RESUMO
BACKGROUND: Rye (Secale cereale L.) is a cereal crop highly tolerant to environmental stresses, including abiotic and biotic stresses (e.g., fungal diseases). Among these fungal diseases, leaf rust (LR) is a major threat to rye production. Despite extensive research, the genetic basis of the rye immune response to LR remains unclear. RESULTS: An RNA-seq analysis was conducted to examine the immune response of three unrelated rye inbred lines (D33, D39, and L318) infected with compatible and incompatible Puccinia recondita f. sp. secalis (Prs) isolates. In total, 877 unique differentially expressed genes (DEGs) were identified at 20 and 36 h post-treatment (hpt). Most of the DEGs were up-regulated. Two lines (D39 and L318) had more up-regulated genes than down-regulated genes, whereas the opposite trend was observed for line D33. The functional classification of the DEGs helped identify the largest gene groups regulated by LR. Notably, these groups included several DEGs encoding cytochrome P450, receptor-like kinases, methylesterases, pathogenesis-related protein-1, xyloglucan endotransglucosylases/hydrolases, and peroxidases. The metabolomic response was highly conserved among the genotypes, with line D33 displaying the most genotype-specific changes in secondary metabolites. The effect of pathogen compatibility on metabolomic changes was less than the effects of the time-points and genotypes. Accordingly, the secondary metabolome of rye is altered by the recognition of the pathogen rather than by a successful infection. The results of the enrichment analysis of the DEGs and differentially accumulated metabolites (DAMs) reflected the involvement of phenylpropanoid and diterpenoid biosynthesis as well as thiamine metabolism in the rye immune response. CONCLUSION: Our work provides novel insights into the genetic and metabolic responses of rye to LR. Numerous immune response-related DEGs and DAMs were identified, thereby clarifying the mechanisms underlying the rye response to compatible and incompatible Prs isolates during the early stages of LR development. The integration of transcriptomic and metabolomic analyses elucidated the contributions of phenylpropanoid biosynthesis and flavonoid pathways to the rye immune response to Prs. This combined analysis of omics data provides valuable insights relevant for future research conducted to enhance rye resistance to LR.
Assuntos
Basidiomycota , Micoses , Puccinia , Transcriptoma , Secale/genética , Secale/microbiologia , Basidiomycota/fisiologia , Metaboloma , Doenças das Plantas/microbiologiaRESUMO
Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1 cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.
Assuntos
Proteínas Fúngicas/fisiologia , RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Northern Blotting , Núcleo Celular/metabolismo , Epitopos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Immunoblotting , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Fenótipo , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , RNA/metabolismo , RNA Polimerase III/química , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Temperatura , Fator de Transcrição TFIIIB , Fatores de Transcrição/química , Fatores de Transcrição TFIII/química , Transcrição GênicaRESUMO
Yeast SUP7' or SUP11 nonsense suppressors have no phenotypic expression in strains deficient in the isopentenylation of A37 in tRNA. Here we show that such strains spontaneously produce cells with a nonsense suppressor phenotype which is related to the cytoplasmically inherited determinant and manifests all the key features of the [PSI+] prion. A screen of a multicopy yeast genomic library for genes that inactivate the [PSI+]-related suppressor phenotype resulted in the isolation of the SSB1 gene. Moreover, we demonstrate that multicopy plasmid encoding the Ssb1 chaperone cures cells of the [PSI+] prion.
Assuntos
Proteínas Fúngicas/genética , Chaperonas Moleculares , Chaperonas Moleculares/genética , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Animais , Clonagem Molecular , Códon sem Sentido , Genes Fúngicos/genética , Genótipo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Fenótipo , Plasmídeos/genética , Príons/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Supressão GenéticaRESUMO
The [PSI(+)] nonsense-suppressor determinant of Saccharomyces cerevisiae results from the ability of Sup35 (eRF3) translation termination factor to undergo prion-like aggregation [1]. Although this process is autocatalytic, in vivo it depends on the chaperone Hsp104, whose lack or overexpression can cure [PSI(+)] [2]. Overproduction of the chaperone protein Ssb1 increased the [PSI(+)] curing by excess Hsp104, although it had no effect on its own, and excess chaperone protein Ssa1 protected [PSI(+)] against Hsp104 [3,4]. We used an artificial [PSI(+)(PS)] based on the Sup35 prion-forming domain from yeast Pichia methanolica [5] to find other prion-curing factors. Both [PSI(+)(PS)] and [PSI(+)] have prion 'strains', differing in their suppressor efficiency and mitotic stability. We show that [PSI(+)(PS)] and a 'weak' strain of [PSI(+)] can be cured by overexpression of chaperones Ssa1, Ssb1 and Ydj1. The ability of different chaperones to cure [PSI(+)(PS)] showed significant prion strain specificity, which could be related to variation in Sup35 prion structure. Our results imply that homologs of these chaperones may be active against mammalian prion and amyloid diseases.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/genética , Chaperonas Moleculares , Fatores de Terminação de Peptídeos , Pichia/genética , Pichia/metabolismoAssuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Príons/fisiologia , Saccharomyces cerevisiae/genética , Proteínas Fúngicas/biossíntese , Genes Fúngicos , Microscopia Eletrônica , Príons/biossíntese , Príons/genética , Príons/isolamento & purificação , Saccharomyces cerevisiae/metabolismoRESUMO
Nam9p is a protein of the mitochondrial ribosome. The respiration-deficient Saccharomyces cerevisiae strain MB43-nam9-1 expresses Nam9-1p containing the point mutation S82L. Respiratory deficiency correlates with a decrease in the steady level of some mitochondrially encoded proteins and the complete lack of mitochondrially encoded cytochrome oxidase subunit 2 (Cox2). De novo synthesis of Cox2 in MB43-nam9-1 is unaffected, indicating that newly synthesized Cox2 is rapidly degraded. Respiratory deficiency of MB43-nam9-1 is overcome by transient overexpression of HSP104, by deletion of HSP104, by transient exposure to guanidine hydrochloride, and by expression of the C-terminal portion of Sup35, indicating an involvement of the yeast prion [PSI(+)]. Respiratory deficiency of MB43-nam9-1 can be reinduced by transfer of cytosol from S. cerevisiae that harbors [PSI(+)]. We conclude that nam9-1 causes respiratory deficiency only in combination with the cytosolic prion [PSI(+)], presenting the first example of a synthetic effect between cytosolic [PSI(+)] and a mutant mitochondrial protein.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/fisiologia , Proteínas Nucleares , Proteínas de Plantas/metabolismo , Príons/fisiologia , Proteínas Repressoras , Proteínas Ribossômicas/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Citosol/metabolismo , DNA Fúngico/genética , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Deleção de Genes , Guanidina/farmacologia , Proteínas de Choque Térmico/deficiência , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiologia , Mitocôndrias/fisiologia , Consumo de Oxigênio/efeitos dos fármacos , Fatores de Terminação de Peptídeos , Fenótipo , Proteínas de Plantas/genética , Mutação Puntual , Príons/genética , Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genéticaRESUMO
In contrast to most other eukaryotic organisms, yeast can survive without respiration. This ability has been exploited to investigate nuclear genes required for expression of mitochondrial DNA. Availability of complete Saccharomyces cerevisiae genomic sequence has provided additional help in detailed molecular analysis. Seven of the eight major products encoded by mitochondrial DNA are hydrophobic subunits of respiratory complexes in the inner membrane. Localization of the translation process in the same cellular compartment ensures synthesis of mitochondrially encoded proteins near sites of their assembly into multimeric respiratory complexes. Association of mitochondrial ribosomes with the membrane is mediated by mRNA-specific translational activators, that are involved in the recognition of initiation codon. The newly synthesized mitochondrial proteins are transferred to membrane by a specific export system. This review discusses the role of membrane-localized factors responsible for quality control and turnover of mitochondrially synthesized subunits as well as for assembly of respiratory complexes.
Assuntos
Transporte de Elétrons , Mitocôndrias/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/fisiologia , Modelos BiológicosAssuntos
Citoplasma/genética , Mitocôndrias/genética , Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas Nucleares Pequenas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fator de Iniciação 4A em Eucariotos , Proteínas Fúngicas/genética , Fatores de Terminação de Peptídeos/genética , Proteínas Ribossômicas/genéticaRESUMO
Alteration of the subcellular distribution of Mod5p-I, a tRNA modification enzyme, member of the sorting isozyme family, affects tRNA-mediated nonsense suppression. Altered suppression efficiency was used to identify MDP genes, which, when mutant, change the mitochondrial/cytosolic distribution of Mod5p-I,KR6. MDP2 is the previously identified VRP1, which encodes verprolin, required for proper organization of the actin cytoskeleton. MDP3 is identical to PAN1, which encodes a protein involved in initiation of translation and actin cytoskeleton organization. We report here the cloning and characterization of wild-type and mutant MDP1 alleles and the isolation and characterization of a multicopy suppressor of mdp1 mutations. MDP1 is identical to RSP5, which encodes ubiquitin-protein ligase, and mdp1 mutations are suppressed by high copy expression of ubiquitin. All four characterized mdp1 mutations cause missense changes located in the hect domain of Rsp5p that is highly conserved among ubiquitin-protein ligases. In addition to its well-known function in protein turnover, ubiquitination has been proposed to play roles in subcellular sorting of proteins via endocytosis and in delivery of proteins to peroxisomes, the endoplasmic reticulum and mitochondria. mdp1, as well as mdp2/vrp1 and mdp3/pan1 mutations, affect endocytosis. Further, mdp1 mutations show synthetic interactions with mdp2/vrp1 and mdp3/pan1. Identification of MDP1 as RSP5, along with our previous identification of MDP2/VRP1 and MDP3/PAN1, implicate interactions of the ubiquitin system, the actin cytoskeleton and protein synthesis in the subcellular distribution of proteins.
Assuntos
Citoplasma/metabolismo , Proteínas Fúngicas/genética , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Complexos Ubiquitina-Proteína Ligase , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Recombinante , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte , Genes Supressores , Dados de Sequência Molecular , MutaçãoRESUMO
Mutation in the MAF1 gene was identified in a screen for decreased efficiency of tRNA suppressor SUP11 in the yeast Saccharomyces cerevisiae (Sc). maf1-1 mutation exerts a dual phenotypic effect: antisuppression and temperature sensitive (ts) respiratory growth. MAF1, cloned by complementation of the ts phenotype of maf1-1, also alleviates the antisuppressor effect. The coding sequence of MAF1 is interrupted by an intron of 80 bp. The putative gene product, Maf1p, is a hydrophilic protein of 395 amino acids (aa) not showing significant similarity to known proteins which indicates that MAF1 encodes a novel protein. Maf1p may play a role in the tRNA biosynthetic pathway since a fragment of the RPO31/RPC160 gene encoding the largest subunit of RNA polymerase III was cloned as a multicopy suppressor of mafl-1.
Assuntos
Alquil e Aril Transferases , Proteínas Fúngicas/genética , Genes Supressores , Mutação , RNA de Transferência , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Clonagem Molecular , Proteínas Fúngicas/biossíntese , Amplificação de Genes , Teste de Complementação Genética , Dados de Sequência Molecular , Fenótipo , Proteínas/genética , RNA Polimerase III/genética , RNA Polimerase III/metabolismoRESUMO
We studied expression of the NAM9 gene of Saccharomyces cerevisiae that was previously reported to code for a mitochondrial ribosomal protein. Increase in NAM9 gene dosage is accompanied by the increase in both mRNA and protein. The levels of the NAM9 transcript and protein are both reduced in cells growing on glucose as compared to cells growing on galactose as a carbon source. Nam9p accumulates to the same level in rho(o) and rho(+) cells. These results confirm previous data indicating diverse regulation of different mitochondrial ribosomal protein genes and suggest that expression of Nam9p is not co-ordinated with the expression of other mitochondrial ribosomal components.
Assuntos
Proteínas Fúngicas/biossíntese , Genes Fúngicos , Proteínas Ribossômicas/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Galactose/metabolismo , Regulação Fúngica da Expressão Gênica , Técnicas de Transferência de Genes , Genoma Fúngico , Glucose/metabolismo , Mutação , Biossíntese de Proteínas , Sequências Reguladoras de Ácido Nucleico , Transcrição GênicaRESUMO
We have cloned and sequenced the GDS1 gene located on the right arm of chromosome XV of Saccharomyces cerevisiae. The gene codes for a 522 amino acid serine-rich protein with no obvious homology to proteins in the database. GDS1 gene was isolated as the multicopy suppressor of the glycerol-deficient phenotype caused by the nam9-1 mutation in the yeast nuclear gene encoding the mitochondrial ribosomal protein homologous to S4 proteins from various organisms. Disruption-deletion of the GDS1 open reading frame leads to a partial impairment of growth on medium containing glycerol as the carbon source, indicating mitochondrial function of the gene product.
Assuntos
Genes Fúngicos , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Fúngicos/genética , Clonagem Molecular , DNA Fúngico/genética , Proteínas Fúngicas/genética , Deleção de Genes , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Fenótipo , Saccharomyces cerevisiae/metabolismoRESUMO
The Saccharomyces cerevisiae MOD5 gene encodes proteins that function in three subcellular locations: mitochondria, the cytoplasm, and nuclei (M. Boguta, L.A. Hunter, W.-C. Shen, E. C. Gillman, N. C. Martin, and A. K. Hopper, Mol. Cell. Biol. 14:2298-2306, 1994; E. C. Gillman, L. B. Slusher, N. C. Martin, and A. K. Hopper, Mol. Cell. Biol. 11:2382-2390, 1991). A mutant allele of MOD5 encoding a protein (Mod5p-I,KR6) located predominantly in mitochondria was constructed. Mutants defective in delivering Mod5p-I,KR6 to mitochondria were sought by selecting cells with increased cytosolic activity of this protein. Twenty-five mutants defining four complementation groups, mdp1, mdp2, mdp3, and mdp4, were found. They are unable to respire at 34 degrees C or to grow on glucose medium at 38 degrees C. Cell fractionation studies showed that mdp1, mdp2, and mdp3 mutants have an altered mitochondrial-cytoplasmic distribution of Mod5p. mdp2 can be suppressed by ACT1, the actin-encoding gene. The actin cytoskeleton organization is also aberrant in mdp2 cells. MDP2 is the same as VRP1 (S. F. H. Donnelly, M. J. Picklington, D. Pallotta, and E. Orr, Mol. Microbiol. 10:585-596, 1993). MDP3 is identical to PAN1, which encodes a protein that interacts with mRNA 3' ends and affects initiation of protein synthesis (A. B. Sachs and J. A. Deardoff, Cell 70:961-973, 1992). These results implicate the actin cytoskeleton and mRNA 3' ends and/or protein synthesis as being as important for protein distribution in S. cerevisiae as they are for distribution of cytosolic proteins in higher eukaryotes. This provides the potential to apply genetic and molecular approaches to study gene products and mechanisms involved in this type of protein distribution. The selection strategy also offers a new approach for identifying gene products involved in the distribution of proteins to their subscellular destinations.
Assuntos
Actinas/metabolismo , Alquil e Aril Transferases , Citoesqueleto/metabolismo , Genes Fúngicos , Mitocôndrias/metabolismo , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Citoplasma/metabolismo , Enzimas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Genes Supressores , Teste de Complementação Genética , Genótipo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Biossíntese de Proteínas , Proteínas/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genéticaRESUMO
The nuclear gene NAM9 from Saccharomyces cerevisiae (Sc) codes for a protein which, on the basis of sequence homology, was previously postulated to be a mitochondrial (mt) equivalent of the Escherichia coli (Ec) S4 ribosomal protein (r-protein) [Boguta et al., Mol. Cell. Biol. 12 (1992) 402-412]. The mt-r character of the NAM9 product is now confirmed by cross-reaction with the antisera for the Sc mt r-proteins. The NAM9-1 mutation, characterized previously as the nuclear suppressor of some ochre mt mit- mutants, is found to be a single nucleotide substitution changing Ser82 to Leu within the part of NAM9 corresponding to the S4 region involved in interaction with the 16S rRNA. This indicates that the mechanism of NAM9-1 suppression could be analogous to the suppression due to ram (ribosomal ambiguity) mutations in the Ec structural gene encoding r-protein S4. The NAM9-1 mutation leads also to defect in respiratory growth in the background of the wild-type mit+ genome.
Assuntos
Núcleo Celular/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Mitocôndrias/genética , Proteínas Nucleares , Proteínas Repressoras , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Supressão Genética , Sequência de Aminoácidos , Sequência de Bases , Compartimento Celular , Reações Cruzadas , Proteínas Fúngicas/imunologia , Dados de Sequência Molecular , Mutação , Proteínas Ribossômicas/imunologia , Homologia de Sequência de AminoácidosRESUMO
MOD5, a gene responsible for the modification of A37 to isopentenyl A37 of both cytosolic and mitochondrial tRNAs, encodes two isozymes. Initiation of translation at the first AUG of the MOD5 open reading frame generates delta 2-isopentenyl pyrophosphate:tRNA isopentanyl transferase I (IPPT-I), which is located predominantly, but not exclusively, in the mitochondria. Initiation of translation at a second AUG generates IPPT-II, which modifies cytoplasmic tRNA. IPPT-II is unable to target to mitochondria. The N-terminal sequence present in IPPT-I and absent in IPPT-II is therefore necessary for mitochondrial targeting. In these studies, we fused MOD5 sequences encoding N-terminal regions to genes encoding passenger proteins, pseudomature COXIV and dihydrofolate reductase, and studied the ability of these chimeric proteins to be imported into mitochondria both in vivo and in vitro. We found that the sequences necessary for mitochondrial import, amino acids 1 to 11, are not sufficient for efficient mitochondrial targeting and that at least some of the amino acids shared by IPPT-I and IPPT-II comprise part of the mitochondrial targeting information. We used indirect immunofluorescence and cell fractionation to locate the MOD5 isozymes in yeast. IPPT-I was found in two subcellular compartments: mitochondria and the cytosol. We also found that IPPT-II had two subcellular locations: nuclei and the cytosol. The nuclear location of this protein is surprising because the A37-->isopentenyl A37 modification had been predicted to occur in the cytoplasm. MOD5 is one of the first genes reported to encode isozymes found in three subcellular compartments.
Assuntos
Alquil e Aril Transferases , Núcleo Celular/enzimologia , Genes Fúngicos , Mitocôndrias/enzimologia , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Transferases/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Citosol/enzimologia , Isoenzimas/análise , Isoenzimas/biossíntese , Isoenzimas/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Fases de Leitura Aberta , Plasmídeos , Proteínas/análise , Proteínas/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae/genética , Transcrição Gênica , Transferases/análiseRESUMO
Two forms of Mod5p, a tRNA modification enzyme, are found in three intracellular compartments, mitochondria, cytoplasm and nucleus, but are encoded by a single MOD5 gene. The two forms of the enzyme, Mod5p-I and Mod5p-II differ at the N-termini and are produced by initiation of translation at different start codons. Mod5p-I does contain a mitochondrial targeting signal and is distributed between mitochondria and cytoplasm, whereas Mod5p-II is found in the cytosol and nucleus (Boguta, M., et al. 1994, Mol. Cell. Biol. 14, 2298-2306). In the present work mutants which mislocalize the Mod5p-I enzyme were isolated. The screen was based on a correlation between the amount of cytosolic protein and the efficiency of tRNA mediated suppression. Identification of mutants is possible because a red pigment accumulates in the cells unable to suppress an ade2-1 nonsense allele. The maf1 mutant, with an altered intracellular localization of the Mod5p-I protein, was isolated. Immunofluorescence data suggest that the mutation causes mislocalization of the Mod5p-I to the nucleus.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Peptídeos/genética , Saccharomyces cerevisiae/metabolismo , Compartimento Celular , Mutação , Peptídeos/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
We report the genetic characterization, molecular cloning, and sequencing of a novel nuclear suppressor, the NAM9 gene from Saccharomyces cerevisiae, which acts on mutations of mitochondrial DNA. The strain NAM9-1 was isolated as a respiration-competent revertant of a mitochondrial mit mutant which carries the V25 ochre mutation in the oxi1 gene. Genetic characterization of the NAM9-1 mutation has shown that it is a nuclear dominant omnipotent suppressor alleviating several mutations in all four mitochondrial genes tested and has suggested its informational, and probably ribosomal, character. The NAM9 gene was cloned by transformation of the recipient oxi1-V25 mutant to respiration competence by using a gene bank from the NAM9-1 rho o strain. Orthogonal-field alternation gel electrophoresis analysis and genetic mapping localized the NAM9 gene on the right arm of chromosome XIV. Sequence analysis of the NAM9 gene showed that it encodes a basic protein of 485 amino acids with a presequence that could target the protein to the mitochondrial matrix. The N-terminal sequence of 200 amino acids of the deduced NAM9 product strongly resembles the S4 ribosomal proteins from chloroplasts and bacteria. Significant although less extensive similarity was found with ribosomal cytoplasmic proteins from lower eucaryotes, including S. cerevisiae. Chromosomal inactivation of the NAM9+ gene is not lethal to the cell but leads to respiration deficiency and loss of mitochondrial DNA integrity. We conclude that the NAM9 gene product is a mitochondrial ribosomal counterpart of S4 ribosomal proteins found in other systems and that the suppressor acts through decreasing the fidelity of translation.
Assuntos
Proteínas Fúngicas/genética , Genes Supressores , Mitocôndrias/metabolismo , Proteínas Nucleares , Proteínas Repressoras , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Bactérias/genética , Sequência de Bases , Cloroplastos/metabolismo , Clonagem Molecular , DNA Fúngico , Eucariotos/genética , Dados de Sequência Molecular , Mutação , Plantas/genética , Saccharomyces cerevisiae/ultraestrutura , Alinhamento de Sequência , Transcrição Gênica , Transformação GenéticaRESUMO
nam3 and R705, yeast nuclear omnipotent suppressors of mitochondrial mit- mutations, reverse the superimposed spectrum of trans-recessive splicing defects by affecting the protein composition of the small mitoribosomal subunit. Analysis of the suppressor's interaction suggests that suppression results from mutations in the mitoribosomal polypeptides. These data indicate an obligatory connection between mitoribosome function and splicing of introns bI2, bI4 and aI1 in yeast mitochondria.
Assuntos
DNA Mitocondrial/genética , Genes Fúngicos , Mitocôndrias/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Supressão Genética , Genes , Mutação , Proteínas Ribossômicas/genéticaRESUMO
Phenotypic suppression by the antibiotic, paromomycin, of the mitochondrial oxi1- -V25 mutation, a mutation which arrests by premature ochre codon the synthesis of the cox II subunit, was studied in isolated yeast mitochondria competent in translation. This antibiotic is known to suppress the mutation in vivo (Dujardin et al. 1984) and allowed in vitro, at concentrations of 20-1100 micrograms per ml. the synthesis of the cox II subunit. This strongly suggests that phenotypic suppression of mit- mutations is due to the direct action of paromomycin on mitochondrial ribosomes. The effect of paromomycin bears a resemblance to the function of the omnipotent nuclear suppressor mutation R705. The nuclear suppression was expressed in isolated mitochondria; suppressor mutation influenced the structure of the mitoribosome. Therefore, it appears that mitoribosomes are indeed the common target in the phenotypical and genetic nuclear suppression of the oxi1-V25 mutation.