bims-cytox1 2024-11-03 papers

bims-cytox1

Biomed News

on Cytochrome oxidase subunit 1

Issue of 2024‒11‒03
twenty papers selected by
Gavin McStay, Liverpool John Moores University

Assays to monitor mitochondrial translation.
In vitro import of mitochondrial precursor proteins into yeast mitochondria.
Oxidative phosphorylation related gene COA6 is a novel indicator for the prognosis and immune response in lung adenocarcinoma.
In organello silencing of mitochondrial gene expression.
Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria.
Analysis of mitochondrial translation using click chemistry.
Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol.
Monitoring mitochondrial protein import by live cell imaging.
The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import.
Quantifying mitochondrial protein import by mePRODmt proteomics.
Monitoring mitochondrial precursor processing and presequence peptide degradation.
Isolation of yeast mitochondria by differential centrifugation.
Subcellular fractionation by differential centrifugation for mitochondrial studies.
Analysis of mitochondrial targeting signal cleavage and protein processing by mass spectrometry.
Analysis of targeting signals for mitochondrial intermembrane space import.
Import of mitochondrial precursor proteins into mitochondria of semi-intact yeast cells.
A novel m.5906G > a variant in MT-CO1 causes MELAS/Leigh overlap syndrome.
Ectopic expression of the mitochondrial protein COXFA4L3 in human sperm acrosome and its potential application in the selection of male infertility treatments.
COX15 deficiency causes oocyte ferroptosis.
Isolation of yeast mitochondria by affinity purification using magnetic beads.

Methods Enzymol. 2024 ;pii: S0076-6879(24)00385-9. [Epub ahead of print]706 519-532

Assays to monitor mitochondrial translation.

Sung-Jun Jung, Martin Ott.

  The complexes of the oxidative phosphorylation (OXPHOS) system found in the mitochondrial inner membrane comprises nuclear and mitochondrial-encoded proteins. The mitochondrial-encoded subunits of the OXPHOS complexes play vital catalytic roles for OXPHOS. These subunits are inserted co-translationally into the inner membrane, where they are matured and assembled with nuclear encoded subunits, requiring a set of OXPHOS assembly and quality control factors. Hence, monitoring the fate of newly synthesized mitochondrial-encoded polypeptides is a basic and essential approach for exploring OXPHOS biogenesis and the related protein quality control processes. Here, we describe a detailed protocol for labeling mitochondrial encoded proteins with 35S-methionine for pulse and pulse/chase experiments, both in vivo and in organello, using the yeast Saccharomyces cerevisiae as the model. These methods enable analyses of the early steps during the biogenesis and turnover of mitochondrial-encoded proteins.

Keywords:  35S-methionine; Mitochondrial translation; isolated mitochondria; protein stability; protein synthesis; yeast

DOI:  https://doi.org/10.1016/bs.mie.2024.07.045
Methods Enzymol. 2024 ;pii: S0076-6879(24)00342-2. [Epub ahead of print]706 347-363

In vitro import of mitochondrial precursor proteins into yeast mitochondria.

Soraya Badrie, Julian Alexander Draken, Dejana Mokranjac.

  Mitochondria contain about 1000 different proteins, only a handful of which are encoded in the mitochondrial genome. The remaining c. 99% of mitochondrial proteins are encoded in the nuclear genome, synthesized on cytosolic ribosomes as precursor proteins with specific mitochondrial targeting signals and are subsequently imported into the organelle. Mitochondrial targeting signals are very diverse and mitochondria therefore also have a number of very sophisticated molecular machines that recognize, import and sort mitochondrial precursor proteins to the different mitochondrial subcompartments. The ability to synthesize mitochondrial precursor proteins in vitro and subsequently import them into isolated mitochondria has revolutionized our understanding of mitochondrial protein import pathways. Here, we describe the basic protocol for synthesis of mitochondrial precursor proteins in vitro and their subsequent import into isolated mitochondria from yeast Saccharomyces cerevisiae, the method which was used to elucidate and characterize the vast majority of mitochondrial protein import pathways.

Keywords:  (35)S-methionine; In vitro import; In vitro transcription and translation; Isolated mitochondria; Protein translocation; Saccharomyces cerevisiae

DOI:  https://doi.org/10.1016/bs.mie.2024.07.016
Sci Rep. 2024 10 29. 14(1): 25970

Oxidative phosphorylation related gene COA6 is a novel indicator for the prognosis and immune response in lung adenocarcinoma.

Wenting Liu, Yantao Jiang, Guoli Li, Dingzhi Huang, Tingting Qin.

  Although the initial research focused on glycolysis, mitochondrial oxidative phosphorylation has become a major target of cancer cells. Cytochrome C oxidase assembly factor 6 (COA6) is a conserved assembly factor necessary for complex IV biogenesis. Nevertheless, the clinical predictive value of COA6, especially its correlation with immune cell infiltration in lung adenocarcinoma (LUAD), has not yet been elucidated. COA6 exhibited higher expression levels in LUAD cells and tumor tissues compared to normal tissues. Additionally, heightened COA6 expression was associated with reduced overall survival (OS) and advanced tumor stage. Apart from its role in mitochondrial respiratory processes, COA6 may be involved in the process of antigen binding, immunoglobulin receptor binding. Interestingly, we observed a positive correlation between COA6 expression and tumor mutational burden (TMB), as well as a significant association with decreased immune cell infiltration. COA6 was linked to resistance against gemcitabine and etoposide. We verified that COA6 was highly expressed in LUAD experimentally and cell proliferation was inhibited after COA6 knockdown. Thus, we conclude that the expression of COA6 was correlated with reduced immune cell infiltration. Additionally, COA6 functioned as a biomarker for drug sensitivity and the prognosis of lung adenocarcinoma.

Keywords:  Biomarker; COA6; Immune infiltration; Lung adenocarcinoma; Prognosis

DOI:  https://doi.org/10.1038/s41598-024-77775-y
Methods Enzymol. 2024 ;pii: S0076-6879(24)00371-9. [Epub ahead of print]706 501-518

In organello silencing of mitochondrial gene expression.

Mats Koschel, Luis Daniel Cruz-Zaragoza.

  Mitochondria contain proteins from two genetic origins. Most mitochondrial proteins are encoded in the nuclear genome, translated in the cytosol, and subsequently imported into the different mitochondrial sub-compartments. A small number is encoded in the mitochondrial DNA (mtDNA). The manipulation of the mtDNA gene expression represents a challenge. Here, we present an in vitro approach using morpholinos chemically linked to a precursor protein to silence gene expression in purified human mitochondria. The protocol is demonstrated with a Jac1-morpholino chimera specifically targeting COX1 mRNA. The chimera import and mitochondrial translation requirements are described in a step-by-step procedure, where the dose-dependent effect of reducing COX1 translation is observed. The affinity and specificity of chimera-mRNA binding also show great applicability to purify transcript-associated proteins by using the imported chimera construct as bait for immunoprecipitation. This new strategy opens up the possibility to address mechanistic questions about gene expression and physiology in mitochondria.

Keywords:  Gene expression; In vitro; Mitochondria; Morpholino; Silencing

DOI:  https://doi.org/10.1016/bs.mie.2024.07.035
Methods Enzymol. 2024 ;pii: S0076-6879(24)00370-7. [Epub ahead of print]706 365-390

Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria.

Jordan J Crameri, Diana Stojanovski.

  Mitochondrial protein import is a complex process governing the delivery of the organelle's proteome. This process, in turn, is essential for maintaining mitochondrial function and cellular homeostasis. Initiated by protein synthesis in the cytoplasm, precursor proteins destined for the mitochondria possess targeting signals that guide them to the mitochondrial surface. At mitochondria, the translocation of proteins across the mitochondrial membranes involves an intricate interplay between translocases, chaperones, and receptors. The mitochondrial import assay offers researchers the opportunity to recapitulate the process of protein import in vitro. The assay has served as an indispensable tool in helping decipher the intricacies of protein translocation into mitochondria, first in fungal models, and subsequently in higher eukaryotic models. In this chapter, we will describe how protein import can be assayed using mammalian mitochondria and provide insight into the types of questions that can be addressed in mammalian mitochondrial biology using this experimental approach.

Keywords:  in vitro; mitochondria; protein import; translocase

DOI:  https://doi.org/10.1016/bs.mie.2024.07.034
Methods Enzymol. 2024 ;pii: S0076-6879(24)00384-7. [Epub ahead of print]706 533-547

Analysis of mitochondrial translation using click chemistry.

Roya Yousefi, Sven Dennerlein.

  Mitochondria contain their own gene expression machinery, which synthesizes core subunits of the oxidative phosphorylation system. Monitoring mitochondrial translation within spatial compartments of cells is difficult. Here we describe a method to visualize mitochondrial translation within defined parts of cells, using a click chemistry approach. This method can be applied to different cell types such as neurons and allows detection of newly synthesized mitochondrial proteins in spatial resolution using microscopy techniques. Furthermore, using click chemistry, mitochondrial translation can also be monitored by standard SDS-PAGE. The described method avenues the analysis of newly synthesized mitochondrial encoded proteins in the cellular context, by avoiding the usage of radioactive components.

Keywords:  Microscopy; Mitochondria; Mitochondrial translation

DOI:  https://doi.org/10.1016/bs.mie.2024.07.044
Methods Enzymol. 2024 ;pii: S0076-6879(24)00356-2. [Epub ahead of print]706 287-311

Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol.

Klaudia K Maruszczak, Agnieszka Chacinska.

  The vast majority of mitochondrial precursor proteins is synthesized in the cytosol and subsequently imported into the organelle with the help of targeting signals that are present within these proteins. Disruptions in mitochondrial import will result in the accumulation of the organellar precursors in the cytosol of the cell. If mislocalized proteins exceed their critical concentrations, they become prone to aggregation. Under certain circumstances, protein aggregation becomes an irreversible process, which eventually endangers cellular health. Impairment in mitochondrial biogenesis and its effect on cellular protein homeostasis were recently linked to neurodegeneration, therefore placing this process in the center of attention. In this chapter, we are presenting a set of techniques that allows to monitor and study mitochondrial precursor protein aggregates upon mitochondrial dysfunction in the cytosol of both yeast and human cells.

Keywords:  Mitochondria; Mitochondrial dysfunction; Mitochondrial import; Protein aggregates

DOI:  https://doi.org/10.1016/bs.mie.2024.07.020
Methods Enzymol. 2024 ;pii: S0076-6879(24)00363-X. [Epub ahead of print]706 437-447

Monitoring mitochondrial protein import by live cell imaging.

F X Reymond Sutandy.

  The majority of mitochondrial proteins are synthesized in the cytosol and must be imported into mitochondria to attain their mature forms and execute their functions. Disruption of mitochondrial functions, whether caused by external or internal stress, may compromise mitochondrial protein import. Therefore, monitoring mitochondrial protein import has become a standard approach to assess mitochondrial health and gain insights into mitochondrial biology, especially during stress. This chapter describes a detailed protocol for monitoring mitochondrial import in live cells using microscopy. Co-localization between mitochondria and a genetic reporter of mitochondrially targeted enhanced GFP (eGFP) is employed to evaluate mitochondrial protein import efficiency under different physiological conditions. Overall, this technique provides a simple and robust approach to assess mitochondrial protein import efficiency within its native cellular environment.

Keywords:  MTS; mitochondria; protein import; stress response

DOI:  https://doi.org/10.1016/bs.mie.2024.07.027
Methods Enzymol. 2024 ;pii: S0076-6879(24)00369-0. [Epub ahead of print]706 407-436

The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import.

Hope I Needs, Youmian Yan, Natalie M Niemi, Ian Collinson.

  The NanoLuc split luciferase assay has proven to be a powerful tool for the analysis of protein translocation. Its flexibility has enabled in vivo, ex vivo, and in vitro studies-including systems reconstituting protein transport from pure components. The assay has been particularly useful in the characterization of bacterial secretion and mitochondrial protein import. In the latter case, MitoLuc has been developed for the investigation of the TIM23-pathway via import into the matrix of isolated yeast mitochondria. Subsequent analysis identified three distinct phases of import, rather than in a single continuous step. The assay has also been developed to monitor import into the mitochondrial matrix of intact cultured cells. This latter innovation has laid the foundations for further analysis of the import process in humans, including the consequences of interactions with cytosolic factors and neighboring organelles. The versatility of the MitoLuc assay is conducive for its adaptation to also monitor import into the inter-membrane space (MIA-pathway), and into the inner-membrane via the TIM22- and TIM23-complexes. Here, we present detailed protocols for the application of MitoLuc to mitochondria isolated from yeast and to those within cultured human cells.

Keywords:  Cell culture; Luciferase; MitoLuc assay; Mitochondrial biogenesis; Mitochondrial protein import; NanoLuc; Protein translocation; Yeast

DOI:  https://doi.org/10.1016/bs.mie.2024.07.033
Methods Enzymol. 2024 ;pii: S0076-6879(24)00347-1. [Epub ahead of print]706 449-474

Quantifying mitochondrial protein import by mePRODmt proteomics.

Süleyman Bozkurt, Bhavesh S Parmar, Christian Münch.

  Mitochondrial protein import is crucial for maintaining cellular health and homeostasis. Disruptions in this process have been linked to various diseases. Traditional methods for studying mitochondrial protein import predominantly focus on individual proteins and lack the dynamic resolution needed to fully appreciate the complexity of mitochondrial proteostasis and protein trafficking. To address these limitations, we developed a technique called mitochondria-specific multiplexed enhanced protein dynamics (mePRODmt). This method is a novel application of the mePROD methodology and utilizes pulsed stable isotope labeling with amino acids in cell culture (pSILAC)-based proteomics approach to study transient mitochondrial protein import. This chapter outlines the mePRODmt protocol, which includes the preparation of heavy SILAC-labeled peptides for boosting overall mitochondrial peptide signals (booster), SILAC labeling of cultured cells under experimental conditions, mitochondria isolation, sample preparation for multiplex proteomics using tandem mass tags (TMT) for isobaric labeling, recommended liquid chromatography-mass spectrometry (LC-MS) settings for reporter ion quantitation and a data analysis pipeline to analyze pSILAC-TMT data.

Keywords:  Mass spectrometry; Mitochondria; Mitochondrial protein import; Proteomics; SILAC; TMT multiplex; Translation; mePROD; pSILAC

DOI:  https://doi.org/10.1016/bs.mie.2024.07.017
Methods Enzymol. 2024 ;pii: S0076-6879(24)00354-9. [Epub ahead of print]706 193-213

Monitoring mitochondrial precursor processing and presequence peptide degradation.

Cansu Kücükköse, F-Nora Vögtle, Annette Flotho.

  The maturation of mitochondrial presequence precursor proteins after their import into the organelle is a complex process that requires the interaction of several mitochondrial proteases. Precursor processing by the mitochondrial presequence proteases is directly coupled to the proteolytic turnover of the cleaved targeting signal by mitochondrial presequence peptidases. Dysfunction of these enzymes is associated with a variety of human diseases, including neurological disorders, cardiomyopathies and renal diseases. In this chapter, we describe experimental approaches to study the activity of the major mitochondrial presequence protease (MPP) and of the presequence peptidases. In vitro assays and soluble mitochondrial extracts allow the assessment and experimental manipulation of peptidase and protease activity using immunoblotting, fluorescence measurements and autoradiography as readouts. In particular, the assays allow manipulation at multiple levels including in vivo, in organello or in soluble extracts/in vitro. Purification of the yeast heterodimeric MPP allows in vitro reconstitution of the initial presequence processing step using radiolabeled precursors as substrates. Application of soluble mitochondrial extracts enables direct assessment of MPP processing and presequence peptide turnover which can be easily manipulated and is uncoupled from protein translocation across the mitochondrial membranes. The techniques presented in this chapter allow in-depth analysis of precursor processing and presequence turnover as well as direct assessment of the impact of patient mutations on the activity of the presequence processing machinery.

Keywords:  Mitochondrial precursor processing; Mitochondrial protein import; Presequence degradation; Presequences; Targeting peptides

DOI:  https://doi.org/10.1016/bs.mie.2024.07.018
Methods Enzymol. 2024 ;pii: S0076-6879(24)00360-4. [Epub ahead of print]706 3-18

Isolation of yeast mitochondria by differential centrifugation.

Kuo Song, Heike Rampelt.

  The isolation of intact and functional mitochondria is a powerful approach to characterize and study this organelle. The classical biochemical method of differential centrifugation is routinely used to isolate mitochondria. This method has several advantages, such as a high yield and easy adaptability. The isolated mitochondria are physiologically active and can be used for a variety of follow-up experiments, for example protein import and respiration measurements. Here, we describe the procedure to purify mitochondria from the budding yeast Saccharomyces cerevisiae. In addition, two approaches are introduced to assess the quality of isolated mitochondria, by limited proteinase K digestion or measurement of the membrane potential.

Keywords:  Mitochondrial preparation; fractionation; limited proteolysis; membrane potential; organelle isolation

DOI:  https://doi.org/10.1016/bs.mie.2024.07.024
Methods Enzymol. 2024 ;pii: S0076-6879(24)00377-X. [Epub ahead of print]706 61-73

Subcellular fractionation by differential centrifugation for mitochondrial studies.

Conny Steiert, Jon V Busto, Laura Melchionda, Nils Wiedemann.

  In addition to fluorescence microscopy, the subcellular fractionation of eukaryotic cells remains one of the central methods for the basic characterization of proteins. Here we describe an optimized procedure for the subcellular fractionation of yeast cells, specifically for mitochondrial studies. Major recommendations are to separate the fractions immediately after each centrifugation step, to carefully discard a significant part of the supernatant fractions which is in the direct vicinity to the pellets and, in addition, to perform an extra homogenization step of the post nuclear supernatant fraction. These principles help to collect supernatant fractions with less cross-contaminations from the corresponding pellets. These approaches are scalable and adaptable for the fractionation of other cell types and are also useful for the characterization of other organelles.

Keywords:  Cell organelles; Cytosol; Endoplasmic reticulum; Microsomes; Mitochondria; Nucleus; Post nuclear supernatant; Saccharomyces cerevisiae; Yeast

DOI:  https://doi.org/10.1016/bs.mie.2024.07.037
Methods Enzymol. 2024 ;pii: S0076-6879(24)00361-6. [Epub ahead of print]706 215-242

Analysis of mitochondrial targeting signal cleavage and protein processing by mass spectrometry.

Fabian Stockert, Henrique Baeta, Pitter F Huesgen.

  The majority of mitochondrial proteins are encoded in the nucleus, synthesized in the cytosol and imported into mitochondria mediated by an N-terminal mitochondrial targeting sequences (MTS). After import, the MTS is cleaved off by the mitochondrial processing peptidase (MPP) and subsets of the imported proteins are further processed by the aminopeptidase intermediate cleaving peptidase 55 (ICP55), the mitochondrial intermediate peptidase (MIP), octapeptidyl aminopeptidase 1 (Oct1) or other proteolytic enzymes. Mutations that impair the mitochondrial processing machinery or mitochondrial protein degradation result in rare but severe human diseases. In addition, aging and various stress conditions are associated with altered proteolysis of mitochondrial proteins. Enrichment of protein terminal peptides in combination with mass spectrometry-based identification and quantification has become the method of choice to study proteolytic processing. Here, we describe an updated step-by-step protocol for the enrichment of N-terminal peptides by Hypersensitive Undecanal-mediated Enrichment of N-Terminal peptides (HUNTER). We describe analysis of mass spectrometry data acquired for HUNTER samples and present a suite of dedicated Python and R scripts for HUNTER quality control, classification of the enriched peptides, annotation of mitochondrial processing sites and quantitative evaluation. The scripts are freely available at https://github.com/FabianStockert/mito_annotation.

Keywords:  Data analysis; Degradomics; Mass spectrometry; Mitochondria; N-terminome; Peptide quantification; Positional annotation; Protein N-termini; Proteolytic processing

DOI:  https://doi.org/10.1016/bs.mie.2024.07.025
Methods Enzymol. 2024 ;pii: S0076-6879(24)00366-5. [Epub ahead of print]706 243-262

Analysis of targeting signals for mitochondrial intermembrane space import.

Kostas Tokatlidis, Amiyo Haider.

  The mitochondrial intermembrane space (IMS) is the smallest sub-mitochondrial compartment, containing only 5%-10% of mitochondrial proteins. Despite its size, it exhibits the most diverse array of protein import mechanisms. These are underpinned by several different types of targeting signals that are quite distinct from targeting signals for other mitochondrial sub-compartments. In this chapter we outlined our current understanding of some of the main IMS import pathways, the primary oxidative protein folding targeting signal, and explore the remarkable variety of alternative import methods. Unlike proteins destined for the matrix or inner membrane (IM), IMS proteins need only traverse the outer mitochondrial membrane. This process doesn't require energy from ATP hydrolysis in the matrix or the IM electrochemical potential. We also examine unconventional IMS import pathways that remain poorly understood, often guided by ill-defined or unknown targeting peptides. Many IMS proteins are implicated in human diseases, making it crucial to comprehend how they reach their functional location within the IMS. The chapter concludes by discussing current insights into how understanding IMS targeting pathways can contribute to improved understanding of a wide range of human disorders.

Keywords:  Chaperones; Disulfide bonds; In vitro protein import; Intermembrane space; MIA pathway; Oxidative folding; Redox; Targeting

DOI:  https://doi.org/10.1016/bs.mie.2024.07.030
Methods Enzymol. 2024 ;pii: S0076-6879(24)00359-8. [Epub ahead of print]706 391-405

Import of mitochondrial precursor proteins into mitochondria of semi-intact yeast cells.

Johannes M Herrmann, Svenja Lenhard, Katja G Hansen.

  Mitochondria import hundreds of different precursor proteins from the cytosol and direct each of these to its specific mitochondrial subcompartment. The import routes and mechanisms by which precursors are transported into the outer membrane, the intermembrane space (IMS), the inner membrane and the matrix have been characterized in depth by use of very powerful in vitro assays. In the 'classical' import assays, radiolabeled precursor proteins are incubated with isolated mitochondria and the protein uptake is monitored by one or more of the following observations: intramitochondrial processing, resistance to externally added proteases, or the formation of disulfide bonds. In this chapter, we describe an alternative import assay which employs semi-intact yeast cells. This assay uses spheroplasts from which the cell wall had been removed by enzymatic digestion before the plasma membrane was partially permeabilized by a freeze-thawing step. Since the organellar architecture is largely maintained in semi-intact cells, this in vitro import assay allows to elucidate the targeting of precursor proteins from the cytoplasm to the mitochondrial surface. Thereby the contribution of other compartments such as the endoplasmic reticulum (ER) can be assessed. Here we describe how semi-intact cells are prepared and used in the in vitro import assay and discuss the pros and cons of this approach.

Keywords:  Intracellular targeting; Mitochondria; Organellar contact sites; Protein import; Protein targeting; Radiolabeled precursor proteins; Spheroplasts

DOI:  https://doi.org/10.1016/bs.mie.2024.07.023
Mol Genet Genomics. 2024 Oct 26. 299(1): 102

A novel m.5906G > a variant in MT-CO1 causes MELAS/Leigh overlap syndrome.

Zhimei Liu, Yaojun Xie, Xiaoting Lou, Xiaofei Zeng, Luyi Zhang, Meng Yu, Junling Wang, Jiuwei Li, Danmin Shen, Hua Li, Suzhou Zhao, Yuwei Zhou, Hezhi Fang, Jianxin Lyu, Yun Yuan, Zhaoxia Wang, Liqin Jin, Fang Fang.

  The MELAS/Leigh overlap syndrome manifests with a blend of clinical and radiographic traits from both MELAS and LS. However, the association of MELAS/Leigh overlap syndrome with MT-CO1 gene variants has not been previously reported. In this study, we report a patient diagnosed with MELAS/Leigh overlap syndrome harboring the m.5906G > A variant in MT-CO1, with biochemical evidence supporting the pathogenicity of the variant. The variant m.5906G > A that led to a synonymous variant in the start codon of MT-CO1 was filtered as the candidate disease-causing variant of the patient. Patient-derived fibroblasts were used to generate a series of monoclonal cells carrying different m.5906G > A variant loads for further functional assays. The oxygen consumption rate, ATP production, mitochondrial membrane potential and lactate assay indicated an impairment of cellular bioenergetics due to the m.5906G > A variant. Blue native PAGE analysis revealed that the m.5906G > A variant caused a deficiency in the content of mitochondrial oxidative phosphorylation complexes. Furthermore, molecular biology assays performed for the pathogenesis, mtDNA copy number, mtDNA-encoded subunits, and recovery capacity of mtDNA were all deficient due to the m.5906G > A variant, which might be caused by mtDNA replication deficiency. Overall, our findings demonstrated the pathogenicity of m.5906G > A variant and proposed a potential pathogenic mechanism, thereby expanding the genetic spectrum of MELAS/Leigh overlap syndrome.

Keywords:  M.5906G > A; MT-CO1 ; MELAS/Leigh overlap syndrome; MtDNA replication

DOI:  https://doi.org/10.1007/s00438-024-02181-y
Reprod Med Biol. 2024 Jan-Dec;23(1):23(1): e12602

Ectopic expression of the mitochondrial protein COXFA4L3 in human sperm acrosome and its potential application in the selection of male infertility treatments.

Yusuke Fujisawa, Sayaka Kikuchi, Fujino Kuba, Kosei Oishi, Soushi Murayama, Tomoya Sugiyama, Reiji Tokito, Hiroe Ueno, Shin-Ichi Kashiwabara, Yasushi Yumura, Yasuyuki Kurihara.

  Purpose: Spermatogenesis requires a large amount of energy, which is primarily produced by the mitochondrial electron transfer chain. Mitochondrial dysfunction affects male infertility, suggesting a relationship between the electron transfer chain and male infertility. COXFA4L3 (C15ORF48) is an emerging subunit protein of cytochrome oxidase specifically expressed in germ cells during spermatogenesis, and it may be involved in male infertility. Therefore, to investigate whether COXFA4L3 could be a marker of mitochondrial dysfunction in the sperm, this study examined the protein expression and localization profile of COXFA4L3 in the sperm of male patients with infertility.Methods: Twenty-seven semen samples from a male infertility clinic at the Reproductive Center of Yokohama City University Medical Center were used to analyze sperm quality parameters and the expression and localization of energy production-related proteins. These data were compared with the outcomes of infertility treatment.
Results: The expression levels of COXFA4L3 varied significantly between samples. Furthermore, COXFA4L3 was ectopically localized to the acrosome.
Conclusions: Ectopic expression of COXFA4L3 and PNA-stained acrosomes may be useful parameters for fertility treatment selection. Assessing the acrosomal localization of COXFA4L3 will expedite pregnancy treatment planning.

Keywords:  cytochrome c oxidase; electron transport chain; male infertility; mitochondria; sperm

DOI:  https://doi.org/10.1002/rmb2.12602
Proc Natl Acad Sci U S A. 2024 Nov 05. 121(45): e2406174121

COX15 deficiency causes oocyte ferroptosis.

Zhihua Zhang, Ran Yu, Qiuwen Shi, Zhi-Jing Wu, Qingchun Li, Jian Mu, Biaobang Chen, Juanzi Shi, Renmin Ni, Ling Wu, Qiaoli Li, Jing Fu, Rong Li, Xiaoxi Sun, Jiucun Wang, Lin He, Yanping Kuang, Qing Sang, Lei Wang.

  Mitochondria play diverse roles in mammalian physiology. The architecture, activity, and physiological functions of mitochondria in oocytes are largely different from those in somatic cells, but the mitochondrial proteins related to oocyte quality and reproductive longevity remain largely unknown. Here, using whole-exome sequencing data from 1,024 women (characterized by oocyte maturation arrest and degenerated or morphologically abnormal oocytes) and 2,868 healthy controls, we performed a population and gene-based burden test for mitochondrial genes and identified a candidate gene, cytochrome c oxidase assembly protein 15 (COX15). We report that biallelic COX15 pathogenic variants cause human oocyte ferroptosis and female infertility in a recessive inheritance pattern. COX15 variants impaired mitochondrial respiration in Saccharomyces cerevisiae and led to reduced protein levels in HeLa cells. Oocyte-specific deletion of Cox15 led to impaired Fe2+ and reactive oxygen species homeostasis that caused mitochondrial dysfunction and ultimately sensitized oocytes to ferroptosis. In addition, ferrostatin-1 (an inhibitor of ferroptosis) could rescue the oocyte ferroptosis phenotype in vitro and ex vivo. Our findings not only provide a genetic diagnostic marker for oocyte development defects but also expand the spectrum of mitochondrial disorders to female infertility and contribute to unique insights into the role of ferroptosis in human oocyte defects.

Keywords:  COX15 deficiency; female infertility; ferroptosis; mitochondrial disorders; oocyte defects

DOI:  https://doi.org/10.1073/pnas.2406174121
Methods Enzymol. 2024 ;pii: S0076-6879(24)00368-9. [Epub ahead of print]706 19-36

Isolation of yeast mitochondria by affinity purification using magnetic beads.

Tzu-Ying Lin, Shih-Hung Chien, Liza A Pon, Pin-Chao Liao.

  Isolated mitochondria have been widely utilized in various model organisms to investigate the diverse functions of the organelle. Techniques such as differential centrifugation, density gradient ultracentrifugation and antibody-coated magnetic beads are employed for isolation of the organelle from whole cells. However, mitochondria isolated using differential centrifugation are often contaminated with other organelles; isolation using density gradient ultracentrifugation can reduce contamination but is time-intensive and requires large amounts of starting materials; and mitochondria isolated using antibody-coated magnetic beads are irreversibly bound to the beads. Here, we provide a step-by-step protocol for the isolation of highly pure mitochondria from Saccharomyces cerevisiae using a magnetic bead affinity purification method that overcomes these limitations. This protocol describes how to isolate mitochondria, tagged by insertion of 6 histidines (6xHis) into the chromosomal copy of the TOM70 (Translocase of outer membrane 70) gene using Ni-NTA (nickel(II) nitrilotriacetic acid) paramagnetic beads, and the subsequent release of mitochondria from the beads using a buffer containing imidazole. We provide examples of expected results, highlighting the purity, integrity and import activity of isolated mitochondria. These affinity-purified mitochondria are intact and functional, containing less contamination with cytosol and other organelles compared to mitochondria isolated by other methods. Our method is adaptable and can be applied to other model organisms that can be genetically manipulated using CRISPR or other methods.

Keywords:  Affinity purification; Budding yeast; Magnetic beads; Mitochondrial import; Mitochondrial isolation

DOI:  https://doi.org/10.1016/bs.mie.2024.07.032