In eukaryotic cells, energy is produced by the coordinated action of the mitochondrial respiratory chain (MRC) and the oxidative phosphorylation system (OXPHOS). Cytochrome c oxidase (COX) is the fourth enzyme of the MRC. COX catalytic activity is mediated by prosthetic groups located in subunits 1 (Cox1) and 2. More than twenty nuclear encoded factors are required for the assembly of the functional enzyme. Cox1 is the center of a regulatory mechanism in which its protein levels depend on the availability of its assembly partners. A key element in the regulatory pathway is the nuclear encoded factor Mss51, a protein essential for COX1 mRNA translation and Cox1 stability. In this thesis work, we show that Mss51 performs these two functions by dynamically interacting with several protein partners, including COX assembly chaperones and the Hsp70 general chaperone Ssc1. We have also characterized functional domains in Mss51. Specifically, we are reporting the presence of two conserved CPX (Val,Leu) heme-binding motifs, essential for in vivo Mss51 functions. Our data supports a system in which the efficiency of Mss51 as a translational activator is regulated by heme levels perhaps in a redox-sensitive manner. This study contributes to the current knowledge and understanding of the COX assembly process by disclosing new mechanisms involved in its intricate regulation.

Uma das razões que fazem de Saccharomyces cerevisiae um organismo modelo é o grau de conservação dos mecanismos celulares que existe entre esta levedura e eucariotos superiores. Porém, mesmo após 21 anos do seqüenciamento do seu genoma, ainda existem mais de 600 ORFs com função desconhecida. Neste trabalho, selecionamos quatro delas para o estudo detalhado. MRPL34 (YDR115w) está presente na subunidade maior do ribossomo mitocondrial de levedura e apresenta similaridade com o gene L34 de E. coli e MRP-L34 de humanos. O mutante Δmrpl34 apresenta DNA mitocondrial (mtDNA) instável e para estudá-lo foram gerados alelos sensíveis à temperatura (ts). Com os ensaios de síntese protéica mitocondrial in vivo foi possível identificar clara diminuição da síntese de proteínas do mutante condicional. Mrpl34p foi identificada no extrato ribossomal, conforme esperado. A desestruturação da subunidade maior do ribossomo mitocondrial, utilizando os mutantes ts, nos forneceu indícios sobre intermediários existentes no seu processo de montagem. Verificamos que a porção N-terminal da proteína é responsável pelo endereçamento à mitocôndria. YPR116w também apresenta alta instabilidade do DNA mitocondrial, desta forma, mutantes termossensíveis foram utilizados nos experimentos. Uma das estratégias utilizadas visou a busca de parceiros genéticos. Verificamos que ylr091wp aumenta a estabilidade do mtDNA de ts- ypr116w, sugerindo atividade supressora. Também averiguamos que o alelo ts-ypr16w apresenta menor quantidade de tRNA mitocondrial, através de ensaios de Northen blot. Duas das ORFs escolhidas (YDL119c e YOR022c) tiveram sua caracterização inicial publicada em 2016, refletindo a importância deste tipo de pesquisa. Vimos que a proteína codificada por YDL119c está localizada na membrana interna da mitocôndria e que o mutante Δyor022c apresenta quantidades reduzidas de cardiolipina, quando crescido à 37ºC. / One of the reasons that turn Saccharomyces cerevisiae a model organism is the degree of conservation of cellular mechanisms that exist between this yeast and higher eukaryotes. However, even after 21 years of sequencing their genome, there are still more than 600 ORFs with unknown function. In this work, we selected four of them for the detailed study. MRPL34 (YDR115w) is present in the major subunit of the yeast mitochondrial ribosome and bears similarity to the L34 gene of E. coli and MRP-L34 from humans. The Δ mrpl34 mutant shows unstable mitochondrial DNA (mtDNA) and to study it, temperature sensitive alleles (ts) were generated. With the mitochondrial protein synthesis assays in vivo, it was possible to identify a clear decrease in the protein synthesis of the conditional mutant. Mrpl34p was identified in the ribosomal extract as expected. The disassembly of the major subunit of the mitochondrial ribosome, using the ts mutants, provided us some clues about intermediates in its assembly process. We have verified that the N-terminal portion of the protein is responsible for addressing the mitochondria. YPR116w also shows high mitochondrial DNA instability, in this way, thermosensitive mutants were used in the experiments. One of the strategies used was the search for genetic partners. We verified that ylr091wp increases the stability of ts-ypr116w mtDNA, suggesting suppressor activity. We also found that the ts-ypr16w allele has a smaller amount of mitochondrial tRNA, through Northen blot assays. Two of the chosen ORFs (YDL119c and YOR022c) had their initial characterization published in 2016, reflecting the importance of this type of research. We have seen that the protein encoded by YDL119c is located on the inner membrane of the mitochondria and that the Δyor022c mutant presents reduced amounts of cardiolipin when grown at 37 ºC.

Uma das razões que fazem de Saccharomyces cerevisiae um organismo modelo é o grau de conservação dos mecanismos celulares que existe entre esta levedura e eucariotos superiores. Porém, mesmo após 21 anos do seqüenciamento do seu genoma, ainda existem mais de 600 ORFs com função desconhecida. Neste trabalho, selecionamos quatro delas para o estudo detalhado. MRPL34 (YDR115w) está presente na subunidade maior do ribossomo mitocondrial de levedura e apresenta similaridade com o gene L34 de E. coli e MRP-L34 de humanos. O mutante Δmrpl34 apresenta DNA mitocondrial (mtDNA) instável e para estudá-lo foram gerados alelos sensíveis à temperatura (ts). Com os ensaios de síntese protéica mitocondrial in vivo foi possível identificar clara diminuição da síntese de proteínas do mutante condicional. Mrpl34p foi identificada no extrato ribossomal, conforme esperado. A desestruturação da subunidade maior do ribossomo mitocondrial, utilizando os mutantes ts, nos forneceu indícios sobre intermediários existentes no seu processo de montagem. Verificamos que a porção N-terminal da proteína é responsável pelo endereçamento à mitocôndria. YPR116w também apresenta alta instabilidade do DNA mitocondrial, desta forma, mutantes termossensíveis foram utilizados nos experimentos. Uma das estratégias utilizadas visou a busca de parceiros genéticos. Verificamos que ylr091wp aumenta a estabilidade do mtDNA de ts- ypr116w, sugerindo atividade supressora. Também averiguamos que o alelo ts-ypr16w apresenta menor quantidade de tRNA mitocondrial, através de ensaios de Northen blot. Duas das ORFs escolhidas (YDL119c e YOR022c) tiveram sua caracterização inicial publicada em 2016, refletindo a importância deste tipo de pesquisa. Vimos que a proteína codificada por YDL119c está localizada na membrana interna da mitocôndria e que o mutante Δyor022c apresenta quantidades reduzidas de cardiolipina, quando crescido à 37ºC. / One of the reasons that turn Saccharomyces cerevisiae a model organism is the degree of conservation of cellular mechanisms that exist between this yeast and higher eukaryotes. However, even after 21 years of sequencing their genome, there are still more than 600 ORFs with unknown function. In this work, we selected four of them for the detailed study. MRPL34 (YDR115w) is present in the major subunit of the yeast mitochondrial ribosome and bears similarity to the L34 gene of E. coli and MRP-L34 from humans. The Δ mrpl34 mutant shows unstable mitochondrial DNA (mtDNA) and to study it, temperature sensitive alleles (ts) were generated. With the mitochondrial protein synthesis assays in vivo, it was possible to identify a clear decrease in the protein synthesis of the conditional mutant. Mrpl34p was identified in the ribosomal extract as expected. The disassembly of the major subunit of the mitochondrial ribosome, using the ts mutants, provided us some clues about intermediates in its assembly process. We have verified that the N-terminal portion of the protein is responsible for addressing the mitochondria. YPR116w also shows high mitochondrial DNA instability, in this way, thermosensitive mutants were used in the experiments. One of the strategies used was the search for genetic partners. We verified that ylr091wp increases the stability of ts-ypr116w mtDNA, suggesting suppressor activity. We also found that the ts-ypr16w allele has a smaller amount of mitochondrial tRNA, through Northen blot assays. Two of the chosen ORFs (YDL119c and YOR022c) had their initial characterization published in 2016, reflecting the importance of this type of research. We have seen that the protein encoded by YDL119c is located on the inner membrane of the mitochondria and that the Δyor022c mutant presents reduced amounts of cardiolipin when grown at 37 ºC.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第21862号 / 人博第891号 / 新制||人||213(附属図書館) / 2018||人博||891(吉田南総合図書館) / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 林 達也, 教授 石原 昭彦, 教授 久代 恵介 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

No description available.

A mitocôndria é um componente essencial para a célula eucariótica, sendo que mutações que comprometam seu funcionamento podem causar as doenças mitocondriais. Estudos a respeito da biogênese mitocondrial para compreender seu funcionamento são importantes para que seja possível elaborar novas formas de tratamento. Saccharomyces cerevisiae é considerada o melhor modelo de estudo de biogênese mitocondrial. Neste trabalho, estudamos o gene nuclear MSC6, de S. cerevisiae, que foi capaz de suprimir a mutação dominante produzida no gene HER2/QRS1, um gene essencial no processo de tradução mitocondrial. A proteína codificada por MSC6 não tinha função conhecida. Verificamos sua presença na matriz mitocondrial, e que a sua ausência prejudica o processo respiratório. Também verificamos uma possível interação de Msc6p com Fmt1p, uma enzima envolvida no início do processo de tradução mitocondrial. Ficou clara a participação de Msc6p no processo traducional mitocondrial, mas novos estudos serão necessários para determinar sua função específica. / Mitochondria is necessary in many cellular processes, therefore, compromised mutations of its operation can cause severe damage to the cell, known as mitochondrial disorders. Thus is necessary the realization of mitochondrial biogenesis studies in order to fully understand its functioning in health and disease. Mitochondria biogenesis studies are favored in Saccharomyces cerevisiae. In this work, we have studied the MSC6 nuclear gene of S. cerevisiae that was able to suppress the HER2/QRS1 dominant mutant, another essential gene for the mitochondrial translation process. Msc6p has a PPR protein motif likely associated to RNA binding but with unknown function. We discovered that Msc6p is localized in the mitochondrial matrix, also that disruption of MSC6 implies in respiratory. We also find a possible interaction between Msc6p and Fmt1p, an enzyme required for mitochondrial translation initiation. In conclusion, is clear the role of MSC6 in the mitochondrial translational process, but further studies are required to indicate the specific function of Msc6p.

This thesis is composed of two topics, for which trypanosomatids and evolution are common denominators. First part deals with phylogenetic relationships among monoxenous trypanosomatids, with emphasis on flagellates parasitizing dipteran hosts, analyzed mainly from biogeographical and evolutionary perspectives. Second part focuses on the trypanosomatid Trypanosoma brucei, causative agent of severe diseases, which serves as a model organism for functional studies of evolutionary conserved mitochondrial proteins, in particular those involved in replication, maintenance and expression of the mitochondrial genome, also termed the kinetoplast. This thesis identified the mtHsp70/mtHsp40 chaperone machinery as an essential component of replication and maintenance of the kinetoplast, and also identified numerous conditions under which mtHsp70 has a tendency to aggregate. Moreover, several conserved proteins, previously identified to be part of the mitochondrial ribosome, were shown to be important for translation of the mitochondrial transcripts.

A mitocôndria é um componente essencial para a célula eucariótica, sendo que mutações que comprometam seu funcionamento podem causar as doenças mitocondriais. Estudos a respeito da biogênese mitocondrial para compreender seu funcionamento são importantes para que seja possível elaborar novas formas de tratamento. Saccharomyces cerevisiae é considerada o melhor modelo de estudo de biogênese mitocondrial. Neste trabalho, estudamos o gene nuclear MSC6, de S. cerevisiae, que foi capaz de suprimir a mutação dominante produzida no gene HER2/QRS1, um gene essencial no processo de tradução mitocondrial. A proteína codificada por MSC6 não tinha função conhecida. Verificamos sua presença na matriz mitocondrial, e que a sua ausência prejudica o processo respiratório. Também verificamos uma possível interação de Msc6p com Fmt1p, uma enzima envolvida no início do processo de tradução mitocondrial. Ficou clara a participação de Msc6p no processo traducional mitocondrial, mas novos estudos serão necessários para determinar sua função específica. / Mitochondria is necessary in many cellular processes, therefore, compromised mutations of its operation can cause severe damage to the cell, known as mitochondrial disorders. Thus is necessary the realization of mitochondrial biogenesis studies in order to fully understand its functioning in health and disease. Mitochondria biogenesis studies are favored in Saccharomyces cerevisiae. In this work, we have studied the MSC6 nuclear gene of S. cerevisiae that was able to suppress the HER2/QRS1 dominant mutant, another essential gene for the mitochondrial translation process. Msc6p has a PPR protein motif likely associated to RNA binding but with unknown function. We discovered that Msc6p is localized in the mitochondrial matrix, also that disruption of MSC6 implies in respiratory. We also find a possible interaction between Msc6p and Fmt1p, an enzyme required for mitochondrial translation initiation. In conclusion, is clear the role of MSC6 in the mitochondrial translational process, but further studies are required to indicate the specific function of Msc6p.

Molecular association is part of many important processes in living cells. Computational methods for calculating binding free energies allows for a quantitative examination of biomolecular structures and hypotheses drawn from biochemical experiments. Here, binding free energy calculations for tRNAs and release factors binding to mRNA codons on the ribosome, sugars binding to lectins and purine analogs binding to the purine riboswitch are presented. The relative affinities between cognate and non-cognate tRNAs for different states involved in codon reading on the ribosome were determined. The calculations show that tRNA discrimination varies between different conformations of the 30S subunit, where the existence of both low and high selectivity states provides an efficient common mechanism for initial selection and proofreading. The simulations reveal a desolvation mechanism for the 30S conformational switch with which the accuracy of peptide bond formation can be amplified. When an mRNA stop codon (UAA, UAG or UGA) is located in the ribosomal A-site release factors bind to the ribosome and the synthesized protein is released. RF1 is specific for UAA and UAG whereas RF2 is specific for UAA and UGA. The free energy calculations and an analysis of the performed simulations show the mechanisms for how RF1 and RF2 are able to read the stop codons with different specificities. Also mitochondrial release factors were investigated. Vertebrate mitochondria have four stop codons, UAA, UAG, AGA and AGG and two release factors mtRF1 and mtRF1a. The calculations show how the specificities of both mtRF1 and mtRF1a agree with RF1 and that none of them are likely to read the non-standard stop codons AGA and AGG. The linear interaction energy method has also been examined for the RSL and PA-IIL lectins and for the purine riboswitch. The standard parameterization of the method works well for RSL, but fails for PA-IIL and the purine riboswitch due to compositions of the active sites in these systems. The development of new parameterizations to overcome these problems leads to a better understanding of both the method and the binding mechanisms in these systems.

This thesis comprises of diverse projects all focused towards analysis of mitochondrial translation in unicellular parasites. As only two mitochondrially encoded genes are required during the life cycle stage when Trypanosoma brucei resides in the bloodstream of a mammalian host, this protist provides a simplified background in which to study mitochondrial translation termination phase. The leading project utilizes T. brucei to examine mitochondrial translation termination factor TbMrf1 by gene knockout. Subsequently, it is suggested that the peptidyl-tRNA hydrolase TbPth4 is able to abate the TbMrf1 knockout phenotype by its ability to rescue mitoribosomes that become stalled when TbMrf1 is absent. Additionally, modifying methyltransferase of TbMrf1, the TbMTQ1, was characterized. And finally, this work contributed to the development of the protein expression regulation method in Leishmania parasites, a protocol for measurement of proton pumping activity of FoF1 ATPase complex in native mitochondria, and optimization of purification protocol for hydrophobic recombinant proteins.