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1.
Elife ; 132024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38363119

RESUMO

The mitochondrial genomes of apicomplexans comprise merely three protein-coding genes, alongside a set of thirty to forty genes encoding small RNAs (sRNAs), many of which exhibit homologies to rRNA from E. coli. The expression status and integration of these short RNAs into ribosomes remains unclear and direct evidence for active ribosomes within apicomplexan mitochondria is still lacking. In this study, we conducted small RNA sequencing on the apicomplexan Toxoplasma gondii to investigate the occurrence and function of mitochondrial sRNAs. To enhance the analysis of sRNA sequencing outcomes, we also re-sequenced the T. gondii mitochondrial genome using an improved organelle enrichment protocol and Nanopore sequencing. It has been established previously that the T. gondii genome comprises 21 sequence blocks that undergo recombination among themselves but that their order is not entirely random. The enhanced coverage of the mitochondrial genome allowed us to characterize block combinations at increased resolution. Employing this refined genome for sRNA mapping, we find that many small RNAs originated from the junction sites between protein-coding blocks and rRNA sequence blocks. Surprisingly, such block border sRNAs were incorporated into polysomes together with canonical rRNA fragments and mRNAs. In conclusion, apicomplexan ribosomes are active within polysomes and are indeed assembled through the integration of sRNAs, including previously undetected sRNAs with merged mRNA-rRNA sequences. Our findings lead to the hypothesis that T. gondii's block-based genome organization enables the dual utilization of mitochondrial sequences as both messenger RNAs and ribosomal RNAs, potentially establishing a link between the regulation of rRNA and mRNA expression.


Assuntos
Genoma Mitocondrial , Pequeno RNA não Traduzido , Ribossomos Mitocondriais/metabolismo , Escherichia coli/genética , RNA Ribossômico/metabolismo , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Recombinação Genética
2.
Nat Commun ; 14(1): 7991, 2023 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-38042949

RESUMO

Mitochondria contain their own genetic information and a dedicated translation system to express it. The mitochondrial ribosome is assembled from mitochondrial-encoded RNA and nuclear-encoded ribosomal proteins. Assembly is coordinated in the mitochondrial matrix by biogenesis factors that transiently associate with the maturing particle. Here, we present a structural snapshot of a large mitoribosomal subunit assembly intermediate containing 7 biogenesis factors including the GTPases GTPBP7 and GTPBP10. Our structure illustrates how GTPBP10 aids the folding of the ribosomal RNA during the biogenesis process, how this process is related to bacterial ribosome biogenesis, and why mitochondria require two biogenesis factors in contrast to only one in bacteria.


Assuntos
Ribossomos Mitocondriais , RNA , Ribossomos Mitocondriais/metabolismo , RNA/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo
3.
Nat Commun ; 14(1): 7217, 2023 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-37940635

RESUMO

Cellular activities are commonly associated with dynamic proteomic changes at the subcellular level. Although several techniques are available to quantify whole-cell protein turnover dynamics, such measurements often lack sufficient spatial resolution at the subcellular level. Herein, we report the development of prox-SILAC method that combines proximity-dependent protein labeling (APEX2/HRP) with metabolic incorporation of stable isotopes (pulse-SILAC) to map newly synthesized proteins with subcellular spatial resolution. We apply prox-SILAC to investigate proteome dynamics in the mitochondrial matrix and the endoplasmic reticulum (ER) lumen. Our analysis reveals a highly heterogeneous distribution in protein turnover dynamics within macromolecular machineries such as the mitochondrial ribosome and respiratory complexes I-V, thus shedding light on their mechanism of hierarchical assembly. Furthermore, we investigate the dynamic changes of ER proteome when cells are challenged with stress or undergoing stimulated differentiation, identifying subsets of proteins with unique patterns of turnover dynamics, which may play key regulatory roles in alleviating stress or promoting differentiation. We envision that prox-SILAC could be broadly applied to profile protein turnover at various subcellular compartments, under both physiological and pathological conditions.


Assuntos
Proteoma , Proteômica , Proteoma/metabolismo , Proteômica/métodos , Mitocôndrias/metabolismo , Ribossomos Mitocondriais/metabolismo , Retículo Endoplasmático/metabolismo
4.
Cell Mol Life Sci ; 80(12): 361, 2023 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-37971521

RESUMO

Mitochondrial translation occurs on the mitochondrial ribosome, also known as the mitoribosome. The assembly of mitoribosomes is a highly coordinated process. During mitoribosome biogenesis, various assembly factors transiently associate with the nascent ribosome, facilitating the accurate and efficient construction of the mitoribosome. However, the specific factors involved in the assembly process, the precise mechanisms, and the cellular compartments involved in this vital process are not yet fully understood. In this study, we discovered a crucial role for GTP-binding protein 8 (GTPBP8) in the assembly of the mitoribosomal large subunit (mt-LSU) and mitochondrial translation. GTPBP8 is identified as a novel GTPase located in the matrix and peripherally bound to the inner mitochondrial membrane. Importantly, GTPBP8 is specifically associated with the mt-LSU during its assembly. Depletion of GTPBP8 leads to an abnormal accumulation of mt-LSU, indicating that GTPBP8 is critical for proper mt-LSU assembly. Furthermore, the absence of GTPBP8 results in reduced levels of fully assembled 55S monosomes. This impaired assembly leads to compromised mitochondrial translation and, consequently, impaired mitochondrial function. The identification of GTPBP8 as an important player in these processes provides new insights into the molecular mechanisms underlying mitochondrial protein synthesis and its regulation.


Assuntos
Mitocôndrias , Membranas Mitocondriais , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Ribossomos Mitocondriais/química , Ribossomos Mitocondriais/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
5.
Nucleic Acids Res ; 51(21): 11797-11812, 2023 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-37823603

RESUMO

The human mitochondrial ribosome contains three [2Fe-2S] clusters whose assembly pathway, role, and implications for mitochondrial and metabolic diseases are unknown. Here, structure-function correlation studies show that the clusters play a structural role during mitoribosome assembly. To uncover the assembly pathway, we have examined the effect of silencing the expression of Fe-S cluster biosynthetic and delivery factors on mitoribosome stability. We find that the mitoribosome receives its [2Fe-2S] clusters from the GLRX5-BOLA3 node. Additionally, the assembly of the small subunit depends on the mitoribosome biogenesis factor METTL17, recently reported containing a [4Fe-4S] cluster, which we propose is inserted via the ISCA1-NFU1 node. Consistently, fibroblasts from subjects suffering from 'multiple mitochondrial dysfunction' syndrome due to mutations in BOLA3 or NFU1 display previously unrecognized attenuation of mitochondrial protein synthesis that contributes to their cellular and pathophysiological phenotypes. Finally, we report that, in addition to their structural role, one of the mitoribosomal [2Fe-2S] clusters and the [4Fe-4S] cluster in mitoribosome assembly factor METTL17 sense changes in the redox environment, thus providing a way to regulate organellar protein synthesis accordingly.


Assuntos
Proteínas Ferro-Enxofre , Doenças Mitocondriais , Ribossomos Mitocondriais , Humanos , Proteínas de Transporte/metabolismo , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Metiltransferases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Enxofre/metabolismo , Doenças Mitocondriais/metabolismo
6.
EMBO Rep ; 24(12): e57228, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-37818824

RESUMO

Mitochondrial diseases are a group of disorders defined by defects in oxidative phosphorylation caused by nuclear- or mitochondrial-encoded gene mutations. A main cellular phenotype of mitochondrial disease mutations is redox imbalances and inflammatory signaling underlying pathogenic signatures of these patients. One method to rescue this cell death vulnerability is the inhibition of mitochondrial translation using tetracyclines. However, the mechanisms whereby tetracyclines promote cell survival are unknown. Here, we show that tetracyclines inhibit the mitochondrial ribosome and promote survival through suppression of endoplasmic reticulum (ER) stress. Tetracyclines increase mitochondrial levels of the mitoribosome quality control factor MALSU1 (Mitochondrial Assembly of Ribosomal Large Subunit 1) and promote its recruitment to the mitoribosome large subunit, where MALSU1 is necessary for tetracycline-induced survival and suppression of ER stress. Glucose starvation induces ER stress to activate the unfolded protein response and IRE1α-mediated cell death that is inhibited by tetracyclines. These studies establish a new interorganelle communication whereby inhibition of the mitoribosome signals to the ER to promote survival, implicating basic mechanisms of cell survival and treatment of mitochondrial diseases.


Assuntos
Doenças Mitocondriais , Ribossomos Mitocondriais , Humanos , Ribossomos Mitocondriais/metabolismo , Ribossomos Mitocondriais/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Sobrevivência Celular , Tetraciclinas/farmacologia , Tetraciclinas/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Estresse do Retículo Endoplasmático/genética , Doenças Mitocondriais/genética
7.
Biomolecules ; 13(6)2023 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-37371470

RESUMO

Insulin-degrading enzyme (IDE) is a highly conserved metalloprotease that is mainly localized in the cytosol. Although IDE can degrade insulin and some other low molecular weight substrates efficiently, its ubiquitous expression suggests additional functions supported by experimental findings, such as a role in stress responses and cellular protein homeostasis. The translation of a long full-length IDE transcript has been reported to result in targeting to mitochondria, but the role of IDE in this compartment is unknown. To obtain initial leads on the function of IDE in mitochondria, we used a proximity biotinylation approach to identify proteins interacting with wild-type and protease-dead IDE targeted to the mitochondrial matrix. We find that IDE interacts with multiple mitochondrial ribosomal proteins as well as with proteins involved in the synthesis and assembly of mitochondrial complex I and IV. The mitochondrial interactomes of wild type and mutant IDE are highly similar and do not reveal any likely proteolytic IDE substrates. We speculate that IDE could adopt similar additional non-proteolytic functions in mitochondria as in the cytosol, acting as a chaperone and contributing to protein homeostasis and stress responses.


Assuntos
Transporte de Elétrons , Insulisina , Ribossomos Mitocondriais , Transporte de Elétrons/fisiologia , Insulina/metabolismo , Insulisina/metabolismo , Mitocôndrias/metabolismo , Ribossomos Mitocondriais/metabolismo , Peptídeo Hidrolases/metabolismo , Humanos
8.
Pathol Res Pract ; 248: 154625, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37343379

RESUMO

Lung cancer has a high fatality rate and incidence rate. At present, the initial and progress mechanism of lung cancer has not been completely elucidated and new therapeutic targets still need to be developed. In this study, the screening process was based on lung cancer expression profile data and survival analysis. Mitochondrial ribosome protein L9 (MRPL9) was upregulated in lung cancer tissues and related to the poor overall survival rate and recurrence-free survival rate of lung cancer patients. Knockdown of MRPL9 inhibited the proliferation, sphere-formation, and migration ability of lung cancer cells. MRPL9 was associated with the c-MYC signaling pathway, and lung cancer patients with high expression of both MRPL9 and MYC had a poor prognosis. Furthermore, c-MYC was associated with the epithelial-mesenchymal transition (EMT) regulatory protein zinc finger E-box binding homeobox 1 (ZEB1) by bioinformatics analysis. The relationship between ZEB1 and c-MYC was further confirmed by interfering with c-MYC expression. MRPL9 is a potential therapeutic target for lung cancer and exerts its biological functions by affecting the transcription factor c-MYC thereby regulating the EMT regulator ZEB1.


Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas c-myc , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ribossomos Mitocondriais/metabolismo , Linhagem Celular Tumoral , Transdução de Sinais , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica/genética , Proliferação de Células/genética , Movimento Celular
9.
Trends Biochem Sci ; 48(7): 629-641, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37169615

RESUMO

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


Assuntos
Proteínas Mitocondriais , Ribossomos Mitocondriais , Humanos , Microscopia Crioeletrônica , Ribossomos Mitocondriais/metabolismo , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Ribossômicas/metabolismo
10.
Methods Mol Biol ; 2661: 3-5, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166628

RESUMO

In this introductory chapter, I will briefly describe how I came to discover the mammalian mitoribosome and will add a few notes on my contribution to the field.


Assuntos
Ribossomos Mitocondriais , Ribossomos , Animais , Ribossomos/genética , Proteínas Ribossômicas , Mamíferos , Proteínas Mitocondriais
11.
Methods Mol Biol ; 2661: 7-21, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166629

RESUMO

The ribosome is among the most complex and ancient cellular macromolecular assemblies that plays a central role in protein biosynthesis in all living cells. Its function of translation of genetic information encoded in messenger RNA into protein molecules also extends to subcellular compartments in eukaryotic cells such as apicoplasts, chloroplasts, and mitochondria. The origin of mitochondria is primarily attributed to an early endosymbiotic event between an alpha-proteobacterium and a primitive (archaeal) eukaryotic cell. The timeline of mitochondrial acquisition, the nature of the host, and their diversification have been studied in great detail and are continually being revised as more genomic and structural data emerge. Recent advancements in high-resolution cryo-EM structure determination have provided architectural details of mitochondrial ribosomes (mitoribosomes) from various species, revealing unprecedented diversifications among them. These structures provide novel insights into the evolution of mitoribosomal structure and function. Here, we present a brief overview of the existing mitoribosomal structures in the context of the eukaryotic evolution tree showing their diversification from their last common ancestor.


Assuntos
Mitocôndrias , Ribossomos Mitocondriais , Ribossomos Mitocondriais/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Ribossomos/metabolismo , Eucariotos/genética , Eucariotos/metabolismo , Células Eucarióticas/metabolismo , Proteínas Mitocondriais/metabolismo , Microscopia Crioeletrônica , Proteínas Ribossômicas/metabolismo
12.
Methods Mol Biol ; 2661: 23-51, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166630

RESUMO

Mitoribosome biogenesis is a complex and energetically costly process that involves RNA elements encoded in the mitochondrial genome and mitoribosomal proteins most frequently encoded in the nuclear genome. The process is catalyzed by extra-ribosomal proteins, nucleus-encoded assembly factors that act in all stages of the assembly process to coordinate the processing and maturation of ribosomal RNAs with the hierarchical association of ribosomal proteins. Biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided hints regarding their assembly. In this general concept chapter, we will briefly describe the current knowledge, mainly regarding the mammalian mitoribosome biogenesis pathway and factors involved, and will emphasize the biological sources and approaches that have been applied to advance the field.


Assuntos
Ribossomos Mitocondriais , Proteínas Ribossômicas , Animais , Ribossomos Mitocondriais/metabolismo , Proteínas Ribossômicas/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Mamíferos/genética , Proteínas Mitocondriais/metabolismo
13.
Methods Mol Biol ; 2661: 53-72, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166631

RESUMO

Mitochondrial protein synthesis is essential for the life of aerobic eukaryotes. Without it, oxidative phosphorylation cannot be coupled. Evolution has shaped a battery of factors and machinery that are key to production of just a handful of critical proteins. In this general concept chapter, we attempt to briefly summarize our current knowledge of the overall process in mitochondria from a variety of species, breaking this down to the four parts of translation: initiation, elongation, termination, and recycling. Where appropriate, we highlight differences between species and emphasize gaps in our understanding. Excitingly, with the current revolution in cryoelectron microscopy and mitochondrial genome editing, it is highly likely that many of these gaps will be resolved in the near future. However, the absence of a faithful in vitro reconstituted system to study mitochondrial translation is still problematic.


Assuntos
Mitocôndrias , Ribossomos Mitocondriais , Ribossomos Mitocondriais/metabolismo , Microscopia Crioeletrônica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Biossíntese de Proteínas , Fosforilação Oxidativa , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo
14.
Methods Mol Biol ; 2661: 89-100, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166633

RESUMO

Single-particle cryoelectron microscopy (cryo-EM) allows structure determination of large macromolecular complexes from conformationally and compositionally heterogeneous mixtures of particles. This technique has been used to reveal the architecture of the mitochondrial ribosome and to visualize transient states that occur during the translation cycle or during mitoribosome biogenesis. Here, we outline an exemplary workflow for the analysis of single-particle cryo-EM data of human mitoribosome samples. In addition, we provide an example dataset which can be used for training purposes alongside the protocol.


Assuntos
Ribossomos Mitocondriais , Imagem Individual de Molécula , Humanos , Microscopia Crioeletrônica/métodos , Substâncias Macromoleculares/química , Processamento de Imagem Assistida por Computador/métodos
15.
Methods Mol Biol ; 2661: 119-132, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166635

RESUMO

Mitochondrial protein biosynthesis is maintained by an interplay between the mitochondrial ribosome (mitoribosome) and a large set of protein interaction partners. This interactome regulates a diverse set of functions, including mitochondrial gene expression, translation, protein quality control, and respiratory chain assembly. Hence, robust methods to biochemically and structurally analyze this molecular machinery are required to understand the sophisticated regulation of mitochondrial protein biosynthesis. In this chapter, we present detailed protocols for immunoprecipitation, sucrose cushions, and linear sucrose gradients to purify and analyze mitoribosomes and their interaction partners.


Assuntos
Ribossomos Mitocondriais , Saccharomyces cerevisiae , Ribossomos Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Mitocôndrias/metabolismo , Biossíntese de Proteínas , Proteínas Mitocondriais/metabolismo , Imunoprecipitação
16.
Methods Mol Biol ; 2661: 101-117, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166634

RESUMO

Faithful expression of the mitochondrial genome is required for the synthesis of the oxidative phosphorylation complexes and cell fitness. In humans, mitochondrial DNA (mtDNA) encodes 13 essential subunits of four oxidative phosphorylation complexes along with tRNAs and rRNAs needed for the translation of these proteins. Protein synthesis occurs on unique ribosomes within the organelle. Over the last decade, the revolution in genetic diagnostics has identified disruptions to the faithful synthesis of these 13 mitochondrial proteins as the largest group of inherited human mitochondrial pathologies. All of the molecular steps required for mitochondrial protein synthesis can be affected, from the genome to protein, including cotranslational quality control. Here, we describe methodologies for the biochemical separation of mitochondrial ribosomes from cultured human cells for RNA and protein analysis. Our method has been optimized to facilitate analysis for low-level sample material and thus does not require prior organelle enrichment.


Assuntos
Ribossomos Mitocondriais , RNA , Humanos , RNA/genética , RNA/metabolismo , Ribossomos Mitocondriais/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Biossíntese de Proteínas , Fosforilação Oxidativa , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo
17.
Methods Mol Biol ; 2661: 133-141, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166636

RESUMO

Cryogenic milling, or cryomilling, involves the use of liquid nitrogen to lower the temperature of the biological material and/or the milling process. When applied to the study of subcellular or suborganellar structures and processes, it allows for their rapid extraction from whole cells frozen in the physiological state of choice. This approach has proven to be useful for the study of yeast mitochondrial ribosomes. Following cryomilling of 100 mL of yeast culture, conveniently tagged mitochondrial ribosomes can be immunoprecipitated and purified in native conditions. These ribosomes are suitable for the application of downstream approaches. These include mitoribosome profiling to analyze the mitochondrial translatome or mass spectrometry analyses to assess the mitoribosome proteome in normal growth conditions or under stress, as described in this method.


Assuntos
Ribossomos Mitocondriais , Saccharomyces cerevisiae , Ribossomos Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Ribossomos/metabolismo , Mitocôndrias/ultraestrutura , Espectrometria de Massas , Proteínas Mitocondriais/metabolismo
18.
Methods Mol Biol ; 2661: 143-161, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166637

RESUMO

The biogenesis of mitoribosomes is an intricate process that relies on the coordinated synthesis of nuclear-encoded mitoribosomal proteins (MRPs) in the cytosol, their translocation across mitochondrial membranes, the transcription of rRNA molecules in the matrix as well as the assembly of the roughly 80 different constituents of the mitoribosome. Numerous chaperones, translocases, processing peptidases, and assembly factors of the cytosol and in mitochondria support this complex reaction. The budding yeast Saccharomyces cerevisiae served as a powerful model organism to unravel the different steps by which MRPs are imported into mitochondria, fold into their native structures, and assemble into functional ribosomes.In this chapter, we provide established protocols to study these different processes experimentally. In particular, we describe methods to purify mitochondria from yeast cells, to import radiolabeled MRPs into isolated mitochondria, and to elucidate the assembly reaction of MRPs by immunoprecipitation. These protocols and the list of dos and don'ts will enable beginners and experienced scientists to study the import and assembly of MRPs.


Assuntos
Saccharomyces cerevisiae , Saccharomycetales , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Ribossomos/metabolismo , Saccharomycetales/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Ribossômicas/metabolismo
19.
Methods Mol Biol ; 2661: 163-191, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166638

RESUMO

Studies of yeast mitoribosome assembly have been historically hampered by the difficulty of generating mitoribosome protein-coding gene deletion strains with a stable mitochondrial genome. The identification of mitochondrial DNA-stabilizing approaches allows for the generation of a complete set of yeast deletion strains covering all mitoribosome proteins and known assembly factors. These strains can be used to analyze the integrity and assembly state of mitoribosomes by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts, coupled to mass spectrometry analysis of mitoribosome composition. Subsequent hierarchical cluster analysis of mitoribosome subassemblies accumulated in mutant strains reveals details regarding the order of protein association during the mitoribosome biogenetic process. These strains also allow the expression of truncated protein variants to probe the role of mitochondrion-specific protein extensions, the relevance of protein cofactors, or the importance of RNA-protein interactions in functional sites of the mitoribosome. In this chapter, we will detail the methodology involved in these studies.


Assuntos
Ribossomos Mitocondriais , Saccharomyces cerevisiae , Ribossomos Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Ribossômicas/metabolismo , DNA Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo
20.
Methods Mol Biol ; 2661: 217-232, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166640

RESUMO

Mitochondria maintain their own translational machinery that is responsible for the synthesis of essential components of the oxidative phosphorylation system. The mammalian mitochondrial translation system differs significantly from its cytosolic and bacterial counterparts. Here, we describe detailed protocols for efficient in vitro reconstitution of the mammalian mitochondrial translation initiation complex, which can be further used for mechanistic analyses of different aspects of mitochondrial translation.


Assuntos
Mitocôndrias , Biossíntese de Proteínas , Animais , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Processamento de Proteína Pós-Traducional , Citosol/metabolismo , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Mamíferos/metabolismo
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