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Agnieszka Chacinska - One of the best experts on this subject based on the ideXlab platform.
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Determinants of the Cytosolic Turnover of Mitochondrial Intermembrane Space Proteins
BMC Biology, 2018Co-Authors: Lukasz Kowalski, Piotr Bragoszewski, Edyta Glow, Anton Khmelinskii, Michael Knop, Agnieszka ChacinskaAbstract:The proteome of mitochondria comprises mostly proteins that originate as precursors in the cytosol. Before import into the organelle, such proteins are exposed to cytosolic quality control mechanisms. Multiple lines of evidence indicate a significant contribution of the major cytosolic protein degradation machinery, the ubiquitin-proteasome system, to the quality control of Mitochondrial proteins. Proteins that are directed to the Mitochondrial Intermembrane Space (IMS) exemplify an entire class of Mitochondrial proteins regulated by proteasomal degradation. However, little is known about how these proteins are selected for degradation. The present study revealed the heterogeneous cytosolic stability of IMS proteins. Using a screening approach, we found that different cytosolic factors are responsible for the degradation of specific IMS proteins, with no single common factor involved in the degradation of all IMS proteins. We found that the Cox12 protein is rapidly degraded when localized to the cytosol, thus providing a sensitive experimental model. Using Cox12, we found that lysine residues but not conserved cysteine residues are among the degron features important for protein ubiquitination. We observed the redundancy of ubiquitination components, with significant roles of Ubc4 E2 ubiquitin-conjugating enzyme and Rsp5 E3 ubiquitin ligase. The amount of ubiquitinated Cox12 was inversely related to Mitochondrial import efficiency. Importantly, we found that precursor protein ubiquitination blocks its import into mitochondria. The present study confirms the involvement of ubiquitin-proteasome system in the quality control of Mitochondrial IMS proteins in the cytosol. Notably, ubiquitination of IMS proteins prohibits their import into mitochondria. Therefore, ubiquitination directly affects the availability of precursor proteins for organelle biogenesis. Importantly, despite their structural similarities, IMS proteins are not selected for degradation in a uniform way. Instead, specific IMS proteins rely on discrete components of the ubiquitination machinery to mediate their clearance by the proteasome.
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Determinants of the cytosolic turnover of Mitochondrial Intermembrane Space proteins
BMC Biology, 2018Co-Authors: Lukasz Kowalski, Piotr Bragoszewski, Edyta Glow, Anton Khmelinskii, Michael Knop, Agnieszka ChacinskaAbstract:BackgroundThe proteome of mitochondria comprises mostly proteins that originate as precursors in the cytosol. Before import into the organelle, such proteins are exposed to cytosolic quality control mechanisms. Multiple lines of evidence indicate a significant contribution of the major cytosolic protein degradation machinery, the ubiquitin-proteasome system, to the quality control of Mitochondrial proteins. Proteins that are directed to the Mitochondrial Intermembrane Space (IMS) exemplify an entire class of Mitochondrial proteins regulated by proteasomal degradation. However, little is known about how these proteins are selected for degradation.ResultsThe present study revealed the heterogeneous cytosolic stability of IMS proteins. Using a screening approach, we found that different cytosolic factors are responsible for the degradation of specific IMS proteins, with no single common factor involved in the degradation of all IMS proteins. We found that the Cox12 protein is rapidly degraded when localized to the cytosol, thus providing a sensitive experimental model. Using Cox12, we found that lysine residues but not conserved cysteine residues are among the degron features important for protein ubiquitination. We observed the redundancy of ubiquitination components, with significant roles of Ubc4 E2 ubiquitin-conjugating enzyme and Rsp5 E3 ubiquitin ligase. The amount of ubiquitinated Cox12 was inversely related to Mitochondrial import efficiency. Importantly, we found that precursor protein ubiquitination blocks its import into mitochondria.ConclusionsThe present study confirms the involvement of ubiquitin-proteasome system in the quality control of Mitochondrial IMS proteins in the cytosol. Notably, ubiquitination of IMS proteins prohibits their import into mitochondria. Therefore, ubiquitination directly affects the availability of precursor proteins for organelle biogenesis. Importantly, despite their structural similarities, IMS proteins are not selected for degradation in a uniform way. Instead, specific IMS proteins rely on discrete components of the ubiquitination machinery to mediate their clearance by the proteasome.
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Retro-translocation of Mitochondrial Intermembrane Space proteins
Proceedings of the National Academy of Sciences, 2015Co-Authors: Piotr Bragoszewski, Agnieszka Gornicka, Michal Wasilewski, Paulina Sakowska, Lena Böttinger, Jian Qiu, Nils Wiedemann, Agnieszka ChacinskaAbstract:The content of Mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the Intermembrane Space by the Mitochondrial Intermembrane Space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the Mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the Mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the Mitochondrial proteome surveillance.
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The Ubiquitin-Proteasome System Regulates Mitochondrial Intermembrane Space Proteins
Molecular and Cellular Biology, 2013Co-Authors: Piotr Bragoszewski, Agnieszka Gornicka, Malgorzata E. Sztolsztener, Agnieszka ChacinskaAbstract:Mitochondrial precursor proteins are synthesized in the cytosol and subsequently imported into mitochondria. The import of Mitochondrial Intermembrane Space proteins is coupled with their oxidative folding and governed by the Mitochondrial Intermembrane Space import and assembly (MIA) pathway. The cytosolic steps that precede Mitochondrial import are not well understood. We identified a role for the ubiquitin-proteasome system in the biogenesis of Intermembrane Space proteins. Interestingly, the function of the ubiquitin-proteasome system is not restricted to conditions of Mitochondrial protein import failure. The ubiquitin-proteasome system persistently removes a fraction of Intermembrane Space proteins under physiological conditions, acting as a negative regulator in the biogenesis of this class of proteins. Thus, the ubiquitin-proteasome system plays an important role in determining the levels of proteins targeted to the Intermembrane Space of mitochondria.
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The MIA pathway: a tight bond between protein transport and oxidative folding in mitochondria.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2012Co-Authors: Diana Stojanovski, Piotr Bragoszewski, Agnieszka ChacinskaAbstract:Many newly synthesized proteins obtain disulfide bonds in the bacterial periplasm, the endoplasmic reticulum (ER) and the Mitochondrial Intermembrane Space. The acquisition of disulfide bonds is critical for the folding, assembly and activity of these proteins. Spontaneous oxidation of thiol groups is inefficient in vivo, therefore cells have developed machineries that catalyse the oxidation of substrate proteins. The identification of the machinery that mediates this process in the Intermembrane Space of mitochondria, known as MIA (Mitochondrial Intermembrane Space assembly), provided a unique mechanism of protein transport. The MIA machinery introduces disulfide bonds into incoming Intermembrane Space precursors and thus tightly couples the process of precursor translocation to precursor oxidation. We discuss our current understanding of the MIA pathway and the mechanisms that oversee thiol-exchange reactions in mitochondria.
Kostas Tokatlidis - One of the best experts on this subject based on the ideXlab platform.
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The biogenesis of Mitochondrial Intermembrane Space proteins.
Biological Chemistry, 2020Co-Authors: Ruairidh Edwards, Sarah Gerlich, Kostas TokatlidisAbstract:The Mitochondrial Intermembrane Space (IMS) houses a large spectrum of proteins with distinct and critical functions. Protein import into this Mitochondrial sub-compartment is underpinned by an intriguing variety of pathways, many of which are still poorly understood. The constricted volume of the IMS and the topological segregation by the inner membrane cristae into a bulk area surrounded by the boundary inner membrane and the lumen within the cristae is an important factor that adds to the complexity of the protein import, folding and assembly processes. We discuss the main import pathways into the IMS, but also how IMS proteins are degraded or even retro-translocated to the cytosol in an integrated network of interactions that is necessary to maintain a healthy balance of IMS proteins under physiological and cellular stress conditions. We conclude this review by highlighting new and exciting perspectives in this area with a view to develop a better understanding of yet unknown, likely unconventional import pathways, how presequence-less proteins can be targeted and the basis for dual localisation in the IMS and the cytosol. Such knowledge is critical to understanding the dynamic changes of the IMS proteome in response to stress, and particularly important for maintaining optimal Mitochondrial fitness.
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Oxidative protein biogenesis and redox regulation in the Mitochondrial Intermembrane Space
Cell and Tissue Research, 2017Co-Authors: Phanee Manganas, Lisa Macpherson, Kostas TokatlidisAbstract:Mitochondria are organelles that play a central role in cellular metabolism, as they are responsible for processes such as iron/sulfur cluster biogenesis, respiration and apoptosis. Here, we describe briefly the various protein import pathways for sorting of Mitochondrial proteins into the different subcompartments, with an emphasis on the targeting to the Intermembrane Space. The discovery of a dedicated redox-controlled pathway in the Intermembrane Space that links protein import to oxidative protein folding raises important questions on the redox regulation of this process. We discuss the salient features of redox regulation in the Intermembrane Space and how such mechanisms may be linked to the more general redox homeostasis balance that is crucial not only for normal cell physiology but also for cellular dysfunction.
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The Mitochondrial Intermembrane Space: a hub for oxidative folding linked to protein biogenesis.
Antioxidants & Redox Signaling, 2013Co-Authors: Afroditi Chatzi, Kostas TokatlidisAbstract:Abstract Significance: The introduction of disulfide bonds in proteins of the Mitochondrial Intermembrane Space (IMS) is fundamental for their folding and assembly. This oxidative folding process depends on the disulfide donor/import receptor Mia40 and the flavin adenine dinucleotide oxidase Erv1 and concerns proteins involved in Mitochondrial biogenesis, respiratory complex assembly, and metal transfer. Recent Advances: The recently determined structural basis of the interaction between Mia40 and some substrates provides a framework for the electron transfer process. A possible proofreading role for the cellular reductant glutathione has been proposed, while other studies suggest the association of Mia40 and Erv1 in dynamic multiprotein complexes in the IMS. Critical Issues: The association of Mia40 with Erv1 and substrates in large multiprotein complexes is critical. Completion of substrate folding by additional disulfide bonds after initial binding to Mia40 remains unclear. Furthermore, a more general ...
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targeting and maturation of erv1 alr in the Mitochondrial Intermembrane Space
ACS Chemical Biology, 2012Co-Authors: Emmanouela Kallergi, Kostas Tokatlidis, Paraskevi Kritsiligkou, Maria Andreadaki, Nitsa Katrakili, Lucia Banci, Charalambos Pozidis, Ivano Bertini, Chiara Cefaro, Simone CiofibaffoniAbstract:The interaction of Mia40 with Erv1/ALR is central to the oxidative protein folding in the Intermembrane Space of mitochondria (IMS) as Erv1/ALR oxidizes reduced Mia40 to restore its functional state. Here we address the role of Mia40 in the import and maturation of Erv1/ALR. The C-terminal FAD-binding domain of Erv1/ALR has an essential role in the import process by creating a transient intermolecular disulfide bond with Mia40. The action of Mia40 is selective for the formation of both intra and intersubunit structural disulfide bonds of Erv1/ALR, but the complete maturation process requires additional binding of FAD. Both of these events must follow a specific sequential order to allow Erv1/ALR to reach the fully functional state, illustrating a new paradigm for protein maturation in the IMS.
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Oxidative protein folding in the Mitochondrial Intermembrane Space
Antioxidants & Redox Signaling, 2010Co-Authors: Dionisia P. Sideris, Kostas TokatlidisAbstract:Abstract Disulfide bond formation is a crucial step for oxidative folding and necessary for the acquisition of a protein's native conformation. Introduction of disulfide bonds is catalyzed in specialized subcellular compartments and requires the coordinated action of specific enzymes. The Intermembrane Space of mitochondria has recently been found to harbor a dedicated machinery that promotes the oxidative folding of substrate proteins by shuttling disulfide bonds. The newly identified oxidative pathway consists of the redox-regulated receptor Mia40 and the sulfhydryl oxidase Erv1. Proteins destined to the Intermembrane Space are trapped by a disulfide relay mechanism that involves an electron cascade from the incoming substrate to Mia40, then on to Erv1, and finally to molecular oxygen via cytochrome c. This thiol–disulfide exchange mechanism is essential for the import and for maintaining the structural stability of the incoming precursors. In this review we describe the mechanistic parameters that defi...
Carla M Koehler - One of the best experts on this subject based on the ideXlab platform.
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Osm1 facilitates the transfer of electrons from Erv1 to fumarate in the redox-regulated import pathway in the Mitochondrial Intermembrane Space
Molecular Biology of the Cell, 2017Co-Authors: Sonya E. Neal, Deepa V. Dabir, Juwina Wijaya, Cennyana Boon, Carla M KoehlerAbstract:Prokaryotes have aerobic and anaerobic electron acceptors for oxidative folding of periplasmic proteins. The Mitochondrial Intermembrane Space has an analogous pathway with the oxidoreductase Mia40 and sulfhydryl oxidase Erv1, termed the Mitochondrial Intermembrane Space assembly (MIA) pathway. The aerobic electron acceptors include oxygen and cytochrome c, but an acceptor that can function under anaerobic conditions has not been identified. Here we show that the fumarate reductase Osm1, which facilitates electron transfer from fumarate to succinate, fills this gap as a new electron acceptor. In addition to microsomes, Osm1 localizes to the Mitochondrial Intermembrane Space and assembles with Erv1 in a complex. In reconstitution studies with reduced Tim13, Mia40, and Erv1, the addition of Osm1 and fumarate completes the disulfide exchange pathway that results in Tim13 oxidation. From in vitro import assays, mitochondria lacking Osm1 display decreased import of MIA substrates, Cmc1 and Tim10. Comparative reconstitution assays support that the Osm1/fumarate couple accepts electrons with similar efficiency to cytochrome c and that the cell has strategies to coordinate expression of the terminal electron acceptors. Thus Osm1/fumarate is a new electron acceptor couple in the Mitochondrial Intermembrane Space that seems to function in both aerobic and anaerobic conditions.
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Redox regulation of protein folding in the Mitochondrial Intermembrane Space.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2009Co-Authors: Carla M Koehler, Heather L. TiensonAbstract:Protein translocation pathways to the Mitochondrial matrix and inner membrane have been well characterized. However, translocation into the Intermembrane Space, which was thought to be simply a modification of the traditional translocation pathways, is complex. The mechanism by which a subset of Intermembrane Space proteins, those with disulfide bonds, are translocated has been largely unknown until recently. Specifically, the Intermembrane Space proteins with disulfide bonds are imported via the Mitochondrial Intermembrane Space assembly (MIA) pathway. Substrates are imported via a disulfide exchange relay with two components Mia40 and Erv1. This new breakthrough has resulted in novel concepts for assembly of proteins in the Intermembrane Space, suggesting that this compartment may be similar to that of the endoplasmic reticulum and the prokaryotic periplasm. As a better understanding of this pathway emerges, new paradigms for thiol-disulfide exchange mechanisms may be developed. Given that the Intermembrane Space is important for disease processes including apoptosis and neurodegeneration, new roles in regulation by oxidation–reduction chemistry seem likely to be relevant.
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a Mitochondrial rhomboid protease
Developmental Cell, 2003Co-Authors: Alexander M Van Der Bliek, Carla M KoehlerAbstract:Abstract Rhomboid proteases are integral membrane proteins, typically associated with cleavage of peptide hormones along the secretory pathway. Recent publications demonstrate that yeast mitochondria contain a rhomboid protease required for the cleavage of two Mitochondrial Intermembrane Space proteins, suggesting that rhomboid proteases play a regulatory role in mitochondria.
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different import pathways through the Mitochondrial Intermembrane Space for inner membrane proteins
The EMBO Journal, 1999Co-Authors: Danielle Leuenberger, Gottfried Schatz, Nikolaus A Bally, Carla M KoehlerAbstract:Earlier work on the protein import system of yeast mitochondria has identified two soluble 70 kDa protein complexes in the Intermembrane Space. One complex contains the essential proteins Tim9p and Tim10p and mediates transport of cytosolically‐made metabolite carrier proteins from the outer to the inner membrane. The other complex contains the non‐essential proteins Tim8p and Tim13p as well as loosely associated Tim9p; its function was unclear, but it interacted structurally or functionally with the Tim9p–Tim10p complex. We now show that the two 70 kDa complexes each mediate the import of a different subset of integral inner membrane proteins and that they can transfer these proteins to one of three different membrane insertion sites: the TIM22 complex, the TIM23 complex or an as yet uncharacterized insertion site. Yeast mitochondria thus use multiple pathways for escorting hydrophobic inner membrane proteins across the aqueous Intermembrane Space.
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How membrane proteins travel across the Mitochondrial Intermembrane Space
Trends in Biochemical Sciences, 1999Co-Authors: Carla M Koehler, Sabeeha S. Merchant, Gottfried SchatzAbstract:A newly discovered family of small proteins in the yeast Mitochondrial Intermembrane Space mediates import of hydrophobic proteins from the cytoplasm into the inner membrane. Loss of one of these chaperone-like proteins from human mitochondria results in a disease that causes deafness, muscle weakness and blindness.
Johannes M Herrmann - One of the best experts on this subject based on the ideXlab platform.
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the Mitochondrial Intermembrane Space facing proteins mcp2 and tgl2 are involved in yeast lipid metabolism
Molecular Biology of the Cell, 2019Co-Authors: Fenja Odendall, Johannes M Herrmann, Sandra Backes, Thomas Langer, Takashi Tatsuta, Uri Weill, Maya Schuldiner, Doron Rapaport, Kai Stefan DimmerAbstract:Mitochondria are unique organelles harboring two distinct membranes, the Mitochondrial inner and outer membrane (MIM and MOM, respectively). Mitochondria comprise only a subset of metabolic pathways for the synthesis of membrane lipids; therefore most lipid species and their precursors have to be imported from other cellular compartments. One such import process is mediated by the ER mitochondria encounter structure (ERMES) complex. Both Mitochondrial membranes surround the hydrophilic Intermembrane Space (IMS). Therefore, additional systems are required that shuttle lipids between the MIM and MOM. Recently, we identified the IMS protein Mcp2 as a high-copy suppressor for cells that lack a functional ERMES complex. To understand better how mitochondria facilitate transport and biogenesis of lipids, we searched for genetic interactions of this suppressor. We found that MCP2 has a negative genetic interaction with the gene TGL2 encoding a neutral lipid hydrolase. We show that this lipase is located in the Intermembrane Space of the mitochondrion and is imported via the Mia40 disulfide relay system. Furthermore, we show a positive genetic interaction of double deletion of MCP2 and PSD1, the gene encoding the enzyme that synthesizes the major amount of cellular phosphatidylethanolamine. Finally, we demonstrate that the nucleotide-binding motifs of the predicted atypical kinase Mcp2 are required for its proper function. Taken together, our data suggest that Mcp2 is involved in Mitochondrial lipid metabolism and an increase of this involvement by overexpression suppresses loss of ERMES.
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Development of the Mitochondrial Intermembrane Space Disulfide Relay Represents a Critical Step in Eukaryotic Evolution
Molecular Biology and Evolution, 2019Co-Authors: Sandra Backes, Valentina Peleh, Sriram G. Garg, Laura Becker, Rudi Glockshuber, Sven B. Gould, Johannes M HerrmannAbstract:The Mitochondrial Intermembrane Space evolved from the bacterial periplasm. Presumably as a consequence of their common origin, most proteins of these compartments are stabilized by structural disulfide bonds. The molecular machineries that mediate oxidative protein folding in bacteria and mitochondria, however, appear to share no common ancestry. Here we tested whether the enzymes Erv1 and Mia40 of the yeast Mitochondrial disulfide relay could be functionally replaced by corresponding components of other compartments. We found that the sulfhydryl oxidase Erv1 could be replaced by the Ero1 oxidase or the protein disulfide isomerase from the endoplasmic reticulum, however at the cost of respiration deficiency. In contrast to Erv1, the Mitochondrial oxidoreductase Mia40 proved to be indispensable and could not be replaced by thioredoxin-like enzymes, including the cytoplasmic reductase thioredoxin, the periplasmic dithiol oxidase DsbA, and Pdi1. From our studies we conclude that the profound inertness against glutathione, its slow oxidation kinetics and its high affinity to substrates renders Mia40 a unique and essential component of Mitochondrial biogenesis. Evidently, the development of a specific Mitochondrial disulfide relay system represented a crucial step in the evolution of the eukaryotic cell.
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Proteasomal degradation competes with Mia40-mediated import into mitochondria
BMC, 2018Co-Authors: Eva Zöller, Todd R. Alexander, Johannes M HerrmannAbstract:Abstract Tandem fluorescent protein timers are elegant tools to determine proteolytic stabilities of cytosolic proteins with high spatial and temporal resolution. In a new study published in BMC Biology, Kowalski et al. fused timers to precursors of proteins of the Mitochondrial Intermembrane Space and found that they are under surveillance of the ubiquitin-proteasome system. Ubiquitination at lysine residues of these precursors directly inhibits their translocation into the Intermembrane Space and targets them for proteasomal degradation
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Erv1 of Arabidopsis thaliana can directly oxidize Mitochondrial Intermembrane Space proteins in the absence of redox-active Mia40.
BMC Biology, 2017Co-Authors: Valentina Peleh, Sandra Backes, Flavien Zannini, Nicolas Rouhier, Johannes M HerrmannAbstract:Many proteins of the Mitochondrial Intermembrane Space (IMS) contain structural disulfide bonds formed by the Mitochondrial disulfide relay. In fungi and animals, the sulfhydryl oxidase Erv1 ‘generates’ disulfide bonds that are passed on to the oxidoreductase Mia40, which oxidizes substrate proteins. A different structural organization of plant Erv1 proteins compared to that of animal and fungal orthologs was proposed to explain its inability to complement the corresponding yeast mutant. Herein, we have revisited the biochemical and functional properties of Arabidopsis thaliana Erv1 by both in vitro reconstituted activity assays and complementation of erv1 and mia40 yeast mutants. These mutants were viable, however, they showed severe defects in the biogenesis of IMS proteins. The plant Erv1 was unable to oxidize yeast Mia40 and rather even blocked its activity. Nevertheless, it was able to mediate the import and folding of Mitochondrial proteins. We observed that plant Erv1, unlike its homologs in fungi and animals, can promote protein import and oxidative protein folding in the IMS independently of the oxidoreductase Mia40. In accordance to the absence of Mia40 in many protists, our study suggests that the Mitochondrial disulfide relay evolved in a stepwise reaction from an Erv1-only system to which Mia40 was added in order to improve substrate specificity.
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kinetic control by limiting glutaredoxin amounts enables thiol oxidation in the reducing Mitochondrial Intermembrane Space
Molecular Biology of the Cell, 2015Co-Authors: Kerstin Kojer, Valentina Peleh, Gaetano Calabrese, Johannes M Herrmann, Jan RiemerAbstract:The Mitochondrial Intermembrane Space (IMS) harbors an oxidizing machinery that drives import and folding of small cysteine-containing proteins without targeting signals. The main component of this pathway is the oxidoreductase Mia40, which introduces disulfides into its substrates. We recently showed that the IMS glutathione pool is maintained as reducing as that of the cytosol. It thus remained unclear how equilibration of protein disulfides with the IMS glutathione pool is prevented in order to allow oxidation-driven protein import. Here we demonstrate the presence of glutaredoxins in the IMS and show that limiting amounts of these glutaredoxins provide a kinetic barrier to prevent the thermodynamically feasible reduction of Mia40 substrates by the IMS glutathione pool. Moreover, they allow Mia40 to exist in a predominantly oxidized state. Consequently, overexpression of glutaredoxin 2 in the IMS results in a more reduced Mia40 redox state and a delay in oxidative folding and Mitochondrial import of different Mia40 substrates. Our findings thus indicate that carefully balanced glutaredoxin amounts in the IMS ensure efficient oxidative folding in the reducing environment of this compartment.
Toshiya Endo - One of the best experts on this subject based on the ideXlab platform.
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Myristoyl group-aided protein import into the Mitochondrial Intermembrane Space.
Scientific Reports, 2019Co-Authors: Eri Ueda, Yasushi Tamura, Shin Kawano, Haruka Sakaue, Chika Kakuta, Shunsuke Matsumoto, Toshiya EndoAbstract:The MICOS complex mediates formation of the crista junctions in mitochondria. Here we analyzed the Mitochondrial import pathways for the six yeast MICOS subunits as a step toward understanding of the assembly mechanisms of the MICOS complex. Mic10, Mic12, Mic26, Mic27, and Mic60 used the presequence pathway to reach the Intermembrane Space (IMS). In contrast, Mic19 took the TIM40/MIA pathway, through its CHCH domain, to reach the IMS. Unlike canonical TIM40/MIA substrates, presence of the N-terminal unfolded DUF domain impaired the import efficiency of Mic19, yet N-terminal myristoylation of Mic19 circumvented this effect. The myristoyl group of Mic19 binds to Tom20 of the TOM complex as well as the outer membrane, which may lead to “entropy pushing” of the DUF domain followed by the CHCH domain of Mic19 into the import channel, thereby achieving efficient import.
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Shuttle mission in the Mitochondrial Intermembrane Space.
The EMBO Journal, 2018Co-Authors: Toshiya Endo, Yasushi TamuraAbstract:Lipid trafficking is essential for biogenesis and maintenance of eukaryotic organelles. In this issue of The EMBO Journal , Saita et al (2018) revealed that proteolytic processing by the rhomboid protease PARL in the Mitochondrial inner membrane facilitates partitioning of START domain‐containing protein STARD7 to the cytosol and Mitochondrial Intermembrane Space. STARD7 in the Mitochondrial Intermembrane Space functions as a lipid transfer protein to shuttle phosphatidylcholine from the outer membrane to the inner membrane.
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Structural basis for the disulfide relay system in the Mitochondrial Intermembrane Space.
Antioxidants & Redox Signaling, 2010Co-Authors: Toshiya Endo, Koji Yamano, Shin KawanoAbstract:Abstract Mitochondria contain two biological membranes. Although reducing agents can diffuse from the cytosol into the Intermembrane Space (IMS) between the outer and inner Mitochondrial membranes, the IMS has a dedicated disulfide relay system to introduce disulfide bonds into mainly small and soluble proteins. This system consists of two essential proteins, a disulfide carrier Tim40/Mia40 and a flavin-dependent sulfhydryl oxidase Erv1, high-resolution structures that have recently become available. Tim40/Mia40 transfers disulfide bonds to newly imported IMS proteins by dithiol/disulfide exchange reactions involving mixed disulfide intermediates. Tight folding by introduction of disulfide bonds prevents egress of these small IMS proteins, resulting in their selective retention in the compartment. After disulfide transfer from Tim40/Mia40 to substrate proteins, Tim40/Mia40 is reoxidized again by Erv1, which is then oxidized by electron transfer to either cytochrome c or molecular oxygen. Here we review th...
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structural basis of yeast tim40 mia40 as an oxidative translocator in the Mitochondrial Intermembrane Space
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Shin Kawano, Koji Yamano, Mari Naoé, Takaki Momose, Kayoko Terao, Shuh-ichi Nishikawa, Nobuhisa Watanabe, Toshiya EndoAbstract:The Mitochondrial Intermembrane Space (IMS) contains many small cysteine-bearing proteins, and their passage across the outer membrane and subsequent folding require recognition and disulfide bond transfer by an oxidative translocator Tim40/Mia40 in the inner membrane facing the IMS. Here we determined the crystal structure of the core domain of yeast Mia40 (Mia40C4) as a fusion protein with maltose-binding protein at a resolution of 3 A. The overall structure of Mia40C4 is a fruit-dish-like shape with a hydrophobic concave region, which accommodates a linker segment of the fusion protein in a helical conformation, likely mimicking a bound substrate. Replacement of the hydrophobic residues in this region resulted in growth defects and impaired assembly of a substrate protein. The Cys296-Cys298 disulfide bond is close to the hydrophobic concave region or possible substrate-binding site, so that it can mediate disulfide bond transfer to substrate proteins. These results are consistent with the growth phenotypes of Mia40 mutant cells containing Ser replacement of the conserved cysteine residues.
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Identification of Tim40 That Mediates Protein Sorting to the Mitochondrial Intermembrane Space
Journal of Biological Chemistry, 2004Co-Authors: Mari Naoé, Shuh-ichi Nishikawa, Yukimasa Ohwa, Daigo Ishikawa, Chié Ohshima, Hayashi Yamamoto, Toshiya EndoAbstract:Most Mitochondrial proteins are synthesized in the cytosol, imported into mitochondria, and sorted to one of the four Mitochondrial subcompartments. Here we identified a new inner membrane protein, Tim40, that mediates sorting of small Tim proteins to the Intermembrane Space. Tim40 is essential for yeast cell growth, and its function in vivo requires six conserved Cys residues but not anchoring of the protein to the inner membrane by its N-terminal hydrophobic segment. Depletion of Tim40 impairs the import of small Tim proteins into mitochondria both in vivo and in vitro. In wild-type mitochondria, Tim40 forms a translocation intermediate with small Tim proteins prior to their assembly in the Intermembrane Space in vitro. These results suggest the essential role of Tim40 in sorting/assembly of small Tim proteins.