bims-mitdyn Biomed News
on Mitochondrial dynamics: mechanisms
Issue of 2023–05–14
25 papers selected by
Edmond Chan, Queen’s University, School of Medicine
  1. GAP43-dependent mitochondria transfer from astrocytes enhances glioblastoma tumorigenicity.
  2. Mind the GAP(43) for mitochondria transfer to glioblastomas.
  3. In situ architecture of the ER-mitochondria encounter structure.
  4. FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.
  5. Fuel oxidation under the control of mitochondrial shape and dynamics.
  6. A two-step mitochondrial import pathway couples the disulfide relay with matrix complex I biogenesis.
  7. TIM23 facilitates PINK1 activation by safeguarding against OMA1-mediated degradation in damaged mitochondria.
  8. APOE expression and secretion are modulated by mitochondrial dysfunction.
  9. Depletion of WFS1 compromises mitochondrial function in hiPSC-derived neuronal models of Wolfram syndrome.
  10. Mitochondrial Ion Channels.
  11. Evolution: Mitochondrial Ribosomes Across Species.
  12. Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation.
  13. Translation in Mitochondrial Ribosomes.
  14. Discovery of Mitochondrial Ribosomes.
  15. Metabolic Labeling of Mitochondrial Translation Products in Whole Cells and Isolated Organelles.
  16. Mitoribosome Biogenesis.
  17. Four-Color STED Super-Resolution RNA Fluorescent In Situ Hybridization and Immunocytochemistry to Visualize Mitochondrial mRNAs in Context with Mitochondrial Ribosomes.
  18. Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast.
  19. Human Mitoribosome Profiling: A Re-engineered Approach Tailored to Study Mitochondrial Translation.
  20. Assembly of the Mitochondrial Translation Initiation Complex.
  21. Sucrose Gradient Analysis of Human Mitochondrial Ribosomes and RNA.
  22. Cryo-EM for Structure Determination of Mitochondrial Ribosome Samples.
  23. Digital RNase Footprinting of RNA-Protein Complexes and Ribosomes in Mitochondria.
  24. Reconstitution of Mammalian Mitochondrial Translation System Capable of Long Polypeptide Synthesis.
  25. Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation.
  1. Nat Cancer. 2023 May 11.
    GAP43-dependent mitochondria transfer from astrocytes enhances glioblastoma tumorigenicity.
    Dionysios C Watson, Defne Bayik, Simon Storevik, Shannon Sherwin Moreino, Samuel A Sprowls, Jianhua Han, Mina Thue Augustsson, Adam Lauko, Palavalasa Sravya, Gro Vatne Røsland, Katie Troike, Karl Johan Tronstad, Sabrina Wang, Katharina Sarnow, Kristen Kay, Taral R Lunavat, Daniel J Silver, Sahil Dayal, Justin Vareecal Joseph, Erin Mulkearns-Hubert, Lars Andreas Rømo Ystaas, Gauravi Deshpande, Joris Guyon, Yadi Zhou, Capucine R Magaut, Juliana Seder, Laura Neises, Sarah E Williford, Johannes Meiser, Andrew J Scott, Peter Sajjakulnukit, Jason A Mears, Rolf Bjerkvig, Abhishek Chakraborty, Thomas Daubon, Feixiong Cheng, Costas A Lyssiotis, Daniel R Wahl, Anita B Hjelmeland, Jubayer A Hossain, Hrvoje Miletic, Justin D Lathia.
      The transfer of intact mitochondria between heterogeneous cell types has been confirmed in various settings, including cancer. However, the functional implications of mitochondria transfer on tumor biology are poorly understood. Here we show that mitochondria transfer is a prevalent phenomenon in glioblastoma (GBM), the most frequent and malignant primary brain tumor. We identified horizontal mitochondria transfer from astrocytes as a mechanism that enhances tumorigenesis in GBM. This transfer is dependent on network-forming intercellular connections between GBM cells and astrocytes, which are facilitated by growth-associated protein 43 (GAP43), a protein involved in neuron axon regeneration and astrocyte reactivity. The acquisition of astrocyte mitochondria drives an increase in mitochondrial respiration and upregulation of metabolic pathways linked to proliferation and tumorigenicity. Functionally, uptake of astrocyte mitochondria promotes cell cycle progression to proliferative G2/M phases and enhances self-renewal and tumorigenicity of GBM. Collectively, our findings reveal a host-tumor interaction that drives proliferation and self-renewal of cancer cells, providing opportunities for therapeutic development.
    DOI:  https://doi.org/10.1038/s43018-023-00556-5
  2. Nat Cancer. 2023 May 11.
    Mind the GAP(43) for mitochondria transfer to glioblastomas.
    Martina Semenzato, Luca Scorrano.
      
    DOI:  https://doi.org/10.1038/s43018-023-00564-5
  3. Nature. 2023 May 10.
    In situ architecture of the ER-mitochondria encounter structure.
    Michael R Wozny, Andrea Di Luca, Dustin R Morado, Andrea Picco, Rasha Khaddaj, Pablo Campomanes, Lazar Ivanović, Patrick C Hoffmann, Elizabeth A Miller, Stefano Vanni, Wanda Kukulski.
      The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1-3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4-6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.
    DOI:  https://doi.org/10.1038/s41586-023-06050-3
  4. EMBO J. 2023 May 10. e112767
    FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.
    Giang Thanh Nguyen-Dien, Keri-Lyn Kozul, Yi Cui, Brendan Townsend, Prajakta Gosavi Kulkarni, Soo Siang Ooi, Antonio Marzio, Nissa Carrodus, Steven Zuryn, Michele Pagano, Robert G Parton, Michael Lazarou, S Sean Millard, Robert W Taylor, Brett M Collins, Mathew Jk Jones, Julia K Pagan.
      To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.
    Keywords:  BNIP3; FBXL4; NIX/BNIP3L; mitochondria; mitophagy
    DOI:  https://doi.org/10.15252/embj.2022112767
  5. EMBO J. 2023 May 08. e114129
    Fuel oxidation under the control of mitochondrial shape and dynamics.
    Erin L Seifert, György Hajnóczky.
      How mitochondrial shape and substrate-specific metabolism are related has been a difficult question to address. Here, new work by Ngo et al (2023) reports that mitochondrial shape-long versus fragmented-determines the activity of β-oxidation of long-chain fatty acids, supporting a novel role for mitochondrial fission products as β-oxidation hubs.
    DOI:  https://doi.org/10.15252/embj.2023114129
  6. 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
  7. Cell Rep. 2023 Apr 30. pii: S2211-1247(23)00465-5. [Epub ahead of print] 112454
    TIM23 facilitates PINK1 activation by safeguarding against OMA1-mediated degradation in damaged mitochondria.
    Shiori Akabane, Kiyona Watanabe, Hidetaka Kosako, Shun-Ichi Yamashita, Kohei Nishino, Masahiro Kato, Shiori Sekine, Tomotake Kanki, Noriyuki Matsuda, Toshiya Endo, Toshihiko Oka.
      PINK1 is activated by autophosphorylation and forms a high-molecular-weight complex, thereby initiating the selective removal of damaged mitochondria by autophagy. Other than translocase of the outer mitochondrial membrane complexes, members of PINK1-containing protein complexes remain obscure. By mass spectrometric analysis of PINK1 co-immunoprecipitates, we identify the inner membrane protein TIM23 as a component of the PINK1 complex. TIM23 downregulation decreases PINK1 levels and significantly delays autophosphorylation, indicating that TIM23 promotes PINK1 accumulation in response to depolarization. Moreover, inactivation of the mitochondrial protease OMA1 not only enhances PINK1 accumulation but also represses the reduction in PINK1 levels induced by TIM23 downregulation, suggesting that TIM23 facilitates PINK1 activation by safeguarding against degradation by OMA1. Indeed, deficiencies of pathogenic PINK1 mutants that fail to interact with TIM23 are partially restored by OMA1 inactivation. These findings indicate that TIM23 plays a distinct role in activating mitochondrial autophagy by protecting PINK1.
    Keywords:  CP: Cell biology; OMA1; PINK1; TIM23; mitochondrial quality control
    DOI:  https://doi.org/10.1016/j.celrep.2023.112454
  8. Elife. 2023 May 12. pii: e85779. [Epub ahead of print]12
    APOE expression and secretion are modulated by mitochondrial dysfunction.
    Meghan E Wynne, Oluwaseun Ogunbona, Alicia R Lane, Avanti Gokhale, Stephanie A Zlatic, Chongchong Xu, Zhexing Wen, Duc M Duong, Sruti Rayaprolu, Anna Ivanova, Eric A Ortlund, Eric B Dammer, Nicholas T Seyfried, Blaine R Roberts, Amanda Crocker, Vinit Shanbhag, Michael Petris, Nanami Senoo, Selvaraju Kandasamy, Steven Michael Claypool, Antoni Barrientos, Aliza Wingo, Thomas S Wingo, Srikant Rangaraju, Allan I Levey, Erica Werner, Victor Faundez.
      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.
    Keywords:  cell biology; human; neuroscience
    DOI:  https://doi.org/10.7554/eLife.85779
  9. Stem Cell Reports. 2023 May 09. pii: S2213-6711(23)00136-4. [Epub ahead of print]18(5): 1090-1106
    Depletion of WFS1 compromises mitochondrial function in hiPSC-derived neuronal models of Wolfram syndrome.
    Malgorzata Zatyka, Tatiana R Rosenstock, Congxin Sun, Adina M Palhegyi, Georgina W Hughes, Samuel Lara-Reyna, Dewi Astuti, Alessandro di Maio, Axel Sciauvaud, Miriam E Korsgen, Vesna Stanulovic, Gamze Kocak, Malgorzata Rak, Sandra Pourtoy-Brasselet, Katherine Winter, Thiago Varga, Margot Jarrige, Hélène Polvèche, Joao Correia, Eva-Maria Frickel, Maarten Hoogenkamp, Douglas G Ward, Laetitia Aubry, Timothy Barrett, Sovan Sarkar.
      Mitochondrial dysfunction involving mitochondria-associated ER membrane (MAM) dysregulation is implicated in the pathogenesis of late-onset neurodegenerative diseases, but understanding is limited for rare early-onset conditions. Loss of the MAM-resident protein WFS1 causes Wolfram syndrome (WS), a rare early-onset neurodegenerative disease that has been linked to mitochondrial abnormalities. Here we demonstrate mitochondrial dysfunction in human induced pluripotent stem cell-derived neuronal cells of WS patients. VDAC1 is identified to interact with WFS1, whereas loss of this interaction in WS cells could compromise mitochondrial function. Restoring WFS1 levels in WS cells reinstates WFS1-VDAC1 interaction, which correlates with an increase in MAMs and mitochondrial network that could positively affect mitochondrial function. Genetic rescue by WFS1 overexpression or pharmacological agents modulating mitochondrial function improves the viability and bioenergetics of WS neurons. Our data implicate a role of WFS1 in regulating mitochondrial functionality and highlight a therapeutic intervention for WS and related rare diseases with mitochondrial defects.
    Keywords:  Cyclosporin A; Human induced pluripotent stem cell-derived neurons; Mitochondria-associated ER membrane; Mitochondrial dysfunction; Mitochondrial membrane potential; MnTBAP; Neurodegeneration; VDAC1; WFS1; Wolfram syndrome
    DOI:  https://doi.org/10.1016/j.stemcr.2023.04.002
  10. Annu Rev Biophys. 2023 05 09. 52 229-254
    Mitochondrial Ion Channels.
    Ildiko Szabo, Adam Szewczyk.
      Mitochondria are involved in multiple cellular tasks, such as ATP synthesis, metabolism, metabolite and ion transport, regulation of apoptosis, inflammation, signaling, and inheritance of mitochondrial DNA. The majority of the correct functioning of mitochondria is based on the large electrochemical proton gradient, whose component, the inner mitochondrial membrane potential, is strictly controlled by ion transport through mitochondrial membranes. Consequently, mitochondrial function is critically dependent on ion homeostasis, the disturbance of which leads to abnormal cell functions. Therefore, the discovery of mitochondrial ion channels influencing ion permeability through the membrane has defined a new dimension of the function of ion channels in different cell types, mainly linked to the important tasks that mitochondrial ion channels perform in cell life and death. This review summarizes studies on animal mitochondrial ion channels with special focus on their biophysical properties, molecular identity, and regulation. Additionally, the potential of mitochondrial ion channels as therapeutic targets for several diseases is briefly discussed.
    Keywords:  calcium channels; chloride channels; mitochondria; mitochondrial megachannel; porin; potassium channels
    DOI:  https://doi.org/10.1146/annurev-biophys-092622-094853
  11. Methods Mol Biol. 2023 ;2661 7-21
    Evolution: Mitochondrial Ribosomes Across Species.
    Rajendra K Agrawal, Soneya Majumdar.
      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.
    Keywords:  Cryo-EM structures; Evolution; MRPs; Mitochondrial rRNAs; Mitochondrial ribosomes
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_2
  12. 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
  13. 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
  14. Methods Mol Biol. 2023 ;2661 3-5
    Discovery of Mitochondrial Ribosomes.
    Thomas W O'Brien.
      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.
    Keywords:  Human MRP genes; Human ribosome purification; Mitochondrial disease; Mitochondrial ribosomal proteins; Mitochondrial translation
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_1
  15. 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
  16. 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
  17. Methods Mol Biol. 2023 ;2661 303-316
    Four-Color STED Super-Resolution RNA Fluorescent In Situ Hybridization and Immunocytochemistry to Visualize Mitochondrial mRNAs in Context with Mitochondrial Ribosomes.
    Christin A Albus, Rolando Berlinguer-Palmini, Zofia M Chrzanowska-Lightowlers, Robert N Lightowlers.
      High-resolution imaging has enabled scientists to explore the mitochondrial network at remarkable resolution. This has been exploited to help increase our knowledge of how mitochondrial gene expression is compartmentalized in cultured cells. Here, we provide detailed methodology to simultaneously visualize up to four components including mtDNA-encoded transcripts, submitochondrial marker proteins, mitoribosomal subunits, or core members of the translational apparatus using STED super-resolution nanoscopy.
    Keywords:  Mitochondrial mRNA; RNA in situ hybridization; STED super-resolution; Super-resolution nanoscopy
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_17
  18. 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
  19. 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
  20. 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
  21. 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
  22. Methods Mol Biol. 2023 ;2661 89-100
    Cryo-EM for Structure Determination of Mitochondrial Ribosome Samples.
    Hauke S Hillen.
      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.
    Keywords:  Mitochondrial ribosome; image processing; sample preparation; single-particle cryo-EM
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_6
  23. Methods Mol Biol. 2023 ;2661 317-328
    Digital RNase Footprinting of RNA-Protein Complexes and Ribosomes in Mitochondria.
    Danielle L Rudler, Stefan J Siira, Oliver Rackham, Aleksandra Filipovska.
      RNA-binding proteins and mitochondrial ribosomes have been found to be linchpins of mitochondrial gene expression in health and disease. The expanding repertoire of proteins that bind and regulate the mitochondrial transcriptome has necessitated the development of new tools and methods to examine their molecular functions. Next-generation sequencing technologies have advanced the RNA biology field through application of high-throughput methods to study RNA-protein interactions. Here we describe a digital RNase footprinting method to analyze protein and ribosome interactions with mitochondrially encoded transcripts that provides insight into their mechanisms and minimal binding sites. We provide details on RNase digestion and next-generation sequencing, along with computational analyses and visualization of the binding targets within the mitochondrial transcriptome.
    Keywords:  Bioinformatics; Footprinting; Mitoribosome; RNA-Seq; RNA-binding proteins; mtDNA
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_18
  24. 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
  25. Methods Mol Biol. 2023 ;2661 75-88
    Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation.
    Lena Thärichen, Robert Englmeier, Friedrich Förster.
      Cryoelectron tomography is a method to image biological samples three-dimensionally at molecular resolution. This modality provides insights into intracellular processes in their physiological settings. Obtaining a high-quality sample for cryoelectron tomography on mitochondria, however, can be challenging. In this chapter, we describe the crucial steps from sample preparation to data acquisition enabling studies of mitochondrial translation in situ by cryoelectron tomography. We provide detailed protocols for yeast and human mitochondria preparations yielding a high concentration of intact mitochondrial vesicles on cryo-EM grids. In addition, we describe a workflow for particle identification and spatial mapping in context of the organelle.
    Keywords:  Cryoelectron tomography; Mitochondria; Ribosome; Translation
    DOI:  https://doi.org/10.1007/978-1-0716-3171-3_5
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