RESUMO
The insertion of genes into mitochondria by biolistic transformation is currently only possible in the yeast Saccharomyces cerevisiae and the algae Chlamydomonas reinhardtii. The fact that S. cerevisiae mitochondria can exist with partial (ρ- mutants) or complete deletions (ρ0 mutants) of mitochondrial DNA (mtDNA), without requiring a specific origin of replication, enables the propagation of exogenous sequences. Additionally, mtDNA in this organism undergoes efficient homologous recombination, making it well-suited for genetic manipulation. In this review, we present a summarized historical overview of the development of biolistic transformation and discuss iconic applications of the technique. We also provide a detailed example on how to obtain transformants with recombined foreign DNA in their mitochondrial genome.
Assuntos
DNA Mitocondrial , Saccharomyces cerevisiae , DNA Mitocondrial/genética , Saccharomyces cerevisiae/genética , Biolística/métodos , Transformação Genética , Mitocôndrias/genéticaRESUMO
Mitochondrial translation is a highly regulated process, and newly synthesized mitochondrial products must first associate with several nuclear-encoded auxiliary factors to form oxidative phosphorylation complexes. The output of mitochondrial products should therefore be in stoichiometric equilibrium with the nuclear-encoded products to prevent unnecessary energy expense or the accumulation of pro-oxidant assembly modules. In the mitochondrial DNA of Saccharomyces cerevisiae, COX1 encodes subunit 1 of the cytochrome c oxidase and COB the central core of the cytochrome bc1 electron transfer complex; however, factors regulating the expression of these mitochondrial products are not completely described. Here, we identified Mrx9p as a new factor that controls COX1 and COB expression. We isolated MRX9 in a screen for mitochondrial factors that cause poor accumulation of newly synthesized Cox1p and compromised transition to the respiratory metabolism. Northern analyses indicated lower levels of COX1 and COB mature mRNAs accompanied by an accumulation of unprocessed transcripts in the presence of excess Mrx9p. In a strain devoid of mitochondrial introns, MRX9 overexpression did not affect COX1 and COB translation or respiratory adaptation, implying Mrx9p regulates processing of COX1 and COB RNAs. In addition, we found Mrx9p was localized in the mitochondrial inner membrane, facing the matrix, as a portion of it cosedimented with mitoribosome subunits and its removal or overexpression altered Mss51p sedimentation. Finally, we showed accumulation of newly synthesized Cox1p in the absence of Mrx9p was diminished in cox14 null mutants. Taken together, these data indicate a regulatory role of Mrx9p in COX1 RNA processing.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In Saccharomyces cerevisiae, mitoribosomes are composed of a 54S large subunit (mtLSU) and a 37S small subunit (mtSSU). The two subunits altogether contain 73 mitoribosome proteins (MRPs) and two ribosomal RNAs (rRNAs). Although mitoribosomes preserve some similarities with their bacterial counterparts, they have significantly diverged by acquiring new proteins, protein extensions, and new RNA segments, adapting the mitoribosome to the synthesis of highly hydrophobic membrane proteins. In this study, we investigated the functional relevance of mitochondria-specific protein extensions at the C-terminus (C) or N-terminus (N) present in 19 proteins of the mtLSU. The studied mitochondria-specific extensions consist of long tails and loops extending from globular domains that mainly interact with mitochondria-specific proteins and 21S rRNA moieties extensions. The expression of variants devoid of extensions in uL4 (C), uL5 (N), uL13 (N), uL13 (C), uL16 (C), bL17 (N), bL17 (C), bL21 (24), uL22 (N), uL23 (N), uL23 (C), uL24 (C), bL27 (C), bL28 (N), bL28 (C), uL29 (N), uL29 (C), uL30 (C), bL31 (C), and bL32 (C) did not rescue the mitochondrial protein synthesis capacities and respiratory growth of the respective null mutants. On the contrary, the truncated form of the mitoribosome exit tunnel protein uL24 (N) yields a partially functional mitoribosome. Also, the removal of mitochondria-specific sequences from uL1 (N), uL3 (N), uL16 (N), bL9 (N), bL19 (C), uL29 (C), and bL31 (N) did not affect the mitoribosome function and respiratory growth. The collection of mutants described here provides new means to study and evaluate defective assembly modules in the mitoribosome biogenesis process.
Assuntos
Mitocôndrias , Ribossomos Mitocondriais , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Ribossomos Mitocondriais/química , Ribossomos Mitocondriais/metabolismo , Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Coenzyme Q (CoQ) is an essential molecule that consists of a highly substituted benzene ring attached to a polyprenyl tail anchored in the inner mitochondrial membrane. CoQ transfers electrons from NADH dehydrogenase and succinate dehydrogenase complexes toward ubiquinol-cytochrome c reductase, and that allows aerobic growth of cells. In Saccharomyces cerevisiae, the synthesis of CoQ depends on fourteen proteins Coq1p-Co11p, Yah1p, Arh1p, and Hfd1p. Some of these proteins are components of CoQ synthome. Using ab initio molecular modeling and site-directed mutagenesis, we identified the functional residues of the O-methyltransferase Coq3p, which depends on S-adenosylmethionine for catalysis and is necessary for two O-methylation steps required for CoQ maturation. Conserved residues as well as those that coevolved in the protein structure were found to have important roles in respiratory growth, CoQ biosynthesis, and also in the stability of CoQ synthome proteins. Finally, a multiple sequence alignment showed that S. cerevisiae Coq3p has a 45 amino acid residues insertion that is poorly conserved or absent in oleaginous yeast, cells that can store up to 20% of their dry weight as lipids. These results point to the Coq3p structural determinants of its biological and catalytic function and could contribute to the development of lipid-producing yeast for biotechnology.
Assuntos
Metiltransferases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Membranas Mitocondriais , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The ease with which the unicellular yeast Saccharomyces cerevisiae can be manipulated genetically and biochemically has established this organism as a good model for the study of human mitochondrial diseases. The combined use of biochemical and molecular genetic tools has been instrumental in elucidating the functions of numerous yeast nuclear gene products with human homologs that affect a large number of metabolic and biological processes, including those housed in mitochondria. These include structural and catalytic subunits of enzymes and protein factors that impinge on the biogenesis of the respiratory chain. This article will review what is currently known about the genetics and clinical phenotypes of mitochondrial diseases of the respiratory chain and ATP synthase, with special emphasis on the contribution of information gained from pet mutants with mutations in nuclear genes that impair mitochondrial respiration. Our intent is to provide the yeast mitochondrial specialist with basic knowledge of human mitochondrial pathologies and the human specialist with information on how genes that directly and indirectly affect respiration were identified and characterized in yeast.
RESUMO
Mitoribosome biogenesis is an expensive metabolic process that is essential to maintain cellular respiratory capacity and requires the stoichiometric accumulation of rRNAs and proteins encoded in two distinct genomes. In yeast, the ribosomal protein Var1, alias uS3m, is mitochondrion-encoded. uS3m is a protein universally present in all ribosomes, where it forms part of the small subunit (SSU) mRNA entry channel and plays a pivotal role in ribosome loading onto the mRNA. However, despite its critical functional role, very little is known concerning VAR1 gene expression. Here, we demonstrate that the protein Sov1 is an in bona fide VAR1 mRNA translational activator and additionally interacts with newly synthesized Var1 polypeptide. Moreover, we show that Sov1 assists the late steps of mtSSU biogenesis involving the incorporation of Var1, an event necessary for uS14 and mS46 assembly. Notably, we have uncovered a translational regulatory mechanism by which Sov1 fine-tunes Var1 synthesis with its assembly into the mitoribosome.
Assuntos
Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Ribossomos Mitocondriais/metabolismo , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Mitocondrial/genética , Regulação Fúngica da Expressão Gênica/genética , RNA Mensageiro/genéticaRESUMO
Coenzyme Q (Q n ) is a vital lipid component of the electron transport chain that functions in cellular energy metabolism and as a membrane antioxidant. In the yeast Saccharomyces cerevisiae, coq1-coq9 deletion mutants are respiratory-incompetent, sensitive to lipid peroxidation stress, and unable to synthesize Q6 The yeast coq10 deletion mutant is also respiratory-deficient and sensitive to lipid peroxidation, yet it continues to produce Q6 at an impaired rate. Thus, Coq10 is required for the function of Q6 in respiration and as an antioxidant and is believed to chaperone Q6 from its site of synthesis to the respiratory complexes. In several fungi, Coq10 is encoded as a fusion polypeptide with Coq11, a recently identified protein of unknown function required for efficient Q6 biosynthesis. Because "fused" proteins are often involved in similar biochemical pathways, here we examined the putative functional relationship between Coq10 and Coq11 in yeast. We used plate growth and Seahorse assays and LC-MS/MS analysis to show that COQ11 deletion rescues respiratory deficiency, sensitivity to lipid peroxidation, and decreased Q6 biosynthesis of the coq10Δ mutant. Additionally, immunoblotting indicated that yeast coq11Δ mutants accumulate increased amounts of certain Coq polypeptides and display a stabilized CoQ synthome. These effects suggest that Coq11 modulates Q6 biosynthesis and that its absence increases mitochondrial Q6 content in the coq10Δcoq11Δ double mutant. This augmented mitochondrial Q6 content counteracts the respiratory deficiency and lipid peroxidation sensitivity phenotypes of the coq10Δ mutant. This study further clarifies the intricate connection between Q6 biosynthesis, trafficking, and function in mitochondrial metabolism.
Assuntos
Deleção de Genes , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Ubiquinona/análogos & derivados , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Mitocôndrias/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Ubiquinona/biossíntese , Ubiquinona/deficiência , Ubiquinona/genética , Ubiquinona/metabolismoRESUMO
Mitochondrial function relies on the activity of oxidative phosphorylation to synthesise ATP and generate an electrochemical gradient across the inner mitochondrial membrane. These coupled processes are mediated by five multi-subunit complexes that reside in this inner membrane. These complexes are the product of both nuclear and mitochondrial gene products. Defects in the function or assembly of these complexes can lead to mitochondrial diseases due to deficits in energy production and mitochondrial functions. Appropriate biogenesis and function are mediated by a complex number of assembly factors that promote maturation of specific complex subunits to form the active oxidative phosphorylation complex. The understanding of the biogenesis of each complex has been informed by studies in both simple eukaryotes such as Saccharomyces cerevisiae and human patients with mitochondrial diseases. These studies reveal each complex assembles through a pathway using specific subunits and assembly factors to form kinetically distinct but related assembly modules. The current understanding of these complexes has embraced the revolutions in genomics and proteomics to further our knowledge on the impact of mitochondrial biology in genetics, medicine, and evolution.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Mamíferos , Mitocôndrias/metabolismo , LevedurasRESUMO
In addition to autophagy, proteasomes are critical for regulating intracellular protein levels and removing misfolded proteins. The 20S proteasome (20SPT), the central catalytic unit, is sometimes flanked by regulatory units at one or both ends. Additionally, proteosomal activation has been associated with increased lifespan in many organisms. Our group previously reported that the gating (open/closed) of the free 20S proteasome is redox controlled, and that S-glutathionylation of two Cys residues (Cys76 and Cys221) in the α5 subunit promotes gate opening. The present study constructed site-directed mutants of these Cys residues, and evaluated the effects these mutations have on proteosome gate opening and yeast cell survival. Notably, the double mutation of both Cys residues (Cys76 and Cys221) rendered the cells nonviable, whereas the lifespan of the yeast carrying the single mutations (α5-C76S or α5-C221S) was attenuated when compared to the wild type counterpart. Furthermore, it was found that α5-C76S or α5-C221S 20SPT were more likely to be found with the gate in a closed conformation. In contrast, a random α5-subunit double mutation (S35P/C221S) promoted gate opening, increased chronological lifespan and provided resistance to oxidative stress. The 20SPT core particle purified from the long-lived strain degraded model proteins (e.g., α-synuclein) more efficiently than preparations obtained from the wild-type counterpart, and also displayed an increased chymotrypsin-like activity. Mass spectrometric analyses of the C76S, C221S, S35P/C221S, S35P and S35P/C76S mutants provided evidence that the highly conserved Cys76 residue of the α5-subunit is the key determinant for gate opening and cellular survival. The present study reveals a sophisticated regulatory mechanism that controls gate opening, which appears to be based on the interactions among multiple residues within the α5-subunit, and consequently impacts the lifespan of yeast.
Assuntos
Cisteína/genética , Longevidade , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Serina/genética , Glutationa/metabolismo , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Proteólise , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Mitochondrial translation normally requires formylation of the initiator tRNA-met, a reaction catalyzed by the enzyme formyltransferase, Fmt1p and MTFMT in Saccharomyces cerevisiae and human mitochondria, respectively. Yeast fmt1 mutants devoid of Fmt1p, however, can synthesize all mitochondrial gene products by initiating translation with a non-formylated methionyl-tRNA. Yeast synthetic respiratory-deficient fmt1 mutants have uncovered several factors suggested to play a role in translation initiation with non-formylated methionyl-tRNA. Here, we present evidence that Msc6p, a member of the pentatricopeptide repeat (PPR) motif family, is another essential factor for mitochondrial translation in fmt1 mutants. The PPR motif is characteristic of RNA-binding proteins found in chloroplasts and plant and fungal mitochondria, and is generally involved in RNA stability and transport. Moreover, in the present study, we show that the respiratory deficiency of fmt1msc6 double mutants can be rescued by overexpression of the yeast mitochondrial initiation factor mIF-2, encoded by IFM1. The role of Msc6p in translational initiation is further supported by pull-down assays showing that it transiently interacts with mIF-2. Altogether, our data indicate that Msc6p is an important factor in mitochondrial translation with an auxiliary function related to the mIF-2-dependent formation of the initiation complex.
Assuntos
Mitocôndrias/metabolismo , Iniciação Traducional da Cadeia Peptídica , Processamento de Proteína Pós-Traducional , RNA de Transferência de Metionina , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Mitochondrial tRNAs are processed at their 5'ends by highly divergent but ubiquitous RNase P. In Saccharomyces cerevisiae, Rpm2p is the protein component of RNase P. Here, we identify four novel genes MTA1, MTA2, GEP5 and PET130 of the Saccharomycetaceae family that are necessary for an efficient processing of mitochondrial tRNAs. Null mutants of mta1, mta2 and gep5 have severely reduced levels of mitochondrial tRNAs; in addition, temperature sensitive (ts) mutants of mta1, mta2, pet130 and gep5 accumulated tRNAs precursor transcripts at the restrictive but not at the permissive temperature. The same mitochondrial tRNAs precursors were also identified in rpm2 ts mutants or in the double ts mutants mta1 rpm2 and mta2 rpm2. The genetic and physical association of these four novel genes corroborate the hypothesis that they have their function associated. Different combinations of mta1, mta2, pet130 and gep5 ts alleles display a synthetic respiratory deficient phenotype, an indication of genetic interactions of the genes. Indeed, Mta1p, Mta2p, Pet130p, and Gep5p are associated with the mitochondrial inner membrane and are all extracted and sediment in sucrose gradients as high molecular weight complexes, where they may be present in a common complex with Rpm2p. This is supported by pull-down assays showing co-immunopurification of Rpm2 with Mta1p.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , RNA Fúngico/biossíntese , RNA Mitocondrial/biossíntese , RNA de Transferência/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , RNA Fúngico/genética , RNA Mitocondrial/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
AMP-activated protein kinase (AMPK) regulates many different metabolic pathways in eukaryote cells including mitochondria biogenesis and energy homeostasis. Here we identify a patient with hypotonia, weakness, delayed milestones and neurological impairment since birth harbouring a novel homozygous mutation in the AMPK catalytic α-subunit 1, encoded by the PRKAA1 gene. The homozygous mutation p.S487L in isoform 1 present in the patient is in a cryptic residue for AMPK activity. In the present study, we performed the characterization of mitochondrial respiratory properties of the patient, in comparison to healthy controls, through the culture of skin fibroblasts in order to understand some of the cellular consequences of the PRKAA1 mutation. In these assays, mitochondrial respiratory complex I showed lower activity, which was followed by a decrement in the mtDNA copy number, which is a probable consequence of the lower expression of PGC-1α and PRKAA1 itself as measured in our quantitative PCRs experiments. Confirming the effect of the patient mutation in respiration, transfection of patient fibroblasts with wild type PRKAA1 partially restore complex I level. The preliminary clinic evaluations of the patient suggested a metabolic defect related to the mitochondrial respiratory function, therefore treatment with CoQ10 supplementation dose started four years ago and a clear improvement in motor skills and strength has been achieved with this treatment.
Assuntos
Proteínas Quinases Ativadas por AMP , Fibroblastos , Homozigoto , Mitocôndrias , Mutação de Sentido Incorreto , Consumo de Oxigênio , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Substituição de Aminoácidos , Pré-Escolar , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismoRESUMO
Saccharomyces cerevisiae mitoribosomes are specialized in the translation of a few number of highly hydrophobic membrane proteins, components of the oxidative phosphorylation system. Mitochondrial characteristics, such as the membrane system and its redox state driven mitoribosomes evolution through great diversion from their bacterial and cytosolic counterparts. Therefore, mitoribosome presents a considerable number of mitochondrial-specific proteins, as well as new protein extensions. In this work we characterize temperature sensitive mutants of the subunit bL34 present in the 54S large subunit. Although bL34 has bacterial homologs, in yeast it has a long 65 aminoacids mitochondrial N-terminal addressing sequence, here we demonstrate that it can be replaced by the mitochondrial addressing sequence of Neurospora crassa ATP9 gene. The bL34 temperature sensitive mutants present lowered translation of mitochondrial COX1 and COX3, which resulted in reduced cytochrome c oxidase activity and respiratory growth deficiency. The sedimentation properties of bL34 in sucrose gradients suggest that similarly to its bacterial homolog, bL34 is also a later participant in the process of mitoribosome biogenesis.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Ribossomos Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutagênese Sítio-Dirigida , Biossíntese de Proteínas , Proteínas RGS/genética , Proteínas RGS/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
One of the hallmarks of Parkinson disease is α-synuclein aggregate deposition that leads to endoplasmic reticulum stress, Golgi fragmentation and impaired energy metabolism with consequent redox imbalance. In the last decade, many studies have used Saccharomyces cerevisiae as a model in order to explore the intracellular consequences of α-synuclein overexpression. In this study we propose to evaluate the respiratory outcome of yeast cells expressing α-synuclein. Cell viability or growth on selective media for respiratory activity was mainly affected in the α-synuclein-expressing cells if they were also treated with menadione, which stimulates reactive oxygen species production. We also tested whether melatonin, a natural antioxidant, would counteract the deleterious effects of α-synuclein and menadione. In fact, melatonin addition improved the respiratory growth of α-synuclein/menadione-challenged cells, presented a general improvement in the enzymatic activity of the respiratory complexes and finally elevated the rate of mitophagy, an important cellular process necessary for the clearance of damaged mitochondria. Altogether, our data confirms that α-synuclein impairs respiration in yeast, which can be rescued by melatonin addition.
Assuntos
Melatonina/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Vitamina K 3/farmacologia , alfa-Sinucleína/farmacologia , Sobrevivência Celular , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Consumo de Oxigênio/fisiologiaRESUMO
Yeast Prx1 is a mitochondrial 1-Cys peroxiredoxin that catalyzes the reduction of endogenously generated H2O2 Prx1 is synthesized on cytosolic ribosomes as a preprotein with a cleavable N-terminal presequence that is the mitochondrial targeting signal, but the mechanisms underlying Prx1 distribution to distinct mitochondrial subcompartments are unknown. Here, we provide direct evidence of the following dual mitochondrial localization of Prx1: a soluble form in the intermembrane space and a form in the matrix weakly associated with the inner mitochondrial membrane. We show that Prx1 sorting into the intermembrane space likely involves the release of the protein precursor within the lipid bilayer of the inner membrane, followed by cleavage by the inner membrane peptidase. We also found that during its import into the matrix compartment, Prx1 is sequentially cleaved by mitochondrial processing peptidase and then by octapeptidyl aminopeptidase 1 (Oct1). Oct1 cleaved eight amino acid residues from the N-terminal region of Prx1 inside the matrix, without interfering with its peroxidase activity in vitro Remarkably, the processing of peroxiredoxin (Prx) proteins by Oct1 appears to be an evolutionarily conserved process because yeast Oct1 could cleave the human mitochondrial peroxiredoxin Prx3 when expressed in Saccharomyces cerevisiae Altogether, the processing of peroxiredoxins by Imp2 or Oct1 likely represents systems that control the localization of Prxs into distinct compartments and thereby contribute to various mitochondrial redox processes.
Assuntos
Metaloproteases/metabolismo , Mitocôndrias/enzimologia , Peroxidases/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Humanos , Metaloproteases/genética , Mitocôndrias/genética , Peroxidases/genética , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Translation of mitochondrial gene products in Saccharomyces cerevisiae depends on mRNA-specific activators that bind to the 5' untranslated regions and promote translation on mitochondrial ribosomes. Here we find that Aep3p, previously shown to stabilize the bicistronic ATP8-ATP6 mRNA and facilitate initiation of translation from unformylated methionine, also activates specifically translation of ATP8 This is supported by several lines of evidence. Temperature-sensitive aep3 mutants are selectively blocked in incorporating [35S]methionine into Atp8p at nonpermissive but not at the permissive temperature. This phenotype is not a consequence of defective transcription or processing of the pre-mRNA. Neither is it explained by turnover of Aep3p, as evidenced by the failure of aep3 mutants to express a recoded ARG8m when this normally nuclear gene is substituted for ATP8 in mitochondrial DNA. Finally, translational of ATP8 mRNA in aep3 mutants is partially rescued by recoded allotopic ATP8 (nATP8) in a high-expression plasmid or in a CEN plasmid in the presence of recessive mutations in genes involved in stability and polyadenylation of RNA.
Assuntos
Proteínas de Membrana/genética , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiões 5' não Traduzidas/genética , DNA Mitocondrial/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras , Mutação , Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Identification of human papillomavirus (HPV) association in head and neck squamous cell carcinoma (HNSCC) is important to identify patients with favorable disease course. However, molecular HPV detection is not universally available. p16 has been proposed as a surrogate marker for HPV infection in HNSCC but, use on its own may result in wrong assignment of some cases to the group of HPV-associated tumors. We have therefore studied 424 HNSCC cases with known p16 and HPV DNA polymerase chain reaction (PCR) status for expression of retinoblastoma protein (pRb) and CyclinD1 by immunohistochemistry using 6-tiered scales (0 to 5) and a combined score (0 to 10). Sixty-one of 424 cases showed overexpression of p16. Of these, 52 cases were HPV DNA-PCR-positive. HPV association strongly correlated with low expression scores for pRb and CyclinD1 individually (scores ≤2) or combined (score sum ≤4), whereas HPV-negative carcinomas showed widely distributed expression scores. High expression scores for pRb or for pRb/CyclinD1 were observed exclusively in HPV DNA-PCR-negative cases. Three of 9 p16-positive/HPV DNA-PCR-negative cases showed high expression of pRb and displayed a high combined pRb/CyclinD1 score. We conclude that HPV-positive HNSCC are characterized by p16 overexpression and low scores for pRb, CyclinD1, and a low combined pRb/CyclinD1 score. High pRb or combined pRb/CyclinD1 scores are strong indicators for HPV-negativity and may justify excluding these cases from further molecular HPV testing. Furthermore p16-positive/HPV DNA-PCR-negative cases show heterogeneous expression of pRb and CyclinD1, including high pRb or high combined pRb/CyclinD1 scores suggesting that at least some of these cases are truly HPV negative.
Assuntos
Carcinoma de Células Escamosas/complicações , Carcinoma de Células Escamosas/diagnóstico , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Neoplasias de Cabeça e Pescoço/complicações , Neoplasias de Cabeça e Pescoço/diagnóstico , Infecções por Papillomavirus/complicações , Proteína do Retinoblastoma/metabolismo , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Imuno-Histoquímica , Masculino , Papillomaviridae/fisiologia , Infecções por Papillomavirus/diagnóstico , Valor Preditivo dos Testes , Proteína do Retinoblastoma/genética , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Recently, a large body of evidences indicates the existence in the mitochondrial matrix of foci that contain different proteins involved in mitochondrial RNA metabolism. Some of these proteins have a pentatricopeptide repeat motif that constitutes their RNA-binding structures. Here we report that MSC6, a mitochondrial pentatricopeptide protein of unknown function, is a multi copy suppressor of mutations in QRS1/HER2 a component of the trimeric complex that catalyzes the transamidation of glutamyl-tRNAQ to glutaminyl-tRNAQ. This is an essential step in mitochondrial translation because of the lack of a specific mitochondrial aminoacyl glutaminyl-tRNA synthetase. MSC6 over-expression did not abolish translation of an aberrant variant form of Cox2p detected in QRS1/HER2 mutants, arguing against a suppression mechanism that bypasses Qrs1p function. A slight decrement of the mitochondrial translation capacity as well as diminished growth on respiratory carbon sources media for respiratory activity was observed in the msc6 null mutant. Additionally, the msc6 null mutant did not display any impairment in RNA transcription, processing or turnover. We concluded that Msc6p is a mitochondrial matrix protein and further studies are required to indicate the specific function of Msc6p in mitochondrial translation.
Assuntos
Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons , Expressão Gênica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Alelos , Aminoacil-tRNA Sintetases/química , Regulação Fúngica da Expressão Gênica , Genótipo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Transcrição GênicaRESUMO
Coenzyme Q (Q) is an isoprenylated benzoquinone electron carrier required for electronic transport in the mitochondrial respiratory chain, shuttling electrons from complexes I and II to complex III. Q synthesis requires proteins termed Coq (Coq1-Coq11). Coq7p is part of the multimeric complex involved in Q synthesis catalyzing the hydroxylation of demethoxy-Q6 (DMQ6), the last monooxygenase step in Q synthesis with a catalytic center containing a carboxylate-bridged di-iron at the active site of the enzyme. Here we indicate a group of Coq7p residues that modulate protein activity: D53, R57, V111 and S114. R57, V111 and S114 are very conserved residues; V111 and S114 are present in separated communities of amino acid correlation analysis. The coq7 double mutant V111G/S114A and S114E show respiratory deficiency at non permissive temperature, DMQ6 accumulation and lower content of Q6. Therefore we conclude that phosphomimetic S114E inhibit Coq7p activity, and propose that S114 phosphorylation is required to move a non-structured loop of 25 amino acids between helix 2 and 3, and that affects the di-iron coordination in Coq7p catalytic center.