Structure and function of MRP20 and MRP49, the nuclear genes for two proteins of the 54 S subunit of the yeast mitochondrial ribosome.

MRP20 and MRP49 are proteins of the large subunit of the mitochondrial ribosome in Saccharomyces cerevisiae. Their genes were identified through immunological screening of a genomic library in the expression vector lambda gt11. Nucleotide sequencing revealed that MRP49 is tightly linked to TPK3 and encodes a 16-kDa, basic protein with no significant relatedness to any other known protein. MRP20 specifies a 263-amino-acid polypeptide with sequence similarity to members of the L23 family of ribosomal proteins. The levels of the mRNAs and proteins for both MRP20 and MRP49 were regulated in response to carbon source. In [rho0] strains lacking mitochondrial rRNA, the levels of the two proteins were reduced severalfold, presumably because the unassembled proteins are unstable. Null mutants of MRP20 converted to [rho-] or [rho0], a characteristic phenotype of mutations in essential genes for mitochondrial translation. Inactivation of MRP49 caused a cold-sensitive respiration-deficient phenotype, indicating that MRP49 is not an essential ribosomal protein. The mrp49 mutants were defective in the assembly of stable 54 S ribosomal subunits at the nonpermissive temperature. With the results presented here, there are now published sequences for 14 yeast mitochondrial ribosomal proteins, only five of which bear discernable relationships to eubacterial ribosomal proteins.     

Characterization of a yeast mitochondrial ribosomal protein structurally related to the mammalian 68-kDa high affinity laminin receptor.

We have cloned the nuclear gene MRP4 coding for a mitochondrial ribosomal protein of the yeast, Saccharomyces cerevisiae. The gene was isolated by complementation of a respiratory-deficient mutant with a pleiotropic defect in mitochondrial gene expression. The nucleotide sequence of MRP4 revealed that it has sequence similarity with Escherichia coli ribosomal protein S2 and related proteins of chloroplast ribosomes from different plants. Further characterization of the MRP4 protein revealed that it is a component of the 37 S subunit of mitochondrial ribosomes. Moreover, the phenotype of cells carrying a disrupted copy of MRP4 is consistent with the MRP4 protein being an essential component of the mitochondrial protein synthetic machinery. Finally, we note that the MRP4 protein and other members of the S2 family of ribosomal proteins have regions of sequence similarity with the mammalian 68-kDa high affinity laminin receptor.     

Incorporation of the yeast mitochondrial ribosomal protein Mrp2 into ribosomal subunits requires the mitochondrially encoded Var1 protein

Mrp2 is a protein component of the small subunit of mitochondrial ribosomes in the yeast Saccharomyces cerevisiae. We have examined the expression of Mrp2 in yeast mutants lacking mitochondrial DNA and found that the steady-state level of Mrp2 is dramatically decreased relative to wild type. These data suggest that the accumulation of Mrp2 depends on the expression of one or more mitochondrial gene products. The mitochondrial genome of S. cerevisiae encodes two components of the small ribosomal subunit, 15S rRNA and the Var1 protein, both of which are necessary for the formation of mature 37S subunits. Several studies have shown that in the absence of Var1 incomplete subunits accumulate, which lack a limited number of ribosomal proteins. Here, we show that Mrp2 is one of the proteins absent from subunits lacking Var1, indicating that Var1 plays an important role in the incorporation of Mrp2 into mitochondrial ribosomal subunits.     

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.     

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.     

Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders

Translation of mitochondrial mRNAs in Saccharomyces cerevisiae depends on mRNA-specific translational activators that recognize the 5′ untranslated leaders (5′-UTLs) of their target mRNAs. We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5′-UTLs of both the COX2 and COX3 mRNAs, indicating a general function in translational activation. One gene, MRP21, encodes a protein with a domain related to the bacterial ribosomal protein S21 and to unidentified proteins of several animals. The other gene, MRP51, encodes a novel protein whose only known homolog is encoded by an unidentified gene in S. kluyveri. Deletion of either MRP21 or MRP51 completely blocked mitochondrial gene expression. Submitochondrial fractionation showed that both Mrp21p and Mrp51p cosediment with the mitochondrial ribosomal small subunit. The suppressor mutations are missense substitutions, and those affecting Mrp21p alter the region homologous to E. coli S21, which is known to interact with mRNAs. Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not generally increase translational efficiency, since some alleles that strongly suppress 5′-UTL mutations fail to suppress initiation codon mutations. We propose that mitochondrial ribosomes themselves recognize a common feature of mRNA 5′-UTLs which, in conjunction with mRNA-specific translational activation, is required for organellar translation initiation.     

Cytosolic Ribosomal Mutations That Abolish Accumulation of Circular Intron in the Mitochondria without Preventing Senescence of Podospora Anserina

The filamentous fungus Podospora anserina presents a degeneration syndrome called Senescence associated with mitochondrial DNA modifications. We show that mutations affecting the two different and interacting cytosolic ribosomal proteins (S7 and S19) systematically and specifically prevent the accumulation of senDNAα (a circular double-stranded DNA plasmid derived from the first intron of the mitochondrial cox1 gene or intron α) without abolishing Senescence nor affecting the accumulation of other usually observed mitochondrial DNA rearrangements. One of the mutant proteins is homologous to the Escherichia coli S4 and Saccharomyces cerevisiae S13 ribosomal proteins, known to be involved in accuracy control of cytosolic translation. The lack of accumulation of senDNAα seems to result from a nontrivial ribosomal alteration unrelated to accuracy control, indicating that S7 and S19 proteins have an additional function. The results strongly suggest that modified expression of nucleus-encoded proteins contributes to Senescence in P. anserina. These data do not fit well with some current models, which propose that intron α plays the role of the cytoplasmic and infectious Determinant of Senescence that was defined in early studies.     

Characterization of the Human Mitochondrial Ribosomal S12 Gene

Translation of mitochondrial encoded mRNAs occurs on mitochondrial ribosomes. The ribosomal RNA components of the mitochondrial ribosomes are coded for by mitochondrial DNA, while all the protein subunits are coded for by nuclear chromosomes. The only mitochondrial protein subunit cloned in mammals is MRPL12, making the study of the role of mitochondrial translation in human disease difficult. We have now cloned the gene for the human mitochondrial ribosomal protein S12, termed RPMS12, based on its homology to theDrosophilatko gene. The gene stretches over 1.7 kb of genomic DNA and maps to chromosome 19q13, near marker D19S881. The mRNA shows three distinct patterns of splicing within the 5′ untranslated region in all tissues examined, one form being predominant over the other two. The coding region of the leader sequence is interrupted in codon 17 by a second intron of 990 bases. The mRNA is predicted to be translated to a prepeptide of 138 amino acids in length and processed to a peptide of 112 aa and a molecular mass of 12.3 kDa. The protein is very basic, with a predicted pIof 10.3, and is highly conserved through evolution. The functional role and map location of the gene make it a candidate gene for susceptibility to aminoglycoside ototoxicity and for the autosomal dominant deafness gene DFNA4.     

A mitochondrial protein from Neurospora crassa detected both on ribosomes and in membrane fractions. Analysis of the gene, the message, and the protein

We have isolated clones representing at least three nuclear genes for mitochondrial ribosomal proteins from Neurospora crassa by screening a lambda gt11 cDNA library with an antiserum against a mixture of these proteins. The cDNA and genomic DNA sequence for one of these genes, mrp-3, was determined. The MRP3 protein was purified by immune-affinity chromatography, using a monoclonal antibody probe, and subjected to amino acid sequence analysis to identify the mature amino terminus and a prospective mitochondrial-targeting presequence. MRP3 was identified as the largest, least basic protein detected from the small subunit of ribosomes which had been salt-washed and fractionated on sucrose gradients. However, the mRNA and protein products of mrp-3 were found to be present in excess over those of other N. crassa mitoribosomal protein genes. Using a solution hybridization/S1 nuclease assay, we found three-fold- more mRNA for mrp-3 than for another mito-ribosomal protein gene. In addition, a 30- to 50-fold excess of non-ribosomal MRP3 protein was discovered. The additional protein was localized in mitochondrial membrane fractions; none was detected in matrix fractions after removal of the ribosomes. An immunologically related protein was detected in ribosome and membrane fractions of mitochondria from Saccharomyces cerevisiae. The functional significance of this dual localization remains an enigma.     

THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to ¹⁴C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-³H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes.     

The human mitochondrial ribosomal protein genes: mapping of 54 genes to the chromosomes and implications for human disorders.

Mitochondria possess their own translational machinery, which is composed of components distinct from their cytoplasmic counterparts. To investigate the possible involvement of mitochondrial ribosomal defects in human disease, we mapped nuclear genes that encode mitochondrial ribosomal proteins (MRPs). We generated sequence-tagged sites (STSs) of individual MRP genes that were able to be detected by PCR. They were placed on an STS content map of the human genome by typing of radiation hybrid panels. We located 54 MRP genes on the STS-content map and assigned these genes to cytogenetic bands of the human chromosomes. Although mitochondria are thought to have originated from bacteria, in which the genes encoding ribosomal proteins are clustered into operons, the mapped MRP genes are widely dispersed throughout the genome, suggesting that transfer of each MRP gene to the nuclear genome occurred individually. We compared the assigned positions with candidate regions for mendelian disorders and found certain genes that might be involved in particular diseases. This map provides a basis for studying possible roles of MRP defects in mitochondrial disorders.     

Identification of the yeast nuclear gene for the mitochondrial homologue of bacterial ribosomal protein L16.

An open reading frame encoding a member of the L16 family of ribosomal proteins is adjacent to the URA7 gene on the left arm of chromosome II in Saccharomyces cerevisiae. The predicted L16-like polypeptide is basic (pl 11.12), contains 232 amino acids (26.52 kDa) and has 36% amino acid sequence identity to E. coli L16. Immunoblot analysis with polyclonal antibodies to the L16-like polypeptide showed specific cross-reaction with a 22,000 Mr mitochondrial polypeptide that co-sediments with the large subunit of the mitochondrial ribosome in sucrose density gradients. The levels of the L16 mRNA and protein varied in response to carbon source. In [rho degree] cells lacking mitochondrial rRNA, the L16 mRNA accumulated at normal levels, but the protein was barely detectable, indicating RNA-dependent accumulation of the L16 protein. Gene disruption experiments demonstrated that the yeast mitochondrial L16 is an essential ribosomal protein in vivo.     

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.     

SSB, Encoding a Ribosome-Associated Chaperone, Is Coordinately Regulated with Ribosomal Protein Genes

Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock the EXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.     

A novel small-subunit ribosomal protein of yeast mitochondria that interacts functionally with an mRNA-specific translational activator.

Mitochondrial translation of the mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the action of three position activator proteins encoded in the nucleus of Saccharomyces cerevisiae. Some mutations affecting one of these activators, PET122, can be suppressed by mutations in an unlinked nuclear gene termed PET123. PET123 function was previously demonstrated to be required for translation of all mitochondrial gene products. We have now generated an antibody against the PET123 protein and have used it to demonstrate that PET123 is a mitochondrial ribosomal protein of the small subunit. PET123 appears to be present at levels comparable to those of other mitochondrial ribosomal proteins, and its accumulation is dependent on the presence of the 15S rRNA gene in mitochondria. Taken together with the previous genetic data, these results strongly support a model in which the mRNA-specific translational activator PET122 works by directly interacting with the small ribosomal subunit to promote translation initiation on the coxIII mRNA.