bims-midmar Biomed News
on Mitochondrial DNA maintenance and replication
Issue of 2021–08–08
seventeen papers selected by
Flavia Söllner, Ludwig-Maximilians University
  1. Mitochondrial Genomic Landscape: A Portrait of the Mitochondrial Genome 40 Years after the First Complete Sequence.
  2. Mitochondrial RNase H1 activity regulates R-loop homeostasis to maintain genome integrity and enable early embryogenesis in Arabidopsis.
  3. Human Mitochondrial RNA Processing and Modifications: Overview.
  4. The Roles of Assembly Factors in Mammalian Mitoribosome Biogenesis.
  5. Functional Diversity of Mitochondrial Peptidyl-tRNA Hydrolase ICT1 in Human Cells.
  6. Instability in NAD+ metabolism leads to impaired cardiac mitochondrial function and communication.
  7. Potential of Mitochondrial Genome Editing for Human Fertility Health.
  8. Mitotherapy: Unraveling a Promising Treatment for Disorders of the Central Nervous System and Other Systemic Conditions.
  9. Fluorescent Materials for Monitoring Mitochondrial Biology.
  10. Alterations in mitochondrial morphology as a key driver of immunity and host defence.
  11. The spectrum of mitochondrial DNA (mtDNA) mutations in pediatric CNS tumors.
  12. Atomic force microscopy in super-resolution.
  13. Excitation light-induced phototoxicity during fluorescence imaging.
  14. Super-resolution microscopy of chromatin fibers and quantitative DNA methylation analysis of DNA fiber preparations.
  15. A fluorescence imaging based-assay to monitor mitophagy in cultured hepatocytes and mouse liver.
  16. iDeepSubMito: identification of protein submitochondrial localization with deep learning.
  17. Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae.
  1. Life (Basel). 2021 Jul 06. pii: 663. [Epub ahead of print]11(7):
    Mitochondrial Genomic Landscape: A Portrait of the Mitochondrial Genome 40 Years after the First Complete Sequence.
    Alessandro Formaggioni, Andrea Luchetti, Federico Plazzi.
      Notwithstanding the initial claims of general conservation, mitochondrial genomes are a largely heterogeneous set of organellar chromosomes which displays a bewildering diversity in terms of structure, architecture, gene content, and functionality. The mitochondrial genome is typically described as a single chromosome, yet many examples of multipartite genomes have been found (for example, among sponges and diplonemeans); the mitochondrial genome is typically depicted as circular, yet many linear genomes are known (for example, among jellyfish, alveolates, and apicomplexans); the chromosome is normally said to be "small", yet there is a huge variation between the smallest and the largest known genomes (found, for example, in ctenophores and vascular plants, respectively); even the gene content is highly unconserved, ranging from the 13 oxidative phosphorylation-related enzymatic subunits encoded by animal mitochondria to the wider set of mitochondrial genes found in jakobids. In the present paper, we compile and describe a large database of 27,873 mitochondrial genomes currently available in GenBank, encompassing the whole eukaryotic domain. We discuss the major features of mitochondrial molecular diversity, with special reference to nucleotide composition and compositional biases; moreover, the database is made publicly available for future analyses on the MoZoo Lab GitHub page.
    Keywords:  Eukaryota; compositional bias; mitochondrial genome; mtDNA architecture; mtDNA expansion; mtDNA structure; nucleotide composition; strand asymmetry
    DOI:  https://doi.org/10.3390/life11070663
  2. PLoS Biol. 2021 Aug;19(8): e3001357
    Mitochondrial RNase H1 activity regulates R-loop homeostasis to maintain genome integrity and enable early embryogenesis in Arabidopsis.
    Lingling Cheng, Wenjie Wang, Yao Yao, Qianwen Sun.
      Plant mitochondrial genomes undergo frequent homologous recombination (HR). Ectopic HR activity is inhibited by the HR surveillance pathway, but the underlying regulatory mechanism is unclear. Here, we show that the mitochondrial RNase H1 AtRNH1B impairs the formation of RNA:DNA hybrids (R-loops) and participates in the HR surveillance pathway in Arabidopsis thaliana. AtRNH1B suppresses ectopic HR at intermediate-sized repeats (IRs) and thus maintains mitochondrial DNA (mtDNA) replication. The RNase H1 AtRNH1C is restricted to the chloroplast; however, when cells lack AtRNH1B, transport of chloroplast AtRNH1C into the mitochondria secures HR surveillance, thus ensuring the integrity of the mitochondrial genome and allowing embryogenesis to proceed. HR surveillance is further regulated by the single-stranded DNA-binding protein ORGANELLAR SINGLE-STRANDED DNA BINDING PROTEIN1 (OSB1), which decreases the formation of R-loops. This study uncovers a facultative dual targeting mechanism between organelles and sheds light on the roles of RNase H1 in organellar genome maintenance and embryogenesis.
    DOI:  https://doi.org/10.1371/journal.pbio.3001357
  3. Int J Mol Sci. 2021 Jul 27. pii: 7999. [Epub ahead of print]22(15):
    Human Mitochondrial RNA Processing and Modifications: Overview.
    Marta Jedynak-Slyvka, Agata Jabczynska, Roman J Szczesny.
      Mitochondria, often referred to as the powerhouses of cells, are vital organelles that are present in almost all eukaryotic organisms, including humans. They are the key energy suppliers as the site of adenosine triphosphate production, and are involved in apoptosis, calcium homeostasis, and regulation of the innate immune response. Abnormalities occurring in mitochondria, such as mitochondrial DNA (mtDNA) mutations and disturbances at any stage of mitochondrial RNA (mtRNA) processing and translation, usually lead to severe mitochondrial diseases. A fundamental line of investigation is to understand the processes that occur in these organelles and their physiological consequences. Despite substantial progress that has been made in the field of mtRNA processing and its regulation, many unknowns and controversies remain. The present review discusses the current state of knowledge of RNA processing in human mitochondria and sheds some light on the unresolved issues.
    Keywords:  RNA decay; RNA modifications; RNA processing; mitochondria; mitochondrial genome; mitochondrial transcription
    DOI:  https://doi.org/10.3390/ijms22157999
  4. Mitochondrion. 2021 Jul 30. pii: S1567-7249(21)00101-X. [Epub ahead of print]
    The Roles of Assembly Factors in Mammalian Mitoribosome Biogenesis.
    Taru Hilander, Christopher B Jackson, Marius Robciuc, Tanzeela Bashir, Hongxia Zhao.
      As ancient bacterial endosymbionts of eukaryotic cells, mitochondria have retained their own circular DNA as well as protein translation system including mitochondrial ribosomes (mitoribosomes). In recent years, methodological advancements in cryoelectron microscopy and mass spectrometry have revealed the extent of the evolutionary divergence of mitoribosomes from their bacterial ancestors and their adaptation to the synthesis of 13 mitochondrial DNA encoded oxidative phosphorylation complex subunits. In addition to the structural data, the first assembly pathway maps of mitoribosomes have started to emerge and concomitantly also the assembly factors involved in this process to achieve fully translational competent particles. These transiently associated factors assist in the intricate assembly process of mitoribosomes by enhancing protein incorporation, ribosomal RNA folding and modification, and by blocking premature or non-native protein binding, for example. This review focuses on summarizing the current understanding of the known mammalian mitoribosome assembly factors and discussing their possible roles in the assembly of small or large mitoribosomal subunits.
    Keywords:  mitochondrial translation; mitoribosome assembly factors; mitoribosome biogenesis
    DOI:  https://doi.org/10.1016/j.mito.2021.07.008
  5. Front Mol Biosci. 2021 ;8 716885
    Functional Diversity of Mitochondrial Peptidyl-tRNA Hydrolase ICT1 in Human Cells.
    I V Chicherin, S V Dukhalin, R A Khannanov, M V Baleva, S A Levitskii, M V Patrushev, P V Sergiev, P Kamenski.
      Mitochondria are energy producing organelles of the eukaryotic cell, involved in the synthesis of key metabolites, calcium homeostasis and apoptosis. Protein biosynthesis in these organelles is a relic of its endosymbiotic origin. While mitochondrial translational factors have homologues among prokaryotes, they possess a number of unique traits. Remarkably as many as four mammalian mitochondrial proteins possess a clear similarity with translation termination factors. The review focuses on the ICT1, which combines several functions. It is a non-canonical termination factor for protein biosynthesis, a rescue factor for stalled mitochondrial ribosomes, a structural protein and a regulator of proliferation, cell cycle, and apoptosis. Such a diversity of roles demonstrates the high functionality of mitochondrial translation associated proteins and their relationship with numerous processes occurring in a living cell.
    Keywords:  apoptosis; cell cycle; mitochondria; proliferation; regulation; ribosome, signaling; termination; translation
    DOI:  https://doi.org/10.3389/fmolb.2021.716885
  6. Elife. 2021 Aug 03. pii: e59828. [Epub ahead of print]10
    Instability in NAD+ metabolism leads to impaired cardiac mitochondrial function and communication.
    Knut H Lauritzen, Maria Belland Olsen, Mohammed Shakil Ahmed, Kuan Yang, Johanne Egge Rinholm, Linda H Bergersen, Qin Ying Esbensen, Lars Jansen Sverkeli, Mathias Ziegler, Håvard Attramadal, Bente Halvorsen, Pål Aukrust, Arne Yndestad.
      Poly(ADP-ribose) polymerase (PARP) enzymes initiate (mt)DNA repair mechanisms and use nicotinamide adenine dinucleotide (NAD+) as energy source. Prolonged PARP activity can drain cellular NAD+ reserves, leading to de-regulation of important molecular processes. Here, we provide evidence of a pathophysiological mechanism that connects mtDNA damage to cardiac dysfunction via reduced NAD+ levels and loss of mitochondrial function and communication. Using a transgenic model, we demonstrate that high levels of mice cardiomyocyte mtDNA damage cause a reduction in NAD+ levels due to extreme DNA repair activity, causing impaired activation of NAD+-dependent SIRT3. In addition, we show that myocardial mtDNA damage in combination with high dosages of nicotinamideriboside (NR) causes an inhibition of sirtuin activity due to accumulation of nicotinamide (NAM), in addition to irregular cardiac mitochondrial morphology. Consequently, high doses of NR should be used with caution, especially when cardiomyopathic symptoms are caused by mitochondrial dysfunction and instability of mtDNA.
    Keywords:  DNA repair; NAD+; SIRT3; biochemistry; cardiovascular disease; chemical biology; human; mitochondrial dna; mouse; nicotinamide riboside
    DOI:  https://doi.org/10.7554/eLife.59828
  7. Front Genet. 2021 ;12 673951
    Potential of Mitochondrial Genome Editing for Human Fertility Health.
    Lin Fu, Yu-Xin Luo, Ying Liu, Hui Liu, Hong-Zhen Li, Yang Yu.
      Mitochondrial DNA (mtDNA) encodes vital proteins and RNAs for the normal functioning of the mitochondria. Mutations in mtDNA leading to mitochondrial dysfunction are relevant to a large spectrum of diseases, including fertility disorders. Since mtDNA undergoes rather complex processes during gametogenesis and fertilization, clarification of the changes and functions of mtDNA and its essential impact on gamete quality and fertility during this process is of great significance. Thanks to the emergence and rapid development of gene editing technology, breakthroughs have been made in mitochondrial genome editing (MGE), offering great potential for the treatment of mtDNA-related diseases. In this review, we summarize the features of mitochondria and their unique genome, emphasizing their inheritance patterns; illustrate the role of mtDNA in gametogenesis and fertilization; and discuss potential therapies based on MGE as well as the outlook in this field.
    Keywords:  gene therapy; human infertility; mitochondria DNA mutation; mitochondrial DNA; mitochondrial genome editing
    DOI:  https://doi.org/10.3389/fgene.2021.673951
  8. Cells. 2021 Jul 20. pii: 1827. [Epub ahead of print]10(7):
    Mitotherapy: Unraveling a Promising Treatment for Disorders of the Central Nervous System and Other Systemic Conditions.
    Gabriel Nascimento-Dos-Santos, Eduardo de-Souza-Ferreira, Rafael Linden, Antonio Galina, Hilda Petrs-Silva.
      Mitochondria are key players of aerobic respiration and the production of adenosine triphosphate and constitute the energetic core of eukaryotic cells. Furthermore, cells rely upon mitochondria homeostasis, the disruption of which is reported in pathological processes such as liver hepatotoxicity, cancer, muscular dystrophy, chronic inflammation, as well as in neurological conditions including Alzheimer's disease, schizophrenia, depression, ischemia and glaucoma. In addition to the well-known spontaneous cell-to-cell transfer of mitochondria, a therapeutic potential of the transplant of isolated, metabolically active mitochondria has been demonstrated in several in vitro and in vivo experimental models of disease. This review explores the striking outcomes achieved by mitotherapy thus far, and the most relevant underlying data regarding isolated mitochondria transplantation, including mechanisms of mitochondria intake, the balance between administration and therapy effectiveness, the relevance of mitochondrial source and purity and the mechanisms by which mitotherapy is gaining ground as a promising therapeutic approach.
    Keywords:  central nervous system; mitochondria; mitotherapy; neurodegeneration; therapy
    DOI:  https://doi.org/10.3390/cells10071827
  9. Materials (Basel). 2021 Jul 27. pii: 4180. [Epub ahead of print]14(15):
    Fluorescent Materials for Monitoring Mitochondrial Biology.
    Yeonjeong Chu, Jisoo Park, Eunha Kim, Sanghee Lee.
      Mitochondria play important roles in diverse cellular processes such as energy production, cellular metabolism, and apoptosis to promote cell death. To investigate mitochondria-associated biological processes such as structure, dynamics, morphological change, metabolism, and mitophagy, there exists a continuous demand for visualizing and monitoring techniques elucidating mitochondrial biology and disease-relevancy. Due to the advantages of high sensitivity and practicality, fluorescence phenomena have been most widely used as scientific techniques for the visualization of biological phenomena and systems. In this review, we briefly overview the different types of fluorescent materials such as chemical probes, peptide- or protein-based probes, and nanomaterials for monitoring mitochondrial biology.
    Keywords:  fluorescence imaging; fluorescent chemical probes; fluorescent nanosensors; mitochondria; mitochondria-targeting peptides
    DOI:  https://doi.org/10.3390/ma14154180
  10. EMBO Rep. 2021 Aug 02. e53086
    Alterations in mitochondrial morphology as a key driver of immunity and host defence.
    Mariana P Cervantes-Silva, Shannon L Cox, Annie M Curtis.
      Mitochondria are dynamic organelles whose architecture changes depending on the cell's energy requirements and other signalling events. These structural changes are collectively known as mitochondrial dynamics. Mitochondrial dynamics are crucial for cellular functions such as differentiation, energy production and cell death. Importantly, it has become clear in recent years that mitochondrial dynamics are a critical control point for immune cell function. Mitochondrial remodelling allows quiescent immune cells to rapidly change their metabolism and become activated, producing mediators, such as cytokines, chemokines and even metabolites to execute an effective immune response. The importance of mitochondrial dynamics in immunity is evident, as numerous pathogens have evolved mechanisms to manipulate host cell mitochondrial remodelling in order to promote their own survival. In this review, we comprehensively address the roles of mitochondrial dynamics in immune cell function, along with modulation of host cell mitochondrial morphology during viral and bacterial infections to facilitate either pathogen survival or host immunity. We also speculate on what the future may hold in terms of therapies targeting mitochondrial morphology for bacterial and viral control.
    Keywords:  bacteria; immune response; mitochondrial dynamics; therapy; virus
    DOI:  https://doi.org/10.15252/embr.202153086
  11. Neurooncol Adv. 2021 Jan-Dec;3(1):3(1): vdab074
    The spectrum of mitochondrial DNA (mtDNA) mutations in pediatric CNS tumors.
    Kristiyana Kaneva, Katrina O'Halloran, Petr Triska, Xiyu Liu, Daria Merkurjev, Moiz Bootwalla, Alex Ryutov, Jennifer A Cotter, Dejerianne Ostrow, Jaclyn A Biegel, Xiaowu Gai.
       Background: We previously established the landscape of mitochondrial DNA (mtDNA) mutations in 23 subtypes of pediatric malignancies, characterized mtDNA mutation profiles among these subtypes, and provided statistically significant evidence for a contributory role of mtDNA mutations to pediatric malignancies.
    Methods: To further delineate the spectrum of mtDNA mutations in pediatric central nervous system (CNS) tumors, we analyzed 545 tumor-normal paired whole-genome sequencing datasets from the Children's Brain Tumor Tissue Consortium.
    Results: Germline mtDNA variants were used to determine the haplogroup, and maternal ancestry, which was not significantly different among tumor types. Among 166 (30.5%) tumors we detected 220 somatic mtDNA mutations, primarily missense mutations (36.8%), as well as 22 loss-of-function mutations. Different pediatric CNS tumor subtypes had distinct mtDNA mutation profiles. The number of mtDNA mutations per tumor ranged from 0.20 (dysembryoplastic neuroepithelial tumor [DNET]) to 0.75 (meningiomas). The average heteroplasmy was 10.7%, ranging from 4.6% in atypical teratoid/rhabdoid tumor (AT/RT) to 26% in diffuse intrinsic pontine glioma. High-grade gliomas had a significant higher number of mtDNA mutations per sample than low-grade gliomas (0.6 vs 0.27) (P = .004), with almost twice as many missense mtDNA mutations per sample (0.24 vs 0.11), and higher average heteroplasmy levels (16% vs 10%). Recurrent mtDNA mutations may represent hotspots which may serve as biologic markers of disease.
    Conclusions: Our findings demonstrate varying contributions of mtDNA mutations in different subtypes of CNS tumors. Sequencing the mtDNA genome may ultimately be used to characterize CNS tumors at diagnosis and monitor disease progression.
    Keywords:  CBTN; CBTTC; CNS tumors; brain tumors; mitochondrial DNA; pediatric
    DOI:  https://doi.org/10.1093/noajnl/vdab074
  12. Nat Methods. 2021 Aug;18(8): 859
    Atomic force microscopy in super-resolution.
    Nina Vogt.
      
    DOI:  https://doi.org/10.1038/s41592-021-01246-9
  13. J Biosci. 2021 ;pii: 78. [Epub ahead of print]46
    Excitation light-induced phototoxicity during fluorescence imaging.
    Abhilasha Ojha, Navin Kumar Ojha.
      Fluorescence microscopy has revolutionized life science research. It is a powerful technique to visualize molecular structures, physiological functions, and dynamic processes in living cells, tissues, and organisms. Excitation-light and/or excited-fluorescent proteins-mediated dysregulation in cell physiology are widely known but poorly considered. Although there are vast applications of fluorescence microscopy in research, imaging results may suffer from the excitation-light-induced artefacts. Here, we highlight potential excitation light-induced alterations in cell functions thereby misinterpretation of imaging results and strategies to minimize common artefacts that can be produced during imaging experiments.
  14. J Cell Sci. 2021 Aug 01. pii: jcs258374. [Epub ahead of print]134(15):
    Super-resolution microscopy of chromatin fibers and quantitative DNA methylation analysis of DNA fiber preparations.
    Michal Franek, Agata Kilar, Petr Fojtík, Marie Olšinová, Aleš Benda, Vladimír Rotrekl, Martina Dvořáčková, Jíří Fajkus.
      Analysis of histone variants and epigenetic marks is dominated by genome-wide approaches in the form of chromatin immunoprecipitation-sequencing (ChIP-seq) and related methods. Although uncontested in their value for single-copy genes, mapping the chromatin of DNA repeats is problematic for biochemical techniques that involve averaging of cell populations or analysis of clusters of tandem repeats in a single-cell analysis. Extending chromatin and DNA fibers allows us to study the epigenetics of individual repeats in their specific chromosomal context, and thus constitutes an important tool for gaining a complete understanding of the epigenetic organization of genomes. We report that using an optimized fiber extension protocol is essential in order to obtain more reproducible data and to minimize the clustering of fibers. We also demonstrate that the use of super-resolution microscopy is important for reliable evaluation of the distribution of histone modifications on individual fibers. Furthermore, we introduce a custom script for the analysis of methylation levels on DNA fibers and apply it to map the methylation of telomeres, ribosomal genes and centromeres.
    Keywords:  Chromatin; DNA; Fiber; Methylation; Microscopy
    DOI:  https://doi.org/10.1242/jcs.258374
  15. Liver Res. 2021 Mar;5(1): 16-20
    A fluorescence imaging based-assay to monitor mitophagy in cultured hepatocytes and mouse liver.
    Xiaowen Ma, Wen-Xing Ding.
       Background and aim: Mitophagy is a lysosomal degradation pathway that selectively removes damaged, aged and dysfunctional mitochondria. Recent advances in understanding mitophagy highlight its importance in various physiological and pathological conditions including liver diseases. However, reliable quantitative assays to monitor mitophagy in cultured cells and in tissues are still scarce.
    Methods: We describe a detailed protocol for monitoring mitophagy in primary cultured hepatocytes and mouse livers using cytochrome C oxidase subunit 8 (Cox8)-enhanced green fluorescent protein (EGFP)-mCherry, a dual color fluorescence based-imaging method.
    Results: Mitochondria are visualized in yellow fluorescence due to the merged EGFP and mCherry signal. In contrast, autolysosome enclosed mitochondria are shown as red puncta due to quenching of EGFP green fluorescence in acidic compartments. Quantifying the number of red-only puncta in each cell can obtain a quantitative measure for mitophagy.
    Conclusions: Cox8-EGFP-mCherry assay can specifically target to mitochondria and be used to monitor mitophagy in vitro and in vivo.
    Keywords:  Autophagy; Cox8-EGFP-mCherry; Fluorescence microscopy; Lysosome; Mitochondria
    DOI:  https://doi.org/10.1016/j.livres.2020.12.002
  16. Brief Bioinform. 2021 Jul 30. pii: bbab288. [Epub ahead of print]
    iDeepSubMito: identification of protein submitochondrial localization with deep learning.
    Zilong Hou, Yuning Yang, Hui Li, Ka-Chun Wong, Xiangtao Li.
      Mitochondria are membrane-bound organelles containing over 1000 different proteins involved in mitochondrial function, gene expression and metabolic processes. Accurate localization of those proteins in the mitochondrial compartments is critical to their operation. A few computational methods have been developed for predicting submitochondrial localization from the protein sequences. Unfortunately, most of these computational methods focus on employing biological features or evolutionary information to extract sequence features, which greatly limits the performance of subsequent identification. Moreover, the efficiency of most computational models is still under explored, especially the deep learning feature, which is promising but requires improvement. To address these limitations, we propose a novel computational method called iDeepSubMito to predict the location of mitochondrial proteins to the submitochondrial compartments. First, we adopted a coding scheme using the ProteinELMo to model the probability distribution over the protein sequences and then represent the protein sequences as continuous vectors. Then, we proposed and implemented convolutional neural network architecture based on the bidirectional LSTM with self-attention mechanism, to effectively explore the contextual information and protein sequence semantic features. To demonstrate the effectiveness of our proposed iDeepSubMito, we performed cross-validation on two datasets containing 424 proteins and 570 proteins respectively, and consisting of four different mitochondrial compartments (matrix, inner membrane, outer membrane and intermembrane regions). Experimental results revealed that our method outperformed other computational methods. In addition, we tested iDeepSubMito on the M187, M983 and MitoCarta3.0 to further verify the efficiency of our method. Finally, the motif analysis and the interpretability analysis were conducted to reveal novel insights into subcellular biological functions of mitochondrial proteins. iDeepSubMito source code is available on GitHub at https://github.com/houzl3416/iDeepSubMito.
    Keywords:  deep learning; protein sequences; protein submitochondrial localization
    DOI:  https://doi.org/10.1093/bib/bbab288
  17. J Vis Exp. 2021 Jul 19.
    Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae.
    Fernando Gomes, Helena Turano, Angélica Ramos, Luis E S Netto.
      Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.
    DOI:  https://doi.org/10.3791/62853
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