bims-cytox1 2023-05-14 papers

bims-cytox1

Biomed News

on Cytochrome oxidase subunit 1

Issue of 2023–05–14
sixteen papers selected by
Gavin McStay, Liverpool John Moores University

Protein transport along the presequence pathway.
The ARG8m Reporter for the Study of Yeast Mitochondrial Translation.
Metabolic Labeling of Mitochondrial Translation Products in Whole Cells and Isolated Organelles.
Sucrose Gradient Analysis of Human Mitochondrial Ribosomes and RNA.
Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast.
Reconstitution of Mammalian Mitochondrial Translation System Capable of Long Polypeptide Synthesis.
Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation.
Mitoribosome Biogenesis.
Assembly of the Mitochondrial Translation Initiation Complex.
A two-step mitochondrial import pathway couples the disulfide relay with matrix complex I biogenesis.
Human Mitoribosome Profiling: A Re-engineered Approach Tailored to Study Mitochondrial Translation.
Translation in Mitochondrial Ribosomes.
Newly imported proteins in mitochondria are particularly sensitive to aggregation.
Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway.
Bifenthrin disrupts cytochrome c oxidase activity and reduces mitochondrial DNA copy number through oxidative damage in pool barb (Puntius sophore).
mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations.

Biol Chem. 2023 May 09.

Protein transport along the presequence pathway.

Abhijith Makki, Peter Rehling.

  Most mitochondrial proteins are nuclear-encoded and imported by the protein import machinery based on specific targeting signals. The proteins that carry an amino-terminal targeting signal (presequence) are imported via the presequence import pathway that involves the translocases of the outer and inner membranes - TOM and TIM23 complexes. In this article, we discuss how mitochondrial matrix and inner membrane precursor proteins are imported along the presequence pathway in Saccharomyces cerevisiae with a focus on the dynamics of the TIM23 complex, and further update with some of the key findings that advanced the field in the last few years.

Keywords:  PAM; TIM23 complex; TOM complex; mitochondria; presequence translocase; protein translocation

DOI:  https://doi.org/10.1515/hsz-2023-0133
Methods Mol Biol. 2023 ;2661 281-301

The ARG8m Reporter for the Study of Yeast Mitochondrial Translation.

Daniel Flores-Mireles, Yolanda Camacho-Villasana, Xochitl Pérez-Martínez.

  Mitochondrial translation is an intricate process involving both general and mRNA-specific factors. In addition, in the yeast Saccharomyces cerevisiae, translation of mitochondrial mRNAs is coupled to assembly of nascent polypeptides into the membrane. ARG8m is a reporter gene widely used to study the mechanisms of yeast mitochondrial translation. This reporter is a recodified gene that uses the mitochondrial genetic code and is inserted at the desired locus in the mitochondrial genome. After deletion of the endogenous nuclear gene, this reporter produces Arg8, an enzyme necessary for arginine biosynthesis. Since Arg8 is a soluble protein with no relation to oxidative phosphorylation, it is a reliable reporter to study mitochondrial mRNAs translation and dissect translation form assembly processes. In this chapter, we explain how to insert the ARG8m reporter in the desired spot in the mitochondrial DNA, how to analyze Arg8 synthesis inside mitochondria, and how to follow steady-state levels of the protein. We also explain how to use it to find spontaneous suppressors of translation defects.

Keywords:  ARG8m; ATP synthase; Cytochrome c oxidase; Mitochondria; Mitochondrial DNA; Respiratory complexes; Suppressor; Translation; Yeast; bc1 complex

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_16
Methods Mol Biol. 2023 ;2661 193-215

Metabolic Labeling of Mitochondrial Translation Products in Whole Cells and Isolated Organelles.

Priyanka Maiti, Flavia Fontanesi.

  Mitochondria retain their own genome and translational apparatus that is highly specialized in the synthesis of a handful of proteins, essential components of the oxidative phosphorylation system. During evolution, the players and mechanisms involved in mitochondrial translation have acquired some unique features, which we have only partially disclosed. The study of the mitochondrial translation process has been historically hampered by the lack of an in vitro translational system and has largely relied on the analysis of the incorporation rate of radiolabeled amino acids into mitochondrial proteins in cellulo or in organello. In this chapter, we describe methods to monitor mitochondrial translation by labeling newly synthesized mitochondrial polypeptides with [S35]-methionine in either yeast or mammalian whole cells or isolated mitochondria.

Keywords:  Human cells; Mitochondrial translation; Newly synthesized polypeptides; Protein synthesis; Pulse-chase labeling; Yeast; [S35]-methionine

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_12
Methods Mol Biol. 2023 ;2661 101-117

Sucrose Gradient Analysis of Human Mitochondrial Ribosomes and RNA.

Kah Ying Ng, Brendan J Battersby.

  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.

Keywords:  Human disease; Mitochondria; Mitochondrial disease; RNA; Ribosomes; Sucrose gradient

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_7
Methods Mol Biol. 2023 ;2661 143-161

Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast.

Lea Bertgen, Tamara Flohr, Johannes M Herrmann.

  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.

Keywords:  Immunoprecipitation; Isolation of mitochondria; Mitochondrial protein import; Mitoribosomal protein (MRP); Sample preparation for mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_10
Methods Mol Biol. 2023 ;2661 233-255

Reconstitution of Mammalian Mitochondrial Translation System Capable of Long Polypeptide Synthesis.

Muhoon Lee, Nono Takeuchi-Tomita.

  Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. Here, we describe the in vitro reconstitution of the mammalian mitochondrial translation system, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a heterologous yeast tRNA mixture. The system is capable of translating leaderless mRNAs encoding model proteins, such as nanoluciferase with a molecular weight of 19 kDa, and is readily applicable for in vitro evaluations of mRNAs and nascent peptide chain sequences, as well as factors and small molecules that affect mitochondrial translation.

Keywords:  55S ribosome; In vitro translation; Leaderless mRNA; Mammalian mitochondria; Reconstituted translation system

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_14
Methods Mol Biol. 2023 ;2661 119-132

Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation.

Andreas Aufschnaiter, Andreas Carlström, Martin Ott.

  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.

Keywords:  Immunoprecipitation; Mitochondria; Mitoribosome; Sucrose cushion; Sucrose gradient; Yeast

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_8
Methods Mol Biol. 2023 ;2661 23-51

Mitoribosome Biogenesis.

J Conor Moran, Samuel Del'Olio, Austin Choi, Hui Zhong, Antoni Barrientos.

  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.

Keywords:  Mitochondrial disease; Mitochondrial ribosome; Mitochondrial translation; Mitoribosome assembly; OXPHOS deficiency

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_3
Methods Mol Biol. 2023 ;2661 217-232

Assembly of the Mitochondrial Translation Initiation Complex.

Cristina Remes, Minh Duc Nguyen, Henrik Spahr, Martin Ng, Clark Fritsch, Arpan Bhattacharya, Hong Li, Barry Cooperman, Joanna Rorbach.

  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.

Keywords:  Mitoribosome; Translation; Translation factors; Translation initiation; tRNA purification

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_13
J Cell Biol. 2023 Jul 03. pii: e202210019. [Epub ahead of print]222(7):

A two-step mitochondrial import pathway couples the disulfide relay with matrix complex I biogenesis.

Esra Peker, Konstantin Weiss, Jiyao Song, Christine Zarges, Sarah Gerlich, Volker Boehm, Aleksandra Trifunovic, Thomas Langer, Niels H Gehring, Thomas Becker, Jan Riemer.

Mitochondria critically rely on protein import and its tight regulation. Here, we found that the complex I assembly factor NDUFAF8 follows a two-step import pathway linking IMS and matrix import systems. A weak targeting sequence drives TIM23-dependent NDUFAF8 matrix import, and en route, allows exposure to the IMS disulfide relay, which oxidizes NDUFAF8. Import is closely surveyed by proteases: YME1L prevents accumulation of excess NDUFAF8 in the IMS, while CLPP degrades reduced NDUFAF8 in the matrix. Therefore, NDUFAF8 can only fulfil its function in complex I biogenesis if both oxidation in the IMS and subsequent matrix import work efficiently. We propose that the two-step import pathway for NDUFAF8 allows integration of the activity of matrix complex I biogenesis pathways with the activity of the mitochondrial disulfide relay system in the IMS. Such coordination might not be limited to NDUFAF8 as we identified further proteins that can follow such a two-step import pathway.

DOI: https://doi.org/10.1083/jcb.202210019
Methods Mol Biol. 2023 ;2661 257-280

Human Mitoribosome Profiling: A Re-engineered Approach Tailored to Study Mitochondrial Translation.

Iliana Soto, Mary Couvillion, L Stirling Churchman.

  To understand the human mitochondrial translation process, tools are required to dissect this system at a global scale. The mechanisms and regulation of translation in mitochondria are different from those in the cytosol, and mitochondrial ribosomes have distinct biochemical properties. In this chapter, we describe in detail the modifications we have made to the ribosome profiling approach to adapt it to the unique characteristics of the human mitochondrial ribosome. This approach maximizes the fraction of mitochondrial ribosomes recovered, providing a snapshot of the mitochondrial translation landscape with minimal bias. We also describe the use of mouse lysate as an internal spike-in control for normalization, allowing quantification of global changes in translation across samples. Finally, we outline the bioinformatic pipelines to process the raw reads and identify mitoribosome A sites in the absence of untranslated regions flanking open reading frames. This method offers a subcodon-resolution time-sensitive global approach to explore the mitochondrial translation process in human cells.

Keywords:  Human mitochondrial translation; Mitochondrial ribosome profiling

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_15
Methods Mol Biol. 2023 ;2661 53-72

Translation in Mitochondrial Ribosomes.

Zofia M Chrzanowska-Lightowlers, Robert N Lightowlers.

  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.

Keywords:  Elongation; Initiation; Mitochondria; Mitoribosomes; Protein synthesis; Recycling; Termination; Translation; mt-mRNAs

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_4
Acta Physiol (Oxf). 2023 May 12. e13985

Newly imported proteins in mitochondria are particularly sensitive to aggregation.

Carmela Vazquez-Calvo, Verena Kohler, Johanna L Höög, Sabrina Büttner, Martin Ott.

   AIM: A functional proteome is essential for life and maintained by protein quality control (PQC) systems in the cytosol and organelles. Protein aggregation is an indicator of a decline of PQC linked to aging and disease. Mitochondrial PQC is critical to maintain mitochondrial function and thus cellular fitness. How mitochondria handle aggregated proteins is not well understood. Here we tested how the metabolic status impacts on formation and clearance of aggregates within yeast mitochondria and assessed which proteins are particularly sensitive to denaturation.
METHODS: Confocal microscopy, electron microscopy, immunoblotting and genetics were applied to assess mitochondrial aggregate handling in response to heat shock and ethanol, using the mitochondrial disaggregase Hsp78 as a marker for protein aggregates.
RESULTS: We show that aggregates formed upon heat or ethanol stress with different dynamics depending on the metabolic state. While fermenting cells displayed numerous small aggregates that coalesced into one large foci that was resistant to clearance, respiring cells showed less aggregates and cleared these aggregates more efficiently. Acute inhibition of mitochondrial translation had no effect, while preventing protein import into mitochondria by inhibition of cytosolic translation prevented aggregate formation.
CONCLUSION: Collectively, our data show that the metabolic state of the cells impacts the dynamics of aggregate formation and clearance, and that mainly newly imported and not yet assembled proteins are prone to form aggregates. Because mitochondrial functionality is crucial for cellular metabolism, these results highlight the importance of efficient protein biogenesis to maintain the mitochondrial proteome operational during metabolic adaptations and cellular stress.

Keywords:  Ageing; Aggregates; Cellular stress; Hsp78; Metabolism; Mitochondria; Protein quality control; Proteostasis

DOI:  https://doi.org/10.1111/apha.13985
Methods Mol Biol. 2023 ;2661 163-191

Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway.

Samuel Del'Olio, Antoni Barrientos.

  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.

Keywords:  Clustering analysis; Gradient fractionation; Immunoblotting; Mass spectrometry; Mitochondrial ribosome; Mitoribosome assembly intermediate; Mitoribosome profile; Sucrose gradient; Yeast mitoribosome gene deletion strain

DOI:  https://doi.org/10.1007/978-1-0716-3171-3_11
Chemosphere. 2023 May 06. pii: S0045-6535(23)01115-3. [Epub ahead of print] 138848

Bifenthrin disrupts cytochrome c oxidase activity and reduces mitochondrial DNA copy number through oxidative damage in pool barb (Puntius sophore).

Anwesha Das, Sarbashri Bank, Srilagna Chatterjee, Nirvika Paul, Kunal Sarkar, Arindam Chatterjee, Santanu Chakraborty, Chaitali Banerjee, Anasuya Majumdar, Madhusudan Das, Sudakshina Ghosh.

  Bifenthrin (BF), a synthetic pyrethroid is used worldwide for both agricultural and non-agricultural purposes due to its high insecticidal activity and low toxicity in mammals. However, its improper usage implies a possible risk to aquatic life. The Study was aimed to correlate the association of BF toxicity with mitochondrial DNA copy number variation in edible fish Punitus sophore. The 96-h LC 50 of BF in P. sophore was 3.4 μg/L, fish was treated with sub-lethal doses (0.34 μg/L,0.68 μg/L) of BF for 15 days. The activity and expression level of cytochrome c oxidase (Mt-COI) were measured to assess mitochondrial dysfunction caused by BF. Results showed BF reduced the level of Mt-COI mRNA in treated groups, hindered complex IV activity and increased ROS generation leading to oxidative damage. mtDNAcn was decreased in the muscle, brain and liver after BF treatment. Furthermore, BF induced neurotoxicity in brain and muscle cells through the inhibition of AchE activity. The treated groups showed elevated level of malondialdehyde (MDA) and an imbalance of antioxidant enzymes activity. Molecular docking and simulation analysis also predicted that BF binds to the active sites of the enzyme and restricts the fluctuation of active sites' residues. Hence, outcome of the study suggests reduction of mtDNAcn could be a potential biomarker to assess Bifenthrin induced toxicity in aquatic ecosystem.

Keywords:  Bifenthrin; Oxidative stress; Puntius sophore; Toxicology; mt-DNA copy number

DOI:  https://doi.org/10.1016/j.chemosphere.2023.138848
bioRxiv. 2023 Apr 26. pii: 2023.04.24.538105. [Epub ahead of print]

mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations.

Alexander N Gasparski, Konstadinos Moissoglu, Sandeep Pallikkuth, Sezen Meydan, Nicholas R Guydosh, Stavroula Mili.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that protein targeting can additionally be determined by mRNA location and translation rate, through modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein, through promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting, through promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a 'partner-selection' mechanism that robustly influences protein distribution and function.

DOI: https://doi.org/10.1101/2023.04.24.538105