bims-mitdyn 2024-11-10 papers

bims-mitdyn

Biomed News

on Mitochondrial dynamics: mechanisms

Issue of 2024–11–10
25 papers selected by
Edmond Chan, Queen’s University, School of Medicine

Cellular ATP demand creates metabolically distinct subpopulations of mitochondria.
Transfer of mitochondrial DNA into the nuclear genome during induced DNA breaks.
Cytosolic N6AMT1-dependent translation supports mitochondrial RNA processing.
Mitochondrial elongation impairs breast cancer metastasis.
Mitochondrial nucleoids.
Analysis of protein-protein interaction of the mitochondrial translocase at work by using technically effective BPA photo-crosslinking method.
Monitoring retro-translocation of proteins from the mitochondrial intermembrane space.
TOM-TIM23 supercomplex formation.
Large-scale purification of mitochondrial protein complexes in yeast expression system for structural analyses.
Approaches for the analysis of redox-dependent protein import into mitochondria of mammalian cells.
Generation of TIM chaperone substrate complexes.
Analysis of protein trafficking between mitochondria and the endoplasmic reticulum by fluorescence microscopy.
Monitoring protein phosphorylation at the mitochondrial protein import machinery by PhosTag electrophoresis.
Analysis of mitochondrial translocases TOM and TIM by the patch-clamping technique.
Monitoring α-helical membrane protein insertion into the outer mitochondrial membrane of yeast cells.
Monitoring alpha-helical membrane protein insertion into the outer mitochondrial membrane in mammalian cells.
STED super-resolution microscopy of mitochondrial translocases.
Analysis of mitochondrial biogenesis regulation by oxidative stress.
Methods to analyze the mitochondrial unfolded protein response (UPRmt).
Analysis of mitochondrial protein translocation by disulfide bond formation and cysteine specific crosslinking.
Seahorse assay for the analysis of mitochondrial respiration using Saccharomyces cerevisiae as a model system.
Analysis of mitochondrial protein aggregation and disaggregation.
Analysis of quality control pathways for the translocase of the outer mitochondrial membrane.
Mass spectrometry-based proteomics to study mutants and interactomes of mitochondrial translocation proteins.
Analysis of the mitochondria-associated degradation pathway (MAD) in yeast cells.

Nature. 2024 Nov 06.

Cellular ATP demand creates metabolically distinct subpopulations of mitochondria.

Keun Woo Ryu, Tak Shun Fung, Daphne C Baker, Michelle Saoi, Jinsung Park, Christopher A Febres-Aldana, Rania G Aly, Ruobing Cui, Anurag Sharma, Yi Fu, Olivia L Jones, Xin Cai, H Amalia Pasolli, Justin R Cross, Charles M Rudin, Craig B Thompson.

Mitochondria serve a crucial role in cell growth and proliferation by supporting both ATP synthesis and the production of macromolecular precursors. Whereas oxidative phosphorylation (OXPHOS) depends mainly on the oxidation of intermediates from the tricarboxylic acid cycle, the mitochondrial production of proline and ornithine relies on reductive synthesis1. How these competing metabolic pathways take place in the same organelle is not clear. Here we show that when cellular dependence on OXPHOS increases, pyrroline-5-carboxylate synthase (P5CS)-the rate-limiting enzyme in the reductive synthesis of proline and ornithine-becomes sequestered in a subset of mitochondria that lack cristae and ATP synthase. This sequestration is driven by both the intrinsic ability of P5CS to form filaments and the mitochondrial fusion and fission cycle. Disruption of mitochondrial dynamics, by impeding mitofusin-mediated fusion or dynamin-like-protein-1-mediated fission, impairs the separation of P5CS-containing mitochondria from mitochondria that are enriched in cristae and ATP synthase. Failure to segregate these metabolic pathways through mitochondrial fusion and fission results in cells either sacrificing the capacity for OXPHOS while sustaining the reductive synthesis of proline, or foregoing proline synthesis while preserving adaptive OXPHOS. These findings provide evidence of the key role of mitochondrial fission and fusion in maintaining both oxidative and reductive biosyntheses in response to changing nutrient availability and bioenergetic demand.

DOI: https://doi.org/10.1038/s41586-024-08146-w
Nat Commun. 2024 Nov 01. 15(1): 9438

Transfer of mitochondrial DNA into the nuclear genome during induced DNA breaks.

Jinchun Wu, Yang Liu, Liqiong Ou, Tingting Gan, Zhengrong Zhangding, Shaopeng Yuan, Xinyi Liu, Mengzhu Liu, Jiasheng Li, Jianhang Yin, Changchang Xin, Ye Tian, Jiazhi Hu.

Mitochondria serve as the cellular powerhouse, and their distinct DNA makes them a prospective target for gene editing to treat genetic disorders. However, the impact of genome editing on mitochondrial DNA (mtDNA) stability remains a mystery. Our study reveals previously unknown risks of genome editing that both nuclear and mitochondrial editing cause discernible transfer of mitochondrial DNA segments into the nuclear genome in various cell types including human cell lines, primary T cells, and mouse embryos. Furthermore, drug-induced mitochondrial stresses and mtDNA breaks exacerbate this transfer of mtDNA into the nuclear genome. Notably, we observe that mitochondrial editors, including mitoTALEN and recently developed base editor DdCBE, can also enhance crosstalk between mtDNA and the nuclear genome. Moreover, we provide a practical solution by co-expressing TREX1 or TREX2 exonucleases during DdCBE editing. These findings imply genome instability of mitochondria during induced DNA breaks and explain the origins of mitochondrial-nuclear DNA segments.

DOI: https://doi.org/10.1038/s41467-024-53806-0
Proc Natl Acad Sci U S A. 2024 Nov 19. 121(47): e2414187121

Cytosolic N6AMT1-dependent translation supports mitochondrial RNA processing.

Mads M Foged, Emeline Recazens, Sylvain Chollet, Miriam Lisci, George E Allen, Boris Zinshteyn, Doha Boutguetait, Christian Münch, Vamsi K Mootha, Alexis A Jourdain.

  Mitochondrial biogenesis relies on both the nuclear and mitochondrial genomes, and imbalance in their expression can lead to inborn errors of metabolism, inflammation, and aging. Here, we investigate N6AMT1, a nucleo-cytosolic methyltransferase that exhibits genetic codependency with mitochondria. We determine transcriptional and translational profiles of N6AMT1 and report that it is required for the cytosolic translation of TRMT10C (MRPP1) and PRORP (MRPP3), two subunits of the mitochondrial RNAse P enzyme. In the absence of N6AMT1, or when its catalytic activity is abolished, RNA processing within mitochondria is impaired, leading to the accumulation of unprocessed and double-stranded RNA, thus preventing mitochondrial protein synthesis and oxidative phosphorylation, and leading to an immune response. Our work sheds light on the function of N6AMT1 in protein synthesis and highlights a cytosolic program required for proper mitochondrial biogenesis.

Keywords:  OXPHOS; RNA processing; mitochondria; mitochondrial RNA granules; translation

DOI:  https://doi.org/10.1073/pnas.2414187121
Sci Adv. 2024 Nov 08. 10(45): eadm8212

Mitochondrial elongation impairs breast cancer metastasis.

Lucía Minarrieta, Matthew G Annis, Yannick Audet-Delage, Hellen Kuasne, Alain Pacis, Catherine St-Louis, Alexander Nowakowski, Marco Biondini, Mireille Khacho, Morag Park, Peter M Siegel, Julie St-Pierre.

Mitochondrial dynamics orchestrate many essential cellular functions, including metabolism, which is instrumental in promoting cancer growth and metastatic progression. However, how mitochondrial dynamics influences metastatic progression remains poorly understood. Here, we show that breast cancer cells with low metastatic potential exhibit a more fused mitochondrial network compared to highly metastatic cells. To study the impact of mitochondrial dynamics on metastasis, we promoted mitochondrial elongation in metastatic breast cancer cells by individual genetic deletion of three key regulators of mitochondrial fission (Drp1, Fis1, Mff) or by pharmacological intervention with leflunomide. Omics analyses revealed that mitochondrial elongation causes substantial alterations in metabolic pathways and processes related to cell adhesion. In vivo, enhanced mitochondrial elongation by loss of mitochondrial fission mediators or treatment with leflunomide notably reduced metastasis formation. Furthermore, the transcriptomic signature associated with elongated mitochondria correlated with improved clinical outcome in patients with breast cancer. Overall, our findings highlight mitochondrial dynamics as a potential therapeutic target in breast cancer.

DOI: https://doi.org/10.1126/sciadv.adm8212
Curr Biol. 2024 Nov 04. pii: S0960-9822(24)01346-0. [Epub ahead of print]34(21): R1067-R1068

Mitochondrial nucleoids.

Eve V Kakudji, Samantha C Lewis.

Eve Kakudji and Samantha Lewis discuss the structure and function of mitochondrial nucleoids - large nucleoprotein complexes containing mitochondrial DNA and the regulatory factors necessary for its packaging, replication, transcription, and repair.

DOI: https://doi.org/10.1016/j.cub.2024.09.078
Methods Enzymol. 2024 ;pii: S0076-6879(24)00407-5. [Epub ahead of print]707 237-256

Analysis of protein-protein interaction of the mitochondrial translocase at work by using technically effective BPA photo-crosslinking method.

Edward Germany, Takuya Shiota.

  The BPA photo-crosslinking method exploits the property of p-benzoyl-L-phenylalanine (pBpa), an amino acid containing a photoreactive side chain, and allows for the crosslinking with nearby proteins upon Ultraviolet irradiation. This feature enables the capture of two proteins within a close proximity with high spatial resolution at the level of amino acid residues. In this chapter, we introduce an example of the employment of the BPA photo-crosslinking method to the Translocase of the Outer Mitochondrial membrane complex of mitochondria in Saccharomyces cerevisiae as a model protein translocase. Here in, we provide three procedures (i) the introduction of pBpa into proteins of interest in living yeast cells by in vivo suppressor tRNA system; (ii) analysis of in vivo subunit-subunit interactions intra-complex; and (iii) analysis of translocase channel-substrate interactions in organello. The use of in vivo and in organello crosslinking tools enable the robust analysis of translocases in a near-to physiological condition.

Keywords:  Mitochondrial protein import; Photo-crosslink; Protein-protein interaction; TOM complex; Tom40; Unnatural amino acid

DOI:  https://doi.org/10.1016/bs.mie.2024.07.062
Methods Enzymol. 2024 ;pii: S0076-6879(24)00387-2. [Epub ahead of print]707 173-208

Monitoring retro-translocation of proteins from the mitochondrial intermembrane space.

Magda Krakowczyk, Piotr Bragoszewski.

  Mitochondria play multiple essential roles in eukaryotic cells. To perform their functions, mitochondria require an adequate supply of externally produced proteins and an intact two-membrane structure. The structure of mitochondrial membranes separates these organelles from their cytosolic environment, with proteins that make up the mitochondrial proteome either being embedded into or enveloped by these membranes. From the experimental point of view, the structural properties of mitochondria contribute to the relative ease of isolating these organelles from other cellular components. The ability to isolate intact mitochondria and analyze them in a well-controlled environment opens up the possibility of tracking any proteins that enter or escape the membrane-formed enclosure. This chapter discusses assays that monitor the movement of proteins out of mitochondria through intact membranes. These protocols provide insight into the mechanisms behind mitochondrial protein quality control. It was discovered that the retro-translocation of IMS proteins regulates the protein content of this specific sub-compartment of the organelle. Additionally, proteins can move out of the mitochondria to resolve failed import events. Assays based on isolated mitochondria precisely tackle such intricate 'dance' of proteins crossing mitochondrial membranes during import and export, maintaining the dynamics of the organellar proteome.

Keywords:  Intermembrane space; MIA pathway; Mitochondria; Protease; Protein import; Protein quality control; Protein retro-translocation; Protein transport; Proteostasis

DOI:  https://doi.org/10.1016/bs.mie.2024.07.047
Methods Enzymol. 2024 ;pii: S0076-6879(24)00382-3. [Epub ahead of print]707 3-22

TOM-TIM23 supercomplex formation.

Naintara Jain, Ridhima Gomkale, Peter Rehling.

  Mitochondria import the vast majority of proteins from the cytosol. Protein translocation machineries in outer and inner membranes facilitate precursor recognition and transport. Most mitochondrial proteins utilize N-terminal presequences as targeting signals that eventually direct them across the inner mitochondrial membrane. These precursors are transported by the TOM complex across the outer-, and subsequently by the TIM23 complex across the inner membrane. During this process the translocases align and the polypeptide chain is translocated across both membranes in a coupled manner. A transient precursor-containing TOM-TIM23 supercomplex is formed. This TOM-TIM23 supercomplex provides a fascinating import intermediate which can be stabilized if the precursor contains a tightly folded moiety at the C-terminus that is not able to pass through the TOM complex. Such a supercomplex can be generated during in vitro import, and in vivo. The stabilized TOM-TIM23 supercomplex can be purified for downstream analysis. The possibility of pausing translocation at this step provides a means to understand the mechanisms underlying precursor translocation.

Keywords:  Import; Mitochondria; Supercomplex; TIM23; TOM

DOI:  https://doi.org/10.1016/bs.mie.2024.07.042
Methods Enzymol. 2024 ;pii: S0076-6879(24)00383-5. [Epub ahead of print]707 367-388

Large-scale purification of mitochondrial protein complexes in yeast expression system for structural analyses.

Nanako Kobayashi, Yuta Imai, Shintaro Kita, Shiho Kawai, Yuhei Araiso.

  Recent developments in cryo-electron microscopy techniques have facilitated intensive research into determining protein structures. Nevertheless, the structures of some mitochondrial membrane protein complexes remain undetermined. One possible reason for this research gap is that mitochondrial membrane protein complexes are difficult to overexpress and purify. Even using high-resolution cryo-electron microscopy, structural determination is not possible without first obtaining purified homogeneous proteins. As determining novel structures of protein complexes would provide opportunities to answer many unresolved biological questions, it is important to generalize purification methods, which often become bottlenecks in protein research. In this chapter, we introduce purification methods for mitochondrial membrane protein complexes and mitochondria-localized soluble protein complexes using a yeast expression system. We also describe the recent development of a mitochondrial membrane isolation method that enables the extraction of large amounts of protein complexes for structural analyses.

Keywords:  Mitochondria preparation; Mitochondrial proteins; Multi-gene expression; Protein complex purification; Structural analysis; Yeast expression system

DOI:  https://doi.org/10.1016/bs.mie.2024.07.043
Methods Enzymol. 2024 ;pii: S0076-6879(24)00367-7. [Epub ahead of print]707 637-671

Approaches for the analysis of redox-dependent protein import into mitochondria of mammalian cells.

Julia Racho, Jan Riemer.

  Oxidation of cysteine residues in proteins can take place as part of an enzymatic reaction cycle, during oxidative protein folding or as a consequence of redox signalling or oxidative stress. Following changes in protein thiol redox states allows to investigate the mechanisms underlying thiol-disulphide redox processes. In this book chapter, we provide information and protocols on different methods for redox state determination with a focus on these processes in the context of oxidation-dependent protein import into the mitochondrial intermembrane space. These methods include assessing the cysteine redox state of mature proteins, methods to investigate oxidative protein folding in radioactive pulse chase assays and methods to follow specifically the formation of oxidative folding intermediates between oxidoreductases and substrates.

Keywords:  ALR; MIA40; disulphide bond formation; import; mitochondria; oxidative protein folding; redox

DOI:  https://doi.org/10.1016/bs.mie.2024.07.031
Methods Enzymol. 2024 ;pii: S0076-6879(24)00396-3. [Epub ahead of print]707 391-422

Generation of TIM chaperone substrate complexes.

Undina Guillerm, Iva Sučec, Paul Schanda.

  Holdase chaperones are essential in the mitochondrial membrane-protein biogenesis as they stabilize preproteins and keep them in an import-competent state as they travel through the aqueous cytosol and intermembrane space. The small TIM chaperones of the mitochondrial intermembrane space function within a fine balance of client promiscuity and high affinity binding, while being also able to release their client proteins without significant energy barrier to the downstream insertases/translocases. The tendency of the preproteins to aggregate and the dynamic nature of the preprotein-chaperone complexes makes the preparation of these complexes challenging. Here we present two optimized methods for complex formation of highly hydrophobic precursor proteins and chaperones: a pull-down approach and an in-vitro translation strategy. In the former, attaching the client protein to an affinity resin keeps the individual client protein copies apart from each other and decreases the client self-aggregation probability, thereby favouring complex formation. In the latter approach, a purified chaperone, added to the cell-free protein synthesis, captures the nascent precursor protein. The choice of method will depend on the desired client-chaperone complex amount, or the need for specific labeling scheme.

Keywords:  Cell-free protein production; Chaperone; Mitochondrial protein import; Promiscuous binding; Pull-down assay

DOI:  https://doi.org/10.1016/bs.mie.2024.07.051
Methods Enzymol. 2024 ;pii: S0076-6879(24)00381-1. [Epub ahead of print]707 153-171

Analysis of protein trafficking between mitochondria and the endoplasmic reticulum by fluorescence microscopy.

Shunsuke Matsumoto, Suzuka Ono, Toshiya Endo.

  Precise protein localization is essential for normal cellular functions. However, recent studies have revealed that protein targeting is error-prone, and tail-anchored proteins mistargeted to mitochondria are transferred to the endoplasmic reticulum (ER) by an ATPase Msp1 (yeast)/ATAD1 (human) in the mitochondrial outer membrane for further quality examination in the ER to determine their fate, degradation or re-targeting. Analysis of the inter-organelle transfer of proteins requires a combination of time-lapse fluorescence microscopy and a system to achieve regulation of the protein levels of both transfer substrates and factors regulating the transfer in a coordinated manner at precise timing. This can be achieved by using a promoter switch for expression and acute depletion of involved factors through the degron-based proteasome system. In this chapter, we will describe methods to analyze inter-organelle protein transfer by fluorescence microscope within living yeast cells, by using the example of Msp1-mediated transfer of mistargeted proteins from mitochondria to the ER.

Keywords:  AID degradation system; Endoplasmic reticulum; Fluorescence microscope; GET system; Inter-organelle protein transfer; Mitochondria; Msp1; Tail-anchored protein

DOI:  https://doi.org/10.1016/bs.mie.2024.07.041
Methods Enzymol. 2024 ;pii: S0076-6879(24)00408-7. [Epub ahead of print]707 501-517

Monitoring protein phosphorylation at the mitochondrial protein import machinery by PhosTag electrophoresis.

Fatimah Lami Imam, Chris Meisinger, Adinarayana Marada.

  The mitochondrial import machinery is regulated by several protein kinases that phosphorylate key components. This allows an adjustment of the protein flux to changing cellular demands and allow a dynamic organellar proteome. PhosTag electrophoresis has been proven as highly valuably tool to study these signalling machanisms at the import machinery.

Keywords:  TOM complex; assembly; biogenesis; import machinery; precursor protein; protein kinase; reversible phosphorylation; signalling

DOI:  https://doi.org/10.1016/bs.mie.2024.07.063
Methods Enzymol. 2024 ;pii: S0076-6879(24)00398-7. [Epub ahead of print]707 329-366

Analysis of mitochondrial translocases TOM and TIM by the patch-clamping technique.

María Luisa Campo.

  Mitochondrial protein import and sorting relies on sophisticated molecular machineries or translocases, of which channels are integral. Channels are built upon membrane proteins whose functions are driven by conformational changes. This implies that structural and functional information need to be integrated to gain a deep understanding of their dynamic behavior. Patch-clamp approaches are well suited for this purpose. This chapter provides a detailed description and practical guidance for applying the patch-clamp methodology to the electrophysiological characterization of mitochondrial protein import. Implementing the technique to intact mitochondria, mitoplasts, and reconstituted proteoliposomes, combined with genetically modified yeast strains, expands the scope of these studies. Focused on the TOM, TIM23, and TIM22 translocases, an analysis of the patch-clamp contribution to the field is outlined.

Keywords:  Mitochondria; Mitochondrial protein import; Patch-clamp; Protein import channels; TIM22 translocase; TIM23 translocase; TOM translocase

DOI:  https://doi.org/10.1016/bs.mie.2024.07.053
Methods Enzymol. 2024 ;pii: S0076-6879(24)00400-2. [Epub ahead of print]707 39-62

Monitoring α-helical membrane protein insertion into the outer mitochondrial membrane of yeast cells.

Isabelle Steymans, Thomas Becker.

  Mitochondria are surrounded by two membranes, the outer and inner membrane. The outer membrane contains a few dozen integral membrane proteins that mediate transport, fusion and fission processes, form contact sites and are involved in signaling pathways. There are two different types of outer membrane proteins. A few proteins are membrane-integrated by a transmembrane β-barrel, while other proteins are embedded by single or multiple α-helical membrane segments. All outer membrane proteins are produced on cytosolic ribosomes, but their import mechanisms differ. The translocase of the outer mitochondrial membrane (TOM complex) and the sorting and assembly machinery (SAM complex) import β-barrel proteins. Different import pathways have been reported for proteins with α-helical membrane anchors. The mitochondrial import (MIM) complex is the major insertase for this type of proteins. The in vitro import of radiolabeled precursor proteins into isolated mitochondria is a versatile technique to study protein import into the outer mitochondrial membrane. The import of these proteins does not involve proteolytic processing and is not dependent on the membrane potential. Therefore, the import assay has to be combined with blue native electrophoresis, carbonate extraction or protease accessibility assays to determine the import efficiency. These techniques allow to define import steps, assembly intermediates and study membrane integration. The in vitro import assay has been a central tool to uncover specific import routes and mechanisms.

Keywords:  MIM complex; Mitochondria; Protein sorting; SAM complex; TOM complex

DOI:  https://doi.org/10.1016/bs.mie.2024.07.055
Methods Enzymol. 2024 ;pii: S0076-6879(24)00399-9. [Epub ahead of print]707 63-99

Monitoring alpha-helical membrane protein insertion into the outer mitochondrial membrane in mammalian cells.

Masami Hazu, Alina Guna, Taylor A Stevens, Rebecca M Voorhees.

  Mitochondrial function is dependent on the correct localization and insertion of membrane proteins into the outer mitochondrial membrane (OM). In mammals, the OM contains ∼150 proteins, the majority of which contain α-helical transmembrane domains. This family of α-helical proteins has significantly expanded in metazoans and has evolved to mediate critical signaling and regulatory processes including mitochondrial fusion and fission, mitophagy, apoptosis and aspects of the innate immune response. Recently, the conserved OM protein MTCH2 has been identified as an insertase for α-helical proteins in human mitochondria. However, our understanding of the targeting, insertion, folding and quality control of α-helical OM proteins remains incomplete. Here we highlight three methods to monitor α-helical protein insertion both in human cells and in vitro. First, we describe a versatile split fluorescent reporter system that can be used to monitor the insertion of α-helical proteins into the OM in human cells. Second, we delineate a streamlined approach to isolating functional, insertion competent mitochondria from human cells that are compatible with in vitro import assays. Finally, we explain in detail how to reconstitute the insertion of α-helical proteins in a minimal system, by creating functional proteoliposomes containing purified MTCH2. Together these tools represent an integrated platform to enable the detailed mechanistic analysis of biogenesis of the diverse and physiologically essential α-helical OM proteome.

Keywords:  Insertion assay; MTCH2; Mitochondria; Outer mitochondrial membrane; Proteoliposomes; Split fluorescent reporter; α-helical membrane protein

DOI:  https://doi.org/10.1016/bs.mie.2024.07.054
Methods Enzymol. 2024 ;pii: S0076-6879(24)00397-5. [Epub ahead of print]707 299-327

STED super-resolution microscopy of mitochondrial translocases.

Sarah V Schweighofer, Kaushik Inamdar, Daniel C Jans, Stefan Jakobs.

The mitochondrial translocases of the outer membrane (TOM) and of the inner membrane (TIM) act together to facilitate the import of nuclear-encoded proteins across the mitochondrial membranes. Stimulated Emission Depletion (STED) super-resolution microscopy enables the in situ imaging of such complexes in single cells at sub-diffraction resolution. STED microscopy requires only conventional sample preparation techniques and provides super-resolved raw data without the need for further image processing. In this chapter, we provide a detailed example protocol for STED microscopy of TOM20 and mitochondrial DNA in fixed mammalian cells. The protocol includes instructions on sample preparation for immunolabeling, including cell line selection, fixation, permeabilization, blocking, labeling and mounting, but also recommendations for sample and microscope performance evaluation. The protocol is supplemented by considerations on key factors that influence the quality of the final image and also includes some considerations for the analysis of the acquired images. While the protocol described here is aimed at imaging TOM20 and DNA, it contains all the information for an immediate adaptation to other cellular targets.

DOI: https://doi.org/10.1016/bs.mie.2024.07.052
Methods Enzymol. 2024 ;pii: S0076-6879(24)00405-1. [Epub ahead of print]707 519-539

Analysis of mitochondrial biogenesis regulation by oxidative stress.

Dheeraj Pathak, Thanuja Krishnamoorthy, Naresh Babu V Sepuri.

  Of all the causes of metabolic and neurological disorders, oxidative stress distinguishes itself by its sweeping effect on the dynamic cellular redox homeostasis and, in its wake, exposing the vulnerabilities of the protein machinery of the cell. High levels of Reactive Oxygen Species (ROS) that mitochondria produce during ATP synthesis can damage mtDNA, lipids, and essential mitochondrial proteins. ROS majorly oxidizes cysteine and methionine amino acids in peptides, which can lead to protein unfolding or misfolding of proteins, which ultimately can have a toll on their function. As mitochondrial biogenesis relies on the continuous import of nuclear-encoded proteins into mitochondria mediated by mitochondrial protein import complexes, oxidative stress triggered by mitochondria can rapidly and detrimentally affect mitochondrial biogenesis and homeostasis. Functional Mge1 is a homodimer and acts as a cochaperone and a nucleotide exchange factor of mitochondrial heat shock protein 70 (mHsp70), crucial for mitochondrial protein import. Oxidative stress like ROS, oxidizes Met 155 in Mge1, compromising its ability to dimerize and interact with mHsp70. The cell employs Methionine sulphoxide reductase 2 (Mxr2), a member of the methionine sulphoxide reductase family, to reduce oxidized Met 155 and thereby restore the essential function of Mge1. Oxidation of methionine as a regulated post-translational modification has been gaining traction. Future high throughput studies that can scan the entire mitochondrial proteome to interrogate methionine oxidation and reversal may increase the repertoire of mitochondrial proteins undergoing regulated oxidation and reduction. In this chapter, we describe the methods followed in our laboratory to study the oxidation of Mge1 and its reduction by Mxr2 in vitro.

Keywords:  Cross linking; Methionine oxidation; Methionine sulfoixde reductase 2; Mge1; Mitochondria; Reactive Oxygen Species

DOI:  https://doi.org/10.1016/bs.mie.2024.07.060
Methods Enzymol. 2024 ;pii: S0076-6879(24)00365-3. [Epub ahead of print]707 543-564

Methods to analyze the mitochondrial unfolded protein response (UPRmt).

Avijit Mallick, Cole M Haynes.

  The mitochondrial unfolded protein response (UPRmt) is a mitochondria-to-nuclear signaling pathway that mediates the transcription of genes required to maintain mitochondrial function during development as well as during aging. In this chapter, we describe the approaches and techniques that we and others have used to elucidate the mechanism(s) by which cells detect mitochondrial stress or dysfunction and communicate with the nucleus to induce transcription of a protective stress response. We also describe approaches to evaluate the impact of UPRmt activation on mitochondrial function and mitochondrial biogenesis including imaging-based approaches as well as approaches to evaluate mitochondrial genome (mtDNA) copy number.

Keywords:  Deleterious mtDNA heteroplasmy; Mito-nuclear communication; Mitochondrial biogenesis; Mitochondrial unfolded protein response; Molecular chaperones; MtDNA replication

DOI:  https://doi.org/10.1016/bs.mie.2024.07.029
Methods Enzymol. 2024 ;pii: S0076-6879(24)00402-6. [Epub ahead of print]707 257-298

Analysis of mitochondrial protein translocation by disulfide bond formation and cysteine specific crosslinking.

Laura F Fielden, Jakob D Busch, Caroline Lindau, Jian Qiu, Nils Wiedemann.

  Protein translocation is a highly dynamic process and, in addition, mitochondrial protein import is especially complicated as the majority of nuclear encoded precursor proteins must engage with multiple translocases before they are assembled in the correct mitochondrial subcompartment. In this chapter, we describe assays for engineered disulfide bond formation and cysteine specific crosslinking to analyze the rearrangement of translocase subunits or to probe protein-protein interactions between precursor proteins and translocase subunits. Such assays were used to characterize the translocase of the outer membrane, the presequence translocase of the inner membrane and the sorting and assembly machinery for the biogenesis of β-Barrel proteins. Moreover, these approaches were also employed to determine the translocation path of precursor proteins (identification of import receptors and specific domains required for translocation) as well as the analysis, location and translocase subunit dependence for the formation of β-Barrel proteins. Here we describe how engineered disulfide bond formation and cysteine specific crosslinking assays are planned and performed and discuss important aspects for its application to study mitochondrial protein translocation.

Keywords:  Mitochondria; Precursor protein; Protein biogenesis; Protein import; Protein-protein interaction; SAM complex; TIM complex; TOM complex; Translocase

DOI:  https://doi.org/10.1016/bs.mie.2024.07.057
Methods Enzymol. 2024 ;pii: S0076-6879(24)00406-3. [Epub ahead of print]707 673-683

Seahorse assay for the analysis of mitochondrial respiration using Saccharomyces cerevisiae as a model system.

Abhishek Kumar, Tejashree Pradip Waingankar, Patrick D'Silva.

  Eukaryotic cells require energy to perform diverse cellular functions critical for survival. Mitochondria are multifunctional organelles that generate energy in the form of Adenosine triphosphate by oxidative phosphorylation, emphasizing their importance to eukaryotic cell viability. The ability of mitochondria to consume oxygen for respiration is a key parameter in assessing mitochondrial health. Therefore, developing new techniques to monitor mitochondrial respiration are crucial for advancing our understanding of organelle functioning. Recently, Seahorse technology has emerged as a valuable tool to analyze various aspects of mitochondrial bioenergetics. Although the Seahorse assay is well established in adherent cell lines and other model organisms, it remains challenging to employ it efficiently in yeast, a powerful genetic system for studying mitochondrial biology. In this chapter, we provide a comprehensive methodology for assessing oxygen consumption rate in baker's yeast using Seahorse.

Keywords:  Mitochondrial respiration; Saccharomyces cerevisiae; Seahorse; electron transport chain; oxygen consumption rate

DOI:  https://doi.org/10.1016/bs.mie.2024.07.061
Methods Enzymol. 2024 ;pii: S0076-6879(24)00388-4. [Epub ahead of print]707 475-498

Analysis of mitochondrial protein aggregation and disaggregation.

Wolfgang Voos, Anne Wilkening, Robin Ostermann, Michael Bruderek, Witold Jaworek, Laura Ruland.

  Deficits of mitochondrial functions have been identified in many human pathologies, in particular in age-related human neurodegenerative diseases. Hence, the molecular causes for mitochondrial dysfunction and potential protection mechanisms have become a major topic in modern cell biology. Apart from defects in their structural integrity, problems in mitochondrial protein biogenesis, including polypeptide transport, folding and assembly to active enzymes, all may result in some degree of functional defects of the organelle. An accumulation of misfolded polypeptides inside mitochondria, confounded by the dual source of mitochondrial polypeptides, will result in the formation of protein aggregates. Such aggregate accumulation bears a cell-toxic potential, resulting in mitochondrial and correlated cellular damages, summarized in the term "aggregate proteotoxicity". Here, we discuss methods to analyze protein aggregation in the mitochondrial matrix compartment. We also address techniques to characterize the biochemical mechanisms that reduce aggregate proteotoxicity, the disaggregation or resolubilization of aggregated polypeptides and the sequestration and neutralization of mitochondrial aggregates at specific sites inside a cell.

Keywords:  ATP-dependent proteases; Human cells; Mitochondria; Molecular chaperones; Protein aggregation; Proteotoxicity; Yeast

DOI:  https://doi.org/10.1016/bs.mie.2024.07.048
Methods Enzymol. 2024 ;pii: S0076-6879(24)00395-1. [Epub ahead of print]707 565-584

Analysis of quality control pathways for the translocase of the outer mitochondrial membrane.

Lara Calvo Santos, Fabian den Brave.

  The functionality of mitochondria depends on the import of proteins synthesized on cytosolic ribosomes. Impaired import into mitochondria results in mitochondrial dysfunction and proteotoxic accumulation of precursor proteins in the cytosol. All proteins sorted to inner mitochondrial compartments are imported via the translocase of the outer membrane (TOM) complex. Premature protein folding, a reduction of the mitochondrial membrane potential or defects in translocases can result in precursor arrest during translocation, thereby clogging the TOM channel and blocking protein import. In recent years, different pathways have been identified, which employ the cytosolic ubiquitin-proteasome system in the extraction and turnover of precursor proteins from the TOM complex. Central events in this process are the modification of arrested precursor proteins with ubiquitin, their extraction by AAA-ATPases and subsequent degradation by the 26 S proteasome. Analysis of these processes is largely facilitated by the expression of model proteins that function as efficient "cloggers" of the import machinery. Here we describe the use of such clogger proteins and how their handling by the protein quality control machinery can be monitored. We provide protocols to study the extent of clogging, the ubiquitin-modification of arrested precursor proteins and their turnover by the 26 S proteasome.

Keywords:  Mitochondria; Proteasome; Protein import; Protein quality control; Ubiquitin

DOI:  https://doi.org/10.1016/bs.mie.2024.07.050
Methods Enzymol. 2024 ;pii: S0076-6879(24)00404-X. [Epub ahead of print]707 101-152

Mass spectrometry-based proteomics to study mutants and interactomes of mitochondrial translocation proteins.

Silke Oeljeklaus, Lakshita Sharma, Julian Bender, Bettina Warscheid.

  The multiple functions of mitochondria are governed by their proteome comprising 1000-1500 proteins depending on the organism. However, only few proteins are synthesized inside mitochondria, whereas most are "born" outside mitochondria. To reach their destined location, these mitochondrial proteins follow specific import routes established by a mitochondrial translocase network. A detailed understanding of the role and interplay of the different translocases is imperative to understand mitochondrial biology and how mitochondria are integrated into the cellular network. Mass spectrometry (MS) proved to be effective to study the translocase network regarding composition, functions, interplay, and cellular responses evoked by dysfunction. In this chapter, we provide protocols tailored to MS-enabled functional analysis of mutants and interactomes of mitochondrial translocation proteins. In the first part, we exemplify the MS-based proteomics analysis of translocation mutants for delineating the human mitochondrial importome following depletion of the central translocation protein TOMM40. The protocol comprises metabolic stable isotope labeling, TOMM40 knockdown, preparation of mitochondrial fractions, and sample preparation for liquid chromatography (LC)-MS. For deep MS analysis, prefractionation of peptide mixtures by high pH reversed-phase LC is described. In the second part, we outline an affinity purification MS approach to reveal the association of an orphaned protein with the translocase TIM23. The protocol covers FLAG-tag affinity purification of protein complexes from mitochondrial fractions and downstream sample preparation for interactome analysis. In the last unifying part, we describe methods for LC-MS, data processing, statistical analysis and visualization of quantitative MS data, and provide a Python code for effective, customizable analysis.

Keywords:  Affinity-purification mass spectrometry; Data analysis; Importome; Interactome; Mass spectrometry; Mitochondria; Protein quantification; Proteomics; SILAC; Translocation mutants

DOI:  https://doi.org/10.1016/bs.mie.2024.07.059
Methods Enzymol. 2024 ;pii: S0076-6879(24)00425-7. [Epub ahead of print]707 585-610

Analysis of the mitochondria-associated degradation pathway (MAD) in yeast cells.

Pin-Chao Liao, Liza A Pon.

  Mitochondria are critical for cellular function in health, disease and aging. Mitochondria-associated degradation (MAD), a pathway for quality control of the organelle, recognizes and ubiquitinates unfolded mitochondrial proteins, removes them from the organelle using a conserved segregase complex, which contains an AAA-ATPase Cdc48 and its cofactors, and degrades them using the ubiquitin-proteasome system (UPS). Here, we describe an approach to (1) study the turnover and ubiquitination of candidate MAD substrates, (2) assay retrotranslocation and export of MAD substrates from the mitochondrial matrix in vitro, and (3) study interactions between MAD substrates and Cdc48 using the budding yeast, Saccharomyces cerevisiae, as a model organism.

Keywords:  Affinity purification; Budding yeast; Co-immunoprecipitation; Mitochondrial isolation; Mitochondrial quality control

DOI:  https://doi.org/10.1016/bs.mie.2024.09.001