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

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

10.1101/2021.06.22.449413 ◽

2021 ◽

Author(s):

Mauricio Cardenas-Rodriguez ◽

Phanee Manganas ◽

Emmanouela Kallergi ◽

Ruairidh Edwards ◽

Afroditi Chatzi ◽

...

Keyword(s):

Redox State ◽

Protein Import ◽

Interaction Analysis ◽

Oxidative Capacity ◽

Intermembrane Space ◽

Thioredoxin System ◽

In Vitro Reconstitution ◽

Mitochondrial Intermembrane Space ◽

Mitochondria Biogenesis

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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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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Protein import and oxidative folding in the mitochondrial intermembrane space of intact mammalian cells

Molecular Biology of the Cell ◽

10.1091/mbc.e12-12-0862 ◽

2013 ◽

Vol 24 (14) ◽

pp. 2160-2170 ◽

Cited By ~ 72

Author(s):

Manuel Fischer ◽

Sebastian Horn ◽

Anouar Belkacemi ◽

Kerstin Kojer ◽

Carmelina Petrungaro ◽

...

Keyword(s):

Protein Oxidation ◽

Mammalian Cells ◽

Protein Import ◽

Mitochondrial Protein ◽

Intermembrane Space ◽

Basic Principles ◽

Oxidative Folding ◽

Physiological Conditions ◽

Mitochondrial Intermembrane Space ◽

Kinetics Of

Oxidation of cysteine residues to disulfides drives import of many proteins into the intermembrane space of mitochondria. Recent studies in yeast unraveled the basic principles of mitochondrial protein oxidation, but the kinetics under physiological conditions is unknown. We developed assays to follow protein oxidation in living mammalian cells, which reveal that import and oxidative folding of proteins are kinetically and functionally coupled and depend on the oxidoreductase Mia40, the sulfhydryl oxidase augmenter of liver regeneration (ALR), and the intracellular glutathione pool. Kinetics of substrate oxidation depends on the amount of Mia40 and requires tightly balanced amounts of ALR. Mia40-dependent import of Cox19 in human cells depends on the inner membrane potential. Our observations reveal considerable differences in the velocities of mitochondrial import pathways: whereas preproteins with bipartite targeting sequences are imported within seconds, substrates of Mia40 remain in the cytosol for several minutes and apparently escape premature degradation and oxidation.

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The mitochondrial intermembrane space: the most constricted mitochondrial sub-compartment with the largest variety of protein import pathways

Open Biology ◽

10.1098/rsob.210002 ◽

2021 ◽

Vol 11 (3) ◽

Author(s):

Ruairidh Edwards ◽

Ross Eaglesfield ◽

Kostas Tokatlidis

Keyword(s):

Protein Import ◽

Atp Hydrolysis ◽

Current Knowledge ◽

Intermembrane Space ◽

Inner Membrane ◽

Mitochondrial Proteome ◽

Normal Site ◽

The Matrix ◽

Human Disorders ◽

Mitochondrial Intermembrane Space

The mitochondrial intermembrane space (IMS) is the most constricted sub-mitochondrial compartment, housing only about 5% of the mitochondrial proteome, and yet is endowed with the largest variability of protein import mechanisms. In this review, we summarize our current knowledge of the major IMS import pathway based on the oxidative protein folding pathway and discuss the stunning variability of other IMS protein import pathways. As IMS-localized proteins only have to cross the outer mitochondrial membrane, they do not require energy sources like ATP hydrolysis in the mitochondrial matrix or the inner membrane electrochemical potential which are critical for import into the matrix or insertion into the inner membrane. We also explore several atypical IMS import pathways that are still not very well understood and are guided by poorly defined or completely unknown targeting peptides. Importantly, many of the IMS proteins are linked to several human diseases, and it is therefore crucial to understand how they reach their normal site of function in the IMS. In the final part of this review, we discuss current understanding of how such IMS protein underpin a large spectrum of human disorders.

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