A Thioredoxin reductive mechanism balances the oxidative protein import pathway in the intermembrane space of mitochondria

Mitochondria biogenesis crucially depends on the oxidative folding system in the mitochondrial intermembrane space. The oxidative capacity needs however to be balanced by a reductive pathway for optimal mitochondrial fitness. Here we report that the cytosolic thioredoxin machinery fulfils this critical reductive function by dual localisation in the mitochondrial intermembrane space (IMS) via an unconventional import pathway. We show that the presence of the Thioredoxin system in the IMS mediates a hitherto unknown communication between mitochondria biogenesis and the metabolic state of the cell via the cytosolic pool of NADPH. By a combination of complete in vitro reconstitution with purified components, import assays and protein interaction analysis we find that the IMS-localised thioredoxin machinery critically controls the redox state of Mia40, the key player in the MIA pathway in mitochondria thereby ensuring optimal mitochondria biogenesis. Intriguingly, we find that the IMS thioredoxin system fulfils a previously unknown role in the retrograde release of structurally destabilised proteins into the cytosol and protection against oxidative damage, both of which serve as critical mechanisms of mitochondrial surveillance and quality control.

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Kinetic control by limiting glutaredoxin amounts enables thiol oxidation in the reducing mitochondrial intermembrane space

Molecular Biology of the Cell ◽

10.1091/mbc.e14-10-1422 ◽

2015 ◽

Vol 26 (2) ◽

pp. 195-204 ◽

Cited By ~ 45

Author(s):

Kerstin Kojer ◽

Valentina Peleh ◽

Gaetano Calabrese ◽

Johannes M. Herrmann ◽

Jan Riemer

Keyword(s):

Redox State ◽

Protein Import ◽

Kinetic Control ◽

Intermembrane Space ◽

Mitochondrial Import ◽

Oxidative Folding ◽

Mitochondrial Intermembrane Space ◽

Reducing Environment ◽

Targeting Signals ◽

Main Component

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.

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Dynamic organization of the mitochondrial protein import machinery

Biological Chemistry ◽

10.1515/hsz-2016-0145 ◽

2016 ◽

Vol 397 (11) ◽

pp. 1097-1114 ◽

Cited By ~ 24

Author(s):

Sebastian P. Straub ◽

Sebastian B. Stiller ◽

Nils Wiedemann ◽

Nikolaus Pfanner

Keyword(s):

Mitochondrial Membrane ◽

Protein Import ◽

Mitochondrial Protein ◽

Membrane Dynamics ◽

Intermembrane Space ◽

Mitochondrial Protein Import ◽

Precursor Proteins ◽

Dynamic Cooperation

Abstract Mitochondria contain elaborate machineries for the import of precursor proteins from the cytosol. The translocase of the outer mitochondrial membrane (TOM) performs the initial import of precursor proteins and transfers the precursors to downstream translocases, including the presequence translocase and the carrier translocase of the inner membrane, the mitochondrial import and assembly machinery of the intermembrane space, and the sorting and assembly machinery of the outer membrane. Although the protein translocases can function as separate entities in vitro, recent studies revealed a close and dynamic cooperation of the protein import machineries to facilitate efficient transfer of precursor proteins in vivo. In addition, protein translocases were found to transiently interact with distinct machineries that function in the respiratory chain or in the maintenance of mitochondrial membrane architecture. Mitochondrial protein import is embedded in a regulatory network that ensures protein biogenesis, membrane dynamics, bioenergetic activity and quality control.

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Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding

eLife ◽

10.7554/elife.16177 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 29

Author(s):

Valentina Peleh ◽

Emmanuelle Cordat ◽

Johannes M Herrmann

Keyword(s):

Disulfide Bonds ◽

Protein Import ◽

Protein Translocation ◽

Hydrophobic Interactions ◽

Substrate Binding ◽

Intermembrane Space ◽

Cysteine Motif ◽

Mitochondrial Intermembrane Space ◽

Necessary And Sufficient ◽

Yeast Mutants

Many proteins of the mitochondrial IMS contain conserved cysteines that are oxidized to disulfide bonds during their import. The conserved IMS protein Mia40 is essential for the oxidation and import of these proteins. Mia40 consists of two functional elements: an N-terminal cysteine-proline-cysteine motif conferring substrate oxidation, and a C-terminal hydrophobic pocket for substrate binding. In this study, we generated yeast mutants to dissect both Mia40 activities genetically and biochemically. Thereby we show that the substrate-binding domain of Mia40 is both necessary and sufficient to promote protein import, indicating that trapping by Mia40 drives protein translocation. An oxidase-deficient Mia40 mutant is inviable, but can be partially rescued by the addition of the chemical oxidant diamide. Our results indicate that Mia40 predominantly serves as a trans-site receptor of mitochondria that binds incoming proteins via hydrophobic interactions thereby mediating protein translocation across the outer membrane by a ‘holding trap’ rather than a ‘folding trap’ mechanism.

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The biogenesis of mitochondrial intermembrane space proteins

Biological Chemistry ◽

10.1515/hsz-2020-0114 ◽

2020 ◽

Vol 401 (6-7) ◽

pp. 737-747 ◽

Cited By ~ 3

Author(s):

Ruairidh Edwards ◽

Sarah Gerlich ◽

Kostas Tokatlidis

Keyword(s):

Protein Import ◽

Cellular Stress ◽

Stress Conditions ◽

Intermembrane Space ◽

Dynamic Changes ◽

Inner Membrane ◽

Integrated Network ◽

Response To Stress ◽

Mitochondrial Intermembrane Space

AbstractThe 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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Phosphatidylserine transport by Ups2–Mdm35 in respiration-active mitochondria

The Journal of Cell Biology ◽

10.1083/jcb.201601082 ◽

2016 ◽

Vol 214 (1) ◽

pp. 77-88 ◽

Cited By ~ 44

Author(s):

Non Miyata ◽

Yasunori Watanabe ◽

Yasushi Tamura ◽

Toshiya Endo ◽

Osamu Kuge

Keyword(s):

Yeast Cells ◽

Intermembrane Space ◽

Diauxic Shift ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Mitochondrial Functions ◽

Phospholipid Transfer ◽

Metabolic Transition ◽

Mitochondrial Intermembrane Space

Phosphatidylethanolamine (PE) is an essential phospholipid for mitochondrial functions and is synthesized mainly by phosphatidylserine (PS) decarboxylase at the mitochondrial inner membrane. In Saccharomyces cerevisiae, PS is synthesized in the endoplasmic reticulum (ER), such that mitochondrial PE synthesis requires PS transport from the ER to the mitochondrial inner membrane. Here, we provide evidence that Ups2–Mdm35, a protein complex localized at the mitochondrial intermembrane space, mediates PS transport for PE synthesis in respiration-active mitochondria. UPS2- and MDM35-null mutations greatly attenuated conversion of PS to PE in yeast cells growing logarithmically under nonfermentable conditions, but not fermentable conditions. A recombinant Ups2–Mdm35 fusion protein exhibited phospholipid-transfer activity between liposomes in vitro. Furthermore, UPS2 expression was elevated under nonfermentable conditions and at the diauxic shift, the metabolic transition from glycolysis to oxidative phosphorylation. These results demonstrate that Ups2–Mdm35 functions as a PS transfer protein and enhances mitochondrial PE synthesis in response to the cellular metabolic state.

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Glutathione redox potential in the mitochondrial intermembrane space is linked to the cytosol and impacts the Mia40 redox state

The EMBO Journal ◽

10.1038/emboj.2012.165 ◽

2012 ◽

Vol 31 (14) ◽

pp. 3169-3182 ◽

Cited By ~ 125

Author(s):

Kerstin Kojer ◽

Melanie Bien ◽

Heike Gangel ◽

Bruce Morgan ◽

Tobias P Dick ◽

...

Keyword(s):

Redox Potential ◽

Redox State ◽

Intermembrane Space ◽

Mitochondrial Intermembrane Space ◽

Glutathione Redox

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In Vivo Structure-Function Analysis and Redox Interactomes of Leishmania tarentolae Erv

Microbiology Spectrum ◽

10.1128/spectrum.00809-21 ◽

2021 ◽

Author(s):

Gino L. Turra ◽

Linda Liedgens ◽

Frederik Sommer ◽

Luzia Schneider ◽

David Zimmer ◽

...

Keyword(s):

Protein Import ◽

Function Analysis ◽

Mitochondrial Protein ◽

Intermembrane Space ◽

Mitochondrial Protein Import ◽

Redox Biology ◽

Oxidative Protein Folding ◽

Mitochondrial Intermembrane Space ◽

New Research

The discovery of the redox proteins Mia40/CHCHD4 and Erv1/ALR, as well as the elucidation of their relevance for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals, founded a new research topic in redox biology and mitochondrial protein import. The lack of Mia40/CHCHD4 in protist lineages raises fundamental and controversial questions regarding the conservation and evolution of this essential pathway.

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Mrs5p, an Essential Protein of the Mitochondrial Intermembrane Space, Affects Protein Import into Yeast Mitochondria

Journal of Biological Chemistry ◽

10.1074/jbc.271.29.17219 ◽

1996 ◽

Vol 271 (29) ◽

pp. 17219-17225 ◽

Cited By ~ 49

Author(s):

Ernst Jarosch ◽

Gabriele Tuller ◽

Günther Daum ◽

Martin Waldherr ◽

Alica Voskova ◽

...

Keyword(s):

Protein Import ◽

Intermembrane Space ◽

Yeast Mitochondria ◽

Essential Protein ◽

Mitochondrial Intermembrane Space

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The MIA system for protein import into the mitochondrial intermembrane space

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/j.bbamcr.2007.10.004 ◽

2008 ◽

Vol 1783 (4) ◽

pp. 610-617 ◽

Cited By ~ 43

Author(s):

Diana Stojanovski ◽

Judith M. Müller ◽

Dusanka Milenkovic ◽

Bernard Guiard ◽

Nikolaus Pfanner ◽

...

Keyword(s):

Protein Import ◽

Intermembrane Space ◽

Mitochondrial Intermembrane Space

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Protein trafficking in the mitochondrial intermembrane space: mechanisms and links to human disease

Biochemical Journal ◽

10.1042/bcj20160627 ◽

2017 ◽

Vol 474 (15) ◽

pp. 2533-2545 ◽

Cited By ~ 8

Author(s):

Lisa MacPherson ◽

Kostas Tokatlidis

Keyword(s):

Protein Trafficking ◽

Protein Import ◽

Protein Targeting ◽

Inorganic Ions ◽

Oxygen Metabolism ◽

Intermembrane Space ◽

Diverse Range ◽

Range Of Functions ◽

Mitochondrial Intermembrane Space ◽

Space Mechanisms

Mitochondria fulfill a diverse range of functions in cells including oxygen metabolism, homeostasis of inorganic ions and execution of apoptosis. Biogenesis of mitochondria relies on protein import pathways that are ensured by dedicated multiprotein translocase complexes localized in all sub-compartments of these organelles. The key components and pathways involved in protein targeting and assembly have been characterized in great detail over the last three decades. This includes the oxidative folding machinery in the intermembrane space, which contributes to the redox-dependent control of proteostasis. Here, we focus on several components of this system and discuss recent evidence suggesting links to human proteopathy.

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