bims-mitdyn 2024-11-03 papers

bims-mitdyn

Biomed News

on Mitochondrial dynamics: mechanisms

Issue of 2024–11–03
24 papers selected by
Edmond Chan, Queen’s University, School of Medicine

Activation of the mitochondrial unfolded protein response regulates the dynamic formation of stress granules.
Pathogenic mitochondrial DNA mutations inhibit melanoma metastasis.
PKCδ is an activator of neuronal mitochondrial metabolism that mediates the spacing effect on memory consolidation.
Noncanonical TCA cycle fosters canonical TCA cycle and mitochondrial integrity in acute myeloid leukemia.
Membrane potential stimulates ADP import and ATP export by the mitochondrial ADP/ATP carrier due to its positively charged binding site.
High mitochondrial DNA levels accelerate lung adenocarcinoma progression.
Structure of the turnover-ready state of an ancestral respiratory complex I.
Analysis of mitochondrial translation using click chemistry.
Monitoring mitochondrial protein import by live cell imaging.
Assays to monitor mitochondrial translation.
Import of mitochondrial precursor proteins into mitochondria of semi-intact yeast cells.
Monitoring mitochondrial precursor processing and presequence peptide degradation.
Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria.
Isolation of yeast mitochondria by differential centrifugation.
Analysis of mitochondrial targeting signal cleavage and protein processing by mass spectrometry.
The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import.
Analysis of targeting signals for mitochondrial intermembrane space import.
Quantifying mitochondrial protein import by mePRODmt proteomics.
Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol.
Subcellular fractionation by differential centrifugation for mitochondrial studies.
Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP.
Methods to monitor mitochondrial disulfide bonds.
In vitro import of mitochondrial precursor proteins into yeast mitochondria.
In organello silencing of mitochondrial gene expression.

J Cell Sci. 2024 Oct 28. pii: jcs.263548. [Epub ahead of print]

Activation of the mitochondrial unfolded protein response regulates the dynamic formation of stress granules.

Marta Lopez-Nieto, Zhaozhi Sun, Emily Relton, Rahme Safakli, Brian D Freibaum, J Paul Taylor, Alessia Ruggieri, Ioannis Smyrnias, Nicolas Locker.

  To rapidly adapt to harmful changes to their environment, cells activate the integrated stress response (ISR). This results in an adaptive transcriptional and translational rewiring, and the formation of biomolecular condensates named stress granules (SGs), to resolve stress. In addition to this first line of defence, the mitochondrial unfolded protein response (UPRmt) activates a specific transcriptional programme to maintain mitochondrial homeostasis. We present evidence that SGs and UPRmt pathways are intertwined and communicate. UPRmt induction results in eIF2a phosphorylation and the initial and transient formation of SGs, which subsequently disassemble. The induction of GADD34 during late UPRmt protects cells from prolonged stress by impairing further assembly of SGs. Furthermore, mitochondrial functions and cellular survival are enhanced during UPRmt activation when SGs are absent, suggesting that UPRmt-induced SGs have an adverse effect on mitochondrial homeostasis. These findings point to a novel crosstalk between SGs and the UPRmt that may contribute to restoring mitochondrial functions under stressful conditions.

Keywords:  GADD34; Integrated stress response; Mitochondrial stress response; Stress granules; UPRmt

DOI:  https://doi.org/10.1242/jcs.263548
Sci Adv. 2024 Nov;10(44): eadk8801

Pathogenic mitochondrial DNA mutations inhibit melanoma metastasis.

Spencer D Shelton, Sara House, Luiza Martins Nascentes Melo, Vijayashree Ramesh, Zhenkang Chen, Tao Wei, Xun Wang, Claire B Llamas, Siva Sai Krishna Venigalla, Cameron J Menezes, Gabriele Allies, Jonathan Krystkiewicz, Jonas Rösler, Sven W Meckelmann, Peihua Zhao, Florian Rambow, Dirk Schadendorf, Zhiyu Zhao, Jennifer G Gill, Ralph J DeBerardinis, Sean J Morrison, Alpaslan Tasdogan, Prashant Mishra.

Mitochondrial DNA (mtDNA) mutations are frequent in cancer, yet their precise role in cancer progression remains debated. To functionally evaluate the impact of mtDNA variants on tumor growth and metastasis, we developed an enhanced cytoplasmic hybrid (cybrid) generation protocol and established isogenic human melanoma cybrid lines with wild-type mtDNA or pathogenic mtDNA mutations with partial or complete loss of mitochondrial oxidative function. Cybrids with homoplasmic levels of pathogenic mtDNA reliably established tumors despite dysfunctional oxidative phosphorylation. However, these mtDNA variants disrupted spontaneous metastasis from primary tumors and reduced the abundance of circulating tumor cells. Migration and invasion of tumor cells were reduced, indicating that entry into circulation is a bottleneck for metastasis amid mtDNA dysfunction. Pathogenic mtDNA did not inhibit organ colonization following intravenous injection. In heteroplasmic cybrid tumors, single-cell analyses revealed selection against pathogenic mtDNA during melanoma growth. Collectively, these findings experimentally demonstrate that functional mtDNA is favored during melanoma growth and supports metastatic entry into the blood.

DOI: https://doi.org/10.1126/sciadv.adk8801
Elife. 2024 Oct 30. pii: RP92085. [Epub ahead of print]13

PKCδ is an activator of neuronal mitochondrial metabolism that mediates the spacing effect on memory consolidation.

Typhaine Comyn, Thomas Preat, Alice Pavlowsky, Pierre-Yves Plaçais.

  Relevance-based selectivity and high energy cost are two distinct features of long-term memory (LTM) formation that warrant its default inhibition. Spaced repetition of learning is a highly conserved cognitive mechanism that can lift this inhibition. Here, we questioned how the spacing effect integrates experience selection and energy efficiency at the cellular and molecular levels. We showed in Drosophila that spaced training triggers LTM formation by extending over several hours an increased mitochondrial metabolic activity in neurons of the associative memory center, the mushroom bodies (MBs). We found that this effect is mediated by PKCδ, a member of the so-called 'novel PKC' family of enzymes, which uncovers the critical function of PKCδ in neurons as a regulator of mitochondrial metabolism for LTM. Additionally, PKCδ activation and translocation to mitochondria result from LTM-specific dopamine signaling on MB neurons. By bridging experience-dependent neuronal circuit activity with metabolic modulation of memory-encoding neurons, PKCδ signaling binds the cognitive and metabolic constraints underlying LTM formation into a unified gating mechanism.

Keywords:  D. melanogaster; Drosophila; dopamine; energy; long-term memory; mushroom body; neuroscience

DOI:  https://doi.org/10.7554/eLife.92085
Cancer Sci. 2024 Oct 31.

Noncanonical TCA cycle fosters canonical TCA cycle and mitochondrial integrity in acute myeloid leukemia.

Atsushi Watanabe, Chartsiam Tipgomut, Haruhito Totani, Kentaro Yoshimura, Tomohiko Iwano, Hamed Bashiri, Lee Hui Chua, Chong Yang, Toshio Suda.

  Cancer cells rely on mitochondrial oxidative phosphorylation (OXPHOS) and the noncanonical tricarboxylic acid (TCA) cycle. In this paper, we shed light on the vital role played by the noncanonical TCA cycle in a host-side concession to mitochondria, especially in highly energy-demanding malignant tumor cells. Inhibition of ATP-citrate lyase (ACLY), a key enzyme in the noncanonical TCA cycle, induced apoptosis by increasing reactive oxygen species levels and DNA damage while reducing mitochondrial membrane potential. The mitochondrial membrane citrate transporter inhibitor, CTPI2, synergistically enhanced these effects. ACLY inhibition reduced cytosolic citrate levels and CTPI2 lowered ACLY activity, suggesting that the noncanonical TCA cycle is sustained by a positive feedback mechanism. These inhibitions impaired ATP production, particularly through OXPHOS. Metabolomic analysis of mitochondrial and cytosolic fractions revealed reduced levels of glutathione pathway-related and TCA cycle-related metabolite, except fumarate, in mitochondria following noncanonical TCA cycle inhibition. Despite the efficient energy supply to the cell by mitochondria, this symbiosis poses challenges related to reactive oxygen species and mitochondrial maintenance. In conclusion, the noncanonical TCA cycle is indispensable for the canonical TCA cycle and mitochondrial integrity, contributing to mitochondrial domestication.

Keywords:  ATP‐citrate lyase; antimetabolites; apoptosis; cancer metabolism; cell lines; hematopoietic organ; mitochondria; noncanonical TCA cycle; others; reactive oxygen species

DOI:  https://doi.org/10.1111/cas.16347
Sci Adv. 2024 Nov;10(44): eadp7725

Membrane potential stimulates ADP import and ATP export by the mitochondrial ADP/ATP carrier due to its positively charged binding site.

Vasiliki Mavridou, Martin S King, Andre Bazzone, Roger Springett, Edmund R S Kunji.

The mitochondrial adenosine 5'-diphosphate (ADP)/adenosine 5'-triphosphate (ATP) carrier imports ADP into the mitochondrion and exports ATP to the cell. Here, we demonstrate that 3.3 positive charges are translocated with the negatively charged substrate in each transport step. They can be assigned to three positively charged residues of the central substrate-binding site and two asparagine/arginine pairs. In this way, the membrane potential stimulates not only the ATP4- export step, as a net -0.7 charge is transported, but also the ADP3- import step, as a net +0.3 charge is transported with the electric field. These positive charge movements also inhibit the import of ATP and export of ADP in the presence of a membrane potential, allowing these nucleotides to be maintained at high concentrations in the cytosol and mitochondrial matrix to drive the hydrolysis and synthesis of ATP, respectively. Thus, this is the mechanism by which the membrane potential drives adenine nucleotide exchange with high directional fluxes to fuel the cellular processes.

DOI: https://doi.org/10.1126/sciadv.adp7725
Sci Adv. 2024 Nov;10(44): eadp3481

High mitochondrial DNA levels accelerate lung adenocarcinoma progression.

Mara Mennuni, Stephen E Wilkie, Pauline Michon, David Alsina, Roberta Filograna, Markus Lindberg, David E Sanin, Florian Rosenberger, Alina Schaaf, Erik Larsson, Erika L Pearce, Nils-Göran Larsson.

Lung adenocarcinoma is a common aggressive cancer and a leading cause of mortality worldwide. Here, we report an important in vivo role for mitochondrial DNA (mtDNA) copy number during lung adenocarcinoma progression in the mouse. We found that lung tumors induced by KRASG12D expression have increased mtDNA levels and enhanced mitochondrial respiration. To experimentally assess a possible causative role in tumor progression, we induced lung cancer in transgenic mice with a general increase in mtDNA copy number and found that they developed a larger tumor burden, whereas mtDNA depletion in tumor cells reduced tumor growth. Immune cell populations in the lung and cytokine levels in plasma were not affected by increased mtDNA levels. Analyses of large cancer databases indicate that mtDNA copy number is also important in human lung cancer. Our study thus reports experimental evidence for a tumor-intrinsic causative role for mtDNA in lung cancer progression, which could be exploited for development of future cancer therapies.

DOI: https://doi.org/10.1126/sciadv.adp3481
Nat Commun. 2024 Oct 29. 15(1): 9340

Structure of the turnover-ready state of an ancestral respiratory complex I.

Bozhidar S Ivanov, Hannah R Bridges, Owen D Jarman, Judy Hirst.

Respiratory complex I is pivotal for cellular energy conversion, harnessing energy from NADH:ubiquinone oxidoreduction to drive protons across energy-transducing membranes for ATP synthesis. Despite detailed structural information on complex I, its mechanism of catalysis remains elusive due to lack of accompanying functional data for comprehensive structure-function analyses. Here, we present the 2.3-Å resolution structure of complex I from the α-proteobacterium Paracoccus denitrificans, a close relative of the mitochondrial progenitor, in phospholipid-bilayer nanodiscs. Three eukaryotic-type supernumerary subunits (NDUFS4, NDUFS6 and NDUFA12) plus a novel L-isoaspartyl-O-methyltransferase are bound to the core complex. Importantly, the enzyme is in a single, homogeneous resting state that matches the closed, turnover-ready (active) state of mammalian complex I. Our structure reveals the elements that stabilise the closed state and completes P. denitrificans complex I as a unified platform for combining structure, function and genetics in mechanistic studies.

DOI: https://doi.org/10.1038/s41467-024-53679-3
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)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)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)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
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)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)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)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)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)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)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)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)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)00364-1. [Epub ahead of print]706 75-95

Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP.

Solène Zuttion, Bruno Senger, Chiranjit Panja, Sylvie Friant, Róża Kucharczyk, Hubert Dominique Becker.

  Even if a myriad of approaches has been developed to identify the subcellular localization of a protein, the easiest and fastest way remains to fuse the protein to Green Fluorescent Protein (GFP) and visualize its location using fluorescence microscopy. However, this strategy is not well suited to visualize the organellar pools of proteins that are simultaneously localized both in the cytosol and in organelles because the GFP signal of a cytosolic pool of the protein (cytosolic echoform) will inevitably mask or overlay the GFP signal of the organellar pool of the protein (organellar echoform). To solve this issue, we engineered a dedicated yeast strain expressing a Bi-Genomic Mitochondrial-Split-GFP. This split-GFP is bi-genomic because the first ten ß-strands of GFP (GFPß1-10) are encoded by the mitochondrial genome and translated by mitoribosomes whereas the remaining ß-strand of GFP (GFPß11) is fused to the protein of interest encoded by the nucleus and expressed by cytosolic ribosomes. Consequently, if the GFPß11-tagged protein localizes into mitochondria, GFP will be reconstituted by self-assembly GFPß1-10 and GFPß11 thereby generating a GFP signal restricted to mitochondria and detectable by regular fluorescence microscopy. In addition, because mitochondrial translocases and import mechanisms are evolutionary well conserved, the BiG Mito-Split-GFP yeast strain can be used to probe mitochondrial importability of proteins regardless of their organismal origins and can thus serve to identify unsuspected mitochondrial echoforms readily from any organism.

Keywords:  Dual-localized; Microscopy; Mitochondria; Protein import; Saccharomyces cerevisiae; Split-GFP

DOI:  https://doi.org/10.1016/bs.mie.2024.07.028
Methods Enzymol. 2024 ;pii: S0076-6879(24)00379-3. [Epub ahead of print]706 125-158

Methods to monitor mitochondrial disulfide bonds.

Ben Hur Marins Mussulini, Michal Wasilewski, Agnieszka Chacinska.

  Mitochondria contain numerous proteins that utilize the chemistry of cysteine residues, which can be reversibly oxidized. These proteins are involved in mitochondrial biogenesis, protection against oxidative stress, metabolism, energy transduction to adenosine triphosphate, signaling and cell death among other functions. Many proteins located in the mitochondrial intermembrane space are imported by the mitochondrial import and assembly pathway the activity of which is based on the reversible oxidation of cysteine residues and oxidative trapping of substrates. Oxidative modifications of cysteine residues are particularly difficult to study because of their labile character. Here we present techniques that allow for monitoring the oxidative state of mitochondrial proteins as well as to investigate the mitochondrial import and assembly pathway. This chapter conveys basic concepts on sample preparation and techniques to monitor the redox state of cysteine residues in mitochondrial proteins as well as the strategies to study mitochondrial import and assembly pathway.

Keywords:  Direct thiol trap; Import assay; Indirect thiol trap; MIA40; Mitochondria

DOI:  https://doi.org/10.1016/bs.mie.2024.07.039
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
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