial mutations. Two dominant nuclear (NAM1-1 and NAM2-1) and one mitochondrial (mim2-1) suppressors are more extensively studied in this paper. We have analysed the action spectrum of these suppressors on 433 mit ^- mutations located in various mitochondrial genes and found that they preferentially alleviate the effects of mutations located within intron open reading frames of the cob-box gene. We conclude that these suppressors permit the maturation of cytochrome b mRNA by restoring the synthesis of intron encoded protein(s) catalytically involved in splicing i.e. mRNA-maturase(s) (cf. Lazowska et al. 1980). NAM1-1 is allele specific and gene non-specific: it suppresses mutations located within different introns. NAM2-1 and mim2-1 are intron-specific: they suppress mutations all located in the same (box7) intron of the cobbox gene. Analyses of cytochrome absorption spectra and mitochondrial translation products of cells in which the suppressors are associated with various other mit ^- mutations show that the suppressors restore cytochrome b and/or cytochrome oxidase (cox 1) synthesis, as expected from their growth phenotype. This suppression is, however, only partial: some new polypeptides characteristic of the mit ^- mutations can be still detected in the presence of suppressor. Interestingly enough when box7 specific suppressors NAM2-1 and mim2-1 are associated with a complete cob-box deletion (leading to a total deficiency of cytochrome b and oxidase) partial restoration of cox I synthesis is observed while cytochrome b is still totally absent. These results show that in strains carrying NAM2-1 or mim2-1 the presence of cytochrome b gene is no longer required for the expression of the oxi3 gene pointing out to the possibility of a mutational switch-on of silent genes, whether mitochondrial, mim2-1, or nuclear, NAM2-1. This switch-on would permit the synthesis of an active maturase acting as a substitute for the box7 maturase in order to splice the cytochrome b and oxidase mRNAs.     

Molecular basis of the 'box effect', A maturase deficiency leading to the absence of splicing of two introns located in two split genes of yeast mitochondrial DNA

In the mitochondrial DNA of Saccharomyces cerevisiae, the genes cob-box and oxi3, coding for apocytochrome b and cytochrome oxidase subunit I respectively, are split. Several mutations located in the introns of the cob-box gene prevent the synthesis of cytochrome b and cytochrome oxidase subunit I (this is known as the 'box effect').-We have elucidated the molecular basis of this phenomenon: these mutants are unable to excise the fourth intron of oxi3 from the cytochrome oxidase subunit I pre-mRNA; the absence of a functional bI4 mRNA maturase, a trans-acting factor encoded by the fourth intron of the cob-box gene explains this phenomenon. This maturase was already known to control the excision of the bI4 intron; consequently we have demonstrated that it is necessary for the processing of two introns located in two different genes. Mutations altering this maturase can be corrected, but only partially, by extragenic suppressors located in the mitochondrial (mim2) or in the nuclear (NAM2) genome. The gene product of these two suppressors should, therefore, control (directly or indirectly) the excision of the two introns as the bI4 mRNA maturase normally does.     

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.     

Proteomic analysis of the mammalian mitochondrial ribosome. Identification of protein components in the 28 S small subunit

The mammalian mitochondrial ribosome (mitoribosome) has a highly protein-rich composition with a small sedimentation coefficient of 55 S, consisting of 39 S large and 28 S small subunits. In the previous study, we analyzed 39 S large subunit proteins from bovine mitoribosome (Suzuki, T., Terasaki, M., Takemoto-Hori, C., Hanada, T., Ueda, T., Wada, A., and Watanabe, K. (2001) J. Biol. Chem. 276, 21724-21736). The results suggested structural compensation for the rRNA deficit through proteins of increased molecular mass in the mitoribosome. We report here the identification of 28 S small subunit proteins. Each protein was separated by radical-free high-reducing two-dimensional polyacrylamide gel electrophoresis and analyzed by liquid chromatography/mass spectrometry/mass spectrometry using electrospray ionization/ion trap mass spectrometer to identify cDNA sequence by expressed sequence tag data base searches in silico. Twenty one proteins from the small subunit were identified, including 11 new proteins along with their complete cDNA sequences from human and mouse. In addition to these proteins, three new proteins were also identified in the 55 S mitoribosome. We have clearly identified a mitochondrial homologue of S12, which is a key regulatory protein of translation fidelity and a candidate for the autosomal dominant deafness gene, DFNA4. The apoptosis-related protein DAP3 was found to be a component of the small subunit, indicating a new function for the mitoribosome in programmed cell death. In summary, we have mapped a total of 55 proteins from the 55 S mitoribosome on the two-dimensional polyacrylamide gels.     

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.     

Suppressor analysis of mutations in the 5'-untranslated region of COB mRNA identifies components of general pathways for mitochondrial mRNA processing and decay in Saccharomyces cerevisiae

The cytochrome b gene in Saccharomyces cerevisiae, COB, is encoded by the mitochondrial genome. Nuclear-encoded Cbp1 protein is required specifically for COB mRNA stabilization. Cbp1 interacts with a CCG element in a 64-nucleotide sequence in the 5'-untranslated region of COB mRNA. Mutation of any nucleotide in the CCG causes the same phenotype as cbp1 mutations, i.e., destabilization of both COB precursor and mature message. In this study, eleven nuclear suppressors of single-nucleotide mutations in CCG were isolated and characterized. One dominant suppressor is in CBP1, while the other 10 semidominant suppressors define five distinct linkage groups. One group of four mutations is in PET127, which is required for 5' end processing of several mitochondrial mRNAs. Another mutation is linked to DSS1, which is a subunit of mitochondrial 3' --> 5' exoribonuclease. A mutation linked to the SOC1 gene, previously defined by recessive mutations that suppress cbp1 ts alleles and stabilize many mitochondrial mRNAs, was also isolated. We hypothesize that the products of the two uncharacterized genes also affect mitochondrial RNA turnover.     

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.     

Nuclear and mitochondrial revertants of a mitochondrial mutant with a defect in the ATP synthetase complex

Summary Yeast strain 990 carries a mutation mapping to the oli1 locus of the mitochondrial genome, the gene encoding ATPase subunit 9. DNA sequence analysis indicated a substitution of valine for alanine at residue 22 of the protein. The strain failed to grow on nonfermentable carbon sources such as glycerol at low temperature (20°C). At 28°C the strain grew on nonfermentable carbon sources and was resistant to the antibiotic oligomycin. ATPase activity in mitochondria isolated from 990 was reduced relative to the wild-type strain from which it was derived, but the residual activity was oligomycin resistant. Subunit 9 (the DCCD-binding proteolipid) from the mutant strain exhibited reduced mobility in SDS-polyacrylamide gels relative to the wild-type proteolipid. Ten revertant strains of 990 were analyzed. All restored the ability to grow on glycerol at 20°C. Mitotic segregation data showed that eight of the ten revertants were attributable to mitochondrial genetic events and two were caused by nuclear events since they appeared to be recessive nuclear suppressors. These nuclear mutations retained partial resistance to oligomycin and did not alter the electrophoretic behavior of subunit 9 or any other ATPase subunit. When mitochondrial DNA from each of the revertant strains was hybridized with an oligonucleotide probe covering the oli1 mutation, seven of the mitochondrial revertants were found to be true revertants and one a second mutation at the site of the original 990 mutation. The oli1 gene from this strain contained a substitution of glycine for valine at residue 22. The proteolipid isolated from this strain had increased electrophoretic mobility relative to the wild-type proteolipid.Abbreviations DCCD dicyclohexylcarbodiimide - SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - SMP submitochondrial particles - mit^- mitochondrial point mutant     

Translation initiation in Saccharomyces cerevisiae mitochondria: functional interactions among mitochondrial ribosomal protein Rsm28p, initiation factor 2, methionyl-tRNA-formyltransferase and novel protein Rmd9p

Rsm28p is a dispensable component of the mitochondrial ribosomal small subunit in Saccharomyces cerevisiae that is not related to known proteins found in bacteria. It was identified as a dominant suppressor of certain mitochondrial mutations that reduced translation of the COX2 mRNA. To explore further the function of Rsm28p, we isolated mutations in other genes that caused a synthetic respiratory defective phenotype together with rsm28Delta. These mutations identified three nuclear genes: IFM1, which encodes the mitochondrial translation initiation factor 2 (IF2); FMT1, which encodes the methionyl-tRNA-formyltransferase; and RMD9, a gene of unknown function. The observed genetic interactions strongly suggest that the ribosomal protein Rsm28p and Ifm1p (IF2) have similar and partially overlapping functions in yeast mitochondrial translation initiation. Rmd9p, bearing a TAP-tag, was localized to mitochondria and exhibited roughly equal distribution in soluble and membrane-bound fractions. A small fraction of the Rmd9-TAP sedimented together with presumed monosomes, but not with either individual ribosomal subunit. Thus, Rmd9 is not a ribosomal protein, but may be a novel factor associated with initiating monosomes. The poorly respiring rsm28Delta, rmd9-V363I double mutant did not have a strong translation-defective phenotype, suggesting that Rmd9p may function upstream of translation initiation, perhaps at the level of localization of mitochondrially coded mRNAs.     

Insights into mitoribosomal biogenesis from recent structural studies

The mitochondrial ribosome (mitoribosome) is a multicomponent machine that has unique structural features. Biogenesis of the human mitoribosome includes correct maturation and folding of the mitochondria-encoded RNA components (12S and 16S mt-rRNAs, and mt-tRNAVal) and their assembly together with 82 nucleus-encoded mitoribosomal proteins. This complex process requires the coordinated action of multiple assembly factors. Recent advances in single-particle cryo-electron microscopy (cryo-EM) have provided detailed insights into the specific functions of several mitoribosome assembly factors and have defined their timing. In this review we summarize mitoribosomal small (mtSSU) and large subunit (mtLSU) biogenesis based on structural findings, and we discuss potential crosstalk between mtSSU and mtLSU assembly pathways as well as coordination between mitoribosome biogenesis and other processes involved in mitochondrial gene expression.     

Mitochondrial and nuclear mutations that affect the biogenesis of the mitochondrial ribosomes of yeast. II. Biochemistry.

1. Several nuclear mutants have been isolated which showed thermo- or cryo-sensitive growth on non-fermentable media. Although the original strain carried mitochondrial drug resistance mutations (CR, ER, OR and PR), the resistance to one or several drugs was suppressed in these mutants. Two of them showed a much reduced amount of the mitochondrial small ribosomal subunit (37S) and of the corresponding 16S ribosomal RNA. Two dimensional electrophoretic analysis did not reveal any change in the position of any of the mitochondrial ribosomal proteins. However one of the mitochondrial ribosomal proteins. However one of the mutants showed a striking decrease in the amounts of three ribosomal proteins S3, S4 and S15. 2. Four temperature-sensitive mitochondrial mutations have been localized in the region of the gene coding for the large mitochondrial ribosomal RNA (23S). These mutants all showed a marked anomaly in the mitochondrial large ribosomal subunit (50S) and/or the corresponding 23S ribosomal RNA.     

Mutational study of the rRNA in yeast mitochondria: functional importance of T1696 in the large rRNA gene.

Four intragenic suppressors of a mitochondrial mutation in the 21S rRNA gene have been characterized in S. cerevisiae. The determination of the nature of the nucleotide changes in the suppressor strains showed that a T at position 1696 in the large rRNA gene is essential for correct function of the mitoribosome. The importance of this specific nucleotide and the fact that this mitochondrial mutation can also be suppressed by a mutation in a nuclear gene are in good agreement with a rRNA-r protein interaction in this part of domain IV, which functional importance is demonstrated in vivo by our results.     

A structural model for the large subunit of the mammalian mitochondrial ribosome

Protein translation is essential for all forms of life and is conducted by a macromolecular complex, the ribosome. Evolutionary changes in protein and RNA sequences can affect the 3D organization of structural features in ribosomes in different species. The most dramatic changes occur in animal mitochondria, whose genomes have been reduced and altered significantly. The RNA component of the mitochondrial ribosome (mitoribosome) is reduced in size, with a compensatory increase in protein content. Until recently, it was unclear how these changes affect the 3D structure of the mitoribosome. Here, we present a structural model of the large subunit of the mammalian mitoribosome developed by combining molecular modeling techniques with cryo-electron microscopic data at 12.1A resolution. The model contains 93% of the mitochondrial rRNA sequence and 16 mitochondrial ribosomal proteins in the large subunit of the mitoribosome. Despite the smaller mitochondrial rRNA, the spatial positions of RNA domains known to be involved directly in protein synthesis are essentially the same as in bacterial and archaeal ribosomes. However, the dramatic reduction in rRNA content necessitates evolution of unique structural features to maintain connectivity between RNA domains. The smaller rRNA sequence also limits the likelihood of tRNA binding at the E-site of the mitoribosome, and correlates with the reduced size of D-loops and T-loops in some animal mitochondrial tRNAs, suggesting co-evolution of mitochondrial rRNA and tRNA structures.     

Mitochondrial assembly in respiration-deficient mutants of Saccharomyces cerevisiae. IV. Effects of nuclear amber suppressors on the accumulation of a mitochondrially made subunit of cytochrome c oxidase.

Earlier studies from this laboratory have shown that cytochrome c oxidase from bakers' yeast contains seven subunits, three of which are made in the mitochondrion (Mason, T. L., and Schatz, G. (1973) J. Biol. Chem. 248, 1355). Moreover, a cytochrome c oxidase-less yeast mutant (pet 494-1) was isolated which lacked one of the mitochondrially made subunits (Ebner, E., Mason, T. L., and Schatz, G. (1973) J. Biol. Chem. 248, 5369). Surprisingly, the mutated gene was localized in the nucleus. The results presented here demonstrate that this mutant phenotype can be suppressed by nuclear amber suppressors which affect translation on cytoplasmic ribosomes. This fact was established by two methods, (a) By constructing pet 494-1 strains possessing various amber and ochre markers, isolating respiring revertants from these strains, and demonstrating co-reversion of the amber (but not of the ochre) markers. (b) By coupling the pet 494-1 allele with the well characterized amber suppressor gene SUP 4-3. These data show that suppressor genes located on nuclear chromosomes may control the accumulation of a mitochondrially synthesized polypeptide. The present results also allow some tentative conclusions about the mechanism of the pet 494 mutation. Because it is highly unlikely that the cytoplasmic and the mitochondrial translation system share a common suppressor, the pet 494 locus probably does not code for the missing mitochondrially made subunit, but for a cytoplasmically made protein. This as yet unidentified protein seems to control the synthesis or the integration of the mitochondrially made subunit. Nuclear suppressor genes may thus be useful tools for studying the role of cytoplasmic protein synthesis in mitochondrial formation.     

Long range control circuits within mitochondria and between nucleus and mitochondria

Summary To uncover the functional circuitry both within the mitochondrial genome and between the mitochondrial and the nuclear genome, we have developed a general method for selecting and characterizing genetically suppressor mutations that restore the respiratory capacity of mit ^- mitochondrial mutants.Several hundreds of pseudo-wild type revertants due to a second unlinked mutation which suppresses a target mit ^- mutation were isolated. The suppressor mutations were found located either in the nuclear (abbreviated NAM for nuclear accommodation of mitochondria) or in the mitochondrial genome (abbreviated MIM for mitochondrial-mitochondrial interaction).The specificity of action of various suppressors upon some 250 different mit ^- mutations located in several genes was tested. According to this specificity of action, suppressors were subdivided into two major classes: allele specific or gene specific suppressors. Because the cob-box mitochondrial gene has a mosaic organization, we were able to find a novel third class of extragenic suppressors specific for mit ^- mutations within the introns of this gene.Four examples of suppressors showing various specificities of action illustrate our approach. (1) a nuclear gene controlling specific alleles of different mitochondrial genes; (2) a nuclear gene controlling selectively one intron of a split mitochondrial gene; (3) a mitochondrial gene controlling specific alleles of different mitochondrial genes; (4) a region in one complex mitochondrial gene which controls selectively one intron of another split mitochondrial gene.Different mechanisms of suppression are discussed stressing the alleviation of splicing deficiencies of intron mutations.