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1.
Methods Mol Biol ; 2839: 249-259, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39008259

RESUMO

Thiol-disulfide interconversions are pivotal in the intricate chemistry of biological systems. They play a vital role in governing cellular redox potential and shielding against oxidative harm. These interconversions can also act as molecular switches within an expanding array of redox-regulated proteins, facilitating dynamic and responsive processes. Furthermore, metal-binding proteins often use thiols for coordination. Reverse thiol trapping is a valuable analytical tool to study the redox state of cysteines in biological systems. By selectively capturing and stabilizing free thiol species with an alkylating agent, reverse thiol trapping allows for their subsequent identification and quantification. Various methods can be employed to analyze the trapped thiol adducts, including electrophoresis-based methods, mass spectrometry, nuclear magnetic resonance spectroscopy, and chromatographic techniques. In this chapter, we will focus on describing a simple and sensitive method to sequentially block thiols in their cellular state with a cell-permeant agent (iodoacetamide), and following reduction and denaturation of the samples, trap the native disulfides with a second blocker that shifts the apparent molecular weight of the protein. The oxidation status of proteins for which suitable antibodies are available can then be analyzed by immunoblotting. We present examples of mitochondrial proteins that use cysteine thiols to coordinate metal factors such as iron-sulfur clusters, zinc, and copper.


Assuntos
Proteínas Mitocondriais , Oxirredução , Compostos de Sulfidrila , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Humanos , Iodoacetamida/química , Dissulfetos/química , Dissulfetos/metabolismo , Metais/química , Metais/metabolismo , Cisteína/química , Cisteína/metabolismo
2.
FEBS Open Bio ; 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38849194

RESUMO

Mitoribosome biogenesis is a complex process involving RNA elements encoded in the mitochondrial genome and mitoribosomal proteins typically encoded in the nuclear genome. This process is orchestrated by extra-ribosomal proteins, nucleus-encoded assembly factors, which play roles across all assembly stages to coordinate ribosomal RNA processing and maturation with the sequential association of ribosomal proteins. Both biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided insights into their assembly process. In this article, we will briefly outline the current understanding of mammalian mitoribosome biogenesis pathways and the factors involved. Special attention is devoted to the recent identification of iron-sulfur clusters as structural components of the mitoribosome and a small subunit assembly factor, the existence of redox-sensitive cysteines in mitoribosome proteins and assembly factors, and the role they may play as redox sensor units to regulate mitochondrial translation under stress.

4.
Nat Commun ; 15(1): 4272, 2024 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-38769321

RESUMO

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 Å resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNAVal. The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed us to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transitions in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide a description of the structure and function of the human mitoribosome.


Assuntos
Ribossomos Mitocondriais , RNA de Transferência , Humanos , RNA de Transferência/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , Ribossomos Mitocondriais/metabolismo , Ribossomos Mitocondriais/química , Ligantes , Simulação de Dinâmica Molecular , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Mitocôndrias/metabolismo , RNA Ribossômico/metabolismo , RNA Ribossômico/química , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/química , Guanosina Difosfato/metabolismo , Poliaminas/metabolismo , Poliaminas/química , Ligação Proteica
5.
Biochim Biophys Acta Mol Cell Res ; 1871(4): 119707, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38493895

RESUMO

Ribosomes across species contain subsets of zinc finger proteins that play structural roles by binding to rRNA. While the majority of these zinc fingers belong to the C2-C2 type, the large subunit protein L36 in bacteria and mitochondria exhibits an atypical C2-CH motif. To comprehend the contribution of each coordinating residue in S. cerevisiae bL36m to mitoribosome assembly and function, we engineered and characterized strains carrying single and double mutations in the zinc coordinating residues. Our findings reveal that although all four residues markedly influence protein stability, C to A mutations in C66 and/or C69 have a more pronounced effect compared to those at C82 and H88. Importantly, protein stability directly correlates with the assembly and function of the mitoribosome and the growth rate of yeast in respiratory conditions. Mass spectrometry analysis of large subunit particles indicates that strains deleted for bL36m or expressing mutant variants have defective assembly of the L7/L12 stalk base, limiting their functional competence. Furthermore, we employed a synthetic bL36m protein collection, including both wild-type and mutant proteins, to elucidate their ability to bind zinc. Our data indicate that mutations in C82 and, particularly, H88 allow for some zinc binding albeit inefficient or unstable, explaining the residual accumulation and activity in mitochondria of bL36m variants carrying mutations in these residues. In conclusion, stable zinc binding by bL36m is essential for optimal mitoribosome assembly and function. MS data are available via ProteomeXchange with identifierPXD046465.


Assuntos
Ribossomos Mitocondriais , Saccharomyces cerevisiae , Ribossomos Mitocondriais/química , Ribossomos Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Dedos de Zinco/genética , Subunidades Ribossômicas Maiores/genética , Zinco/metabolismo
7.
bioRxiv ; 2023 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-37961485

RESUMO

The mammalian mitochondrial genome encodes thirteen oxidative phosphorylation system proteins, crucial in aerobic energy transduction. These proteins are translated from 9 monocistronic and 2 bicistronic transcripts, whose native structures remain unexplored, leaving fundamental molecular determinants of mitochondrial gene expression unknown. To address this gap, we developed a mitoDMS-MaPseq approach and used DREEM clustering to resolve the native human mitochondrial mt-mRNA structurome. We gained insights into mt-mRNA biology and translation regulatory mechanisms, including a unique programmed ribosomal frameshifting for the ATP8/ATP6 transcript. Furthermore, absence of the mt-mRNA maintenance factor LRPPRC led to a mitochondrial transcriptome structured differently, with specific mRNA regions exhibiting increased or decreased structuredness. This highlights the role of LRPPRC in maintaining mRNA folding to promote mt-mRNA stabilization and efficient translation. In conclusion, our mt-mRNA folding maps reveal novel mitochondrial gene expression mechanisms, serving as a detailed reference and tool for studying them in different physiological and pathological contexts.

8.
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
9.
EMBO Rep ; 24(11): e57092, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37828827

RESUMO

The mitochondrial respiratory chain (MRC) is a key energy transducer in eukaryotic cells. Four respiratory chain complexes cooperate in the transfer of electrons derived from various metabolic pathways to molecular oxygen, thereby establishing an electrochemical gradient over the inner mitochondrial membrane that powers ATP synthesis. This electron transport relies on mobile electron carries that functionally connect the complexes. While the individual complexes can operate independently, they are in situ organized into large assemblies termed respiratory supercomplexes. Recent structural and functional studies have provided some answers to the question of whether the supercomplex organization confers an advantage for cellular energy conversion. However, the jury is still out, regarding the universality of these claims. In this review, we discuss the current knowledge on the functional significance of MRC supercomplexes, highlight experimental limitations, and suggest potential new strategies to overcome these obstacles.


Assuntos
Mitocôndrias , Membranas Mitocondriais , Membranas Mitocondriais/metabolismo , Transporte de Elétrons , Mitocôndrias/metabolismo
10.
bioRxiv ; 2023 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-37503168

RESUMO

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 Å resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNA Val . The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transition in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide the most complete description so far of the structure and function of the human mitoribosome.

11.
Brain ; 146(10): 4191-4199, 2023 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-37170631

RESUMO

COQ7 encodes a hydroxylase responsible for the penultimate step of coenzyme Q10 (CoQ10) biosynthesis in mitochondria. CoQ10 is essential for multiple cellular functions, including mitochondrial oxidative phosphorylation, lipid metabolism, and reactive oxygen species homeostasis. Mutations in COQ7 have been previously associated with primary CoQ10 deficiency, a clinically heterogeneous multisystemic mitochondrial disorder. We identified COQ7 biallelic variants in nine families diagnosed with distal hereditary motor neuropathy with upper neuron involvement, expending the clinical phenotype associated with defects in this gene. A recurrent p.Met1? change was identified in five families from Brazil with evidence of a founder effect. Fibroblasts isolated from patients revealed a substantial depletion of COQ7 protein levels, indicating protein instability leading to loss of enzyme function. High-performance liquid chromatography assay showed that fibroblasts from patients had reduced levels of CoQ10, and abnormal accumulation of the biosynthetic precursor DMQ10. Accordingly, fibroblasts from patients displayed significantly decreased oxygen consumption rates in patients, suggesting mitochondrial respiration deficiency. Induced pluripotent stem cell-derived motor neurons from patient fibroblasts showed significantly increased levels of extracellular neurofilament light protein, indicating axonal degeneration. Our findings indicate a molecular pathway involving CoQ10 biosynthesis deficiency and mitochondrial dysfunction in patients with distal hereditary motor neuropathy. Further studies will be important to evaluate the potential benefits of CoQ10 supplementation in the clinical outcome of the disease.


Assuntos
Doenças Mitocondriais , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Neurônios Motores/metabolismo , Mutação/genética , Ubiquinona/genética
12.
Elife ; 122023 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-37171075

RESUMO

Mitochondria influence cellular function through both cell-autonomous and non-cell autonomous mechanisms, such as production of paracrine and endocrine factors. Here, we demonstrate that mitochondrial regulation of the secretome is more extensive than previously appreciated, as both genetic and pharmacological disruption of the electron transport chain caused upregulation of the Alzheimer's disease risk factor apolipoprotein E (APOE) and other secretome components. Indirect disruption of the electron transport chain by gene editing of SLC25A mitochondrial membrane transporters as well as direct genetic and pharmacological disruption of either complexes I, III, or the copper-containing complex IV of the electron transport chain elicited upregulation of APOE transcript, protein, and secretion, up to 49-fold. These APOE phenotypes were robustly expressed in diverse cell types and iPSC-derived human astrocytes as part of an inflammatory gene expression program. Moreover, age- and genotype-dependent decline in brain levels of respiratory complex I preceded an increase in APOE in the 5xFAD mouse model. We propose that mitochondria act as novel upstream regulators of APOE-dependent cellular processes in health and disease.


Assuntos
Apolipoproteína E4 , Mitocôndrias , Animais , Humanos , Camundongos , Apolipoproteína E4/genética , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Astrócitos/metabolismo , Genótipo , Mitocôndrias/metabolismo , Mitocôndrias/patologia
13.
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
14.
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
15.
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
16.
Elife ; 112022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36255054

RESUMO

Mammalian carotid body arterial chemoreceptors function as an early warning system for hypoxia, triggering acute life-saving arousal and cardiorespiratory reflexes. To serve this role, carotid body glomus cells are highly sensitive to decreases in oxygen availability. While the mitochondria and plasma membrane signaling proteins have been implicated in oxygen sensing by glomus cells, the mechanism underlying their mitochondrial sensitivity to hypoxia compared to other cells is unknown. Here, we identify HIGD1C, a novel hypoxia-inducible gene domain factor isoform, as an electron transport chain complex IV-interacting protein that is almost exclusively expressed in the carotid body and is therefore not generally necessary for mitochondrial function. Importantly, HIGD1C is required for carotid body oxygen sensing and enhances complex IV sensitivity to hypoxia. Thus, we propose that HIGD1C promotes exquisite oxygen sensing by the carotid body, illustrating how specialized mitochondria can be used as sentinels of metabolic stress to elicit essential adaptive behaviors.


Assuntos
Corpo Carotídeo , Animais , Oxigênio/metabolismo , Células Quimiorreceptoras/metabolismo , Mitocôndrias/metabolismo , Hipóxia/metabolismo , Mamíferos/metabolismo
17.
Genome Biol ; 23(1): 170, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35945592

RESUMO

BACKGROUND: Oxidative phosphorylation (OXPHOS) complexes consist of nuclear and mitochondrial DNA-encoded subunits. Their biogenesis requires cross-compartment gene regulation to mitigate the accumulation of disproportionate subunits. To determine how human cells coordinate mitochondrial and nuclear gene expression processes, we tailored ribosome profiling for the unique features of the human mitoribosome. RESULTS: We resolve features of mitochondrial translation initiation and identify a small ORF in the 3' UTR of MT-ND5. Analysis of ribosome footprints in five cell types reveals that average mitochondrial synthesis levels correspond precisely to cytosolic levels across OXPHOS complexes, and these average rates reflect the relative abundances of the complexes. Balanced mitochondrial and cytosolic synthesis does not rely on rapid feedback between the two translation systems, and imbalance caused by mitochondrial translation deficiency is associated with the induction of proteotoxicity pathways. CONCLUSIONS: Based on our findings, we propose that human OXPHOS complexes are synthesized proportionally to each other, with mitonuclear balance relying on the regulation of OXPHOS subunit translation across cellular compartments, which may represent a proteostasis vulnerability.


Assuntos
Mitocôndrias , Proteínas Mitocondriais , DNA Mitocondrial/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Biossíntese de Proteínas
18.
Nat Commun ; 13(1): 3615, 2022 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-35750769

RESUMO

Mitochondrial cytochrome c oxidase (CcO) or respiratory chain complex IV is a heme aa3-copper oxygen reductase containing metal centers essential for holo-complex biogenesis and enzymatic function that are assembled by subunit-specific metallochaperones. The enzyme has two copper sites located in the catalytic core subunits. The COX1 subunit harbors the CuB site that tightly associates with heme a3 while the COX2 subunit contains the binuclear CuA site. Here, we report that in human cells the CcO copper chaperones form macromolecular assemblies and cooperate with several twin CX9C proteins to control heme a biosynthesis and coordinate copper transfer sequentially to the CuA and CuB sites. These data on CcO illustrate a mechanism that regulates the biogenesis of macromolecular enzymatic assemblies with several catalytic metal redox centers and prevents the accumulation of cytotoxic reactive assembly intermediates.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Oxirredutases , Cobre/metabolismo , Citocromos c/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Heme/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Oxirredutases/metabolismo
19.
Dev Cell ; 57(5): 654-669.e9, 2022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35247316

RESUMO

The response to oxygen availability is a fundamental process concerning metabolism and survival/death in all mitochondria-containing eukaryotes. However, the known oxygen-sensing mechanism in mammalian cells depends on pVHL, which is only found among metazoans but not in other species. Here, we present an alternative oxygen-sensing pathway regulated by ATE1, an enzyme ubiquitously conserved in eukaryotes that influences protein degradation by posttranslational arginylation. We report that ATE1 centrally controls the hypoxic response and glycolysis in mammalian cells by preferentially arginylating HIF1α that is hydroxylated by PHD in the presence of oxygen. Furthermore, the degradation of arginylated HIF1α is independent of pVHL E3 ubiquitin ligase but dependent on the UBR family proteins. Bioinformatic analysis of human tumor data reveals that the ATE1/UBR and pVHL pathways jointly regulate oxygen sensing in a transcription-independent manner with different tissue specificities. Phylogenetic analysis suggests that eukaryotic ATE1 likely evolved during mitochondrial domestication, much earlier than pVHL.


Assuntos
Aminoaciltransferases , Oxigênio , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Animais , Humanos , Mamíferos/metabolismo , Filogenia , Proteólise
20.
Yeast ; 39(3): 208-229, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34713496

RESUMO

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/metabolismo
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