Oxidative folding of small Tims is mediated by site-specific docking onto Mia40 in the mitochondrial intermembrane space

2007 ◽  
Vol 65 (5) ◽  
pp. 1360-1373 ◽  
Author(s):  
Dionisia P. Sideris ◽  
Kostas Tokatlidis
Keyword(s):  
Intermembrane Space ◽  
Oxidative Folding ◽  
Site Specific ◽  
Mitochondrial Intermembrane Space

scholarly journals The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the CytochromecOxidase Assembly Protein Cox19

2014 ◽  
Vol 289 (14) ◽  
pp. 9852-9864 ◽  
Author(s):  
Hugo Fraga ◽  
Joan-Josep Bech-Serra ◽  
Francesc Canals ◽  
Gabriel Ortega ◽  
Oscar Millet ◽  
...  
Keyword(s):  
Folding Pathway ◽  
Intermembrane Space ◽  
Oxidative Folding ◽  
Mitochondrial Intermembrane Space

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

2013 ◽  
Vol 24 (14) ◽  
pp. 2160-2170 ◽  
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.

scholarly journals Mitochondrial protein import: Mia40 facilitates Tim22 translocation into the inner membrane of mitochondria

2013 ◽  
Vol 24 (5) ◽  
pp. 543-554 ◽  
Author(s):  
Lidia Wrobel ◽  
Agata Trojanowska ◽  
Malgorzata E. Sztolsztener ◽  
Agnieszka Chacinska
Keyword(s):  
Disulfide Bonds ◽  
Protein Import ◽  
Noncovalent Interactions ◽  
Mitochondrial Protein ◽  
Central Component ◽  
Intermembrane Space ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
Oxidative Folding ◽  
Mitochondrial Intermembrane Space

The mitochondrial intermembrane space assembly (MIA) pathway is generally considered to be dedicated to the redox-dependent import and biogenesis of proteins localized to the intermembrane space of mitochondria. The oxidoreductase Mia40 is a central component of the pathway responsible for the transfer of disulfide bonds to intermembrane space precursor proteins, causing their oxidative folding. Here we present the first evidence that the function of Mia40 is not restricted to the transport and oxidative folding of intermembrane space proteins. We identify Tim22, a multispanning membrane protein and core component of the TIM22 translocase of inner membrane, as a protein with cysteine residues undergoing oxidation during Tim22 biogenesis. We show that Mia40 is involved in the biogenesis and complex assembly of Tim22. Tim22 forms a disulfide-bonded intermediate with Mia40 upon import into mitochondria. Of interest, Mia40 binds the Tim22 precursor also via noncovalent interactions. We propose that Mia40 not only is responsible for disulfide bond formation, but also assists the Tim22 protein in its integration into the inner membrane of mitochondria.

Oxidative folding competes with mitochondrial import of the small Tim proteins

2008 ◽  
Vol 411 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Bruce Morgan ◽  
Hui Lu
Keyword(s):  
Disulfide Bonds ◽  
Intermembrane Space ◽  
Mitochondrial Import ◽  
Oxidative Folding ◽  
Mutant Proteins ◽  
Standard Redox Potential ◽  
Mitochondrial Intermembrane Space ◽  
Tim Proteins

All small Tim proteins of the mitochondrial intermembrane space contain two conserved CX3C motifs, which form two intramolecular disulfide bonds essential for function, but only the cysteine-reduced, but not oxidized, proteins can be imported into mitochondria. We have shown that Tim10 can be oxidized by glutathione under cytosolic concentrations. However, it was unknown whether oxidative folding of other small Tims can occur under similar conditions and whether oxidative folding competes kinetically with mitochondrial import. In the present study, the effect of glutathione on the cysteine-redox state of Tim9 was investigated, and the standard redox potential of Tim9 was determined to be approx. −0.31 V at pH 7.4 and 25 °C with both the wild-type and Tim9F43W mutant proteins, using reverse-phase HPLC and fluorescence approaches. The results show that reduced Tim9 can be oxidized by glutathione under cytosolic concentrations. Next, we studied the rate of mitochondrial import and oxidative folding of Tim9 under identical conditions. The rate of import was approx. 3-fold slower than that of oxidative folding of Tim9, resulting in approx. 20% of the precursor protein being imported into an excess amount of mitochondria. A similar correlation between import and oxidative folding was obtained for Tim10. Therefore we conclude that oxidative folding and mitochondrial import are kinetically competitive processes. The efficiency of mitochondrial import of the small Tim proteins is controlled, at least partially in vitro, by the rate of oxidative folding, suggesting that a cofactor is required to stabilize the cysteine residues of the precursors from oxidation in vivo.

scholarly journals A novel intermembrane space–targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding

2009 ◽  
Vol 187 (7) ◽  
pp. 1007-1022 ◽  
Author(s):  
Dionisia P. Sideris ◽  
Nikos Petrakis ◽  
Nitsa Katrakili ◽  
Despina Mikropoulou ◽  
Angelo Gallo ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Dual Function ◽  
Intermembrane Space ◽  
Amphipathic Helix ◽  
Specific Mechanism ◽  
Oxidative Folding ◽  
Hydrophobic Residues ◽  
Targeting Signal ◽  
Mitochondrial Intermembrane Space ◽  
Internal Peptide

Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step–specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys.

Oxidative folding: recent developments

2011 ◽  
Vol 2 (5) ◽  
pp. 379-390 ◽  
Author(s):  
András Szarka ◽  
Gábor Bánhegyi
Keyword(s):  
Endoplasmic Reticulum ◽  
Disulfide Bonds ◽  
Redox Regulation ◽  
Disulfide Bridge ◽  
Intermembrane Space ◽  
Redox Systems ◽  
Oxidative Folding ◽  
Recent Developments ◽  
Mitochondrial Intermembrane Space ◽  
Structure Stabilization

AbstractDisulfide bond formation in proteins is an effective tool of both structure stabilization and redox regulation. The prokaryotic periplasm and the endoplasmic reticulum of eukaryotes were long considered as the only compartments for enzyme mediated formation of stable disulfide bonds. Recently, the mitochondrial intermembrane space has emerged as the third protein-oxidizing compartment. The classic view on the mechanism of oxidative folding in the endoplasmic reticulum has also been reshaped by new observations. Moreover, besides the structure stabilizing function, reversible disulfide bridge formation in some proteins of the endoplasmic reticulum, seems to play a regulatory role. This review briefly summarizes the present knowledge of the redox systems supporting oxidative folding, emphasizing recent developments.

scholarly journals In vivo evidence for cooperation of Mia40 and Erv1 in the oxidation of mitochondrial proteins

2012 ◽  
Vol 23 (20) ◽  
pp. 3957-3969 ◽  
Author(s):  
Lena Böttinger ◽  
Agnieszka Gornicka ◽  
Tomasz Czerwik ◽  
Piotr Bragoszewski ◽  
Adrianna Loniewska-Lwowska ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Complex Dynamics ◽  
Intermembrane Space ◽  
Oxidative Folding ◽  
Formation Pathway ◽  
Substrate Complex ◽  
Substrate Protein ◽  
In Organello ◽  
Mitochondrial Intermembrane Space

The intermembrane space of mitochondria accommodates the essential mitochondrial intermembrane space assembly (MIA) machinery that catalyzes oxidative folding of proteins. The disulfide bond formation pathway is based on a relay of reactions involving disulfide transfer from the sulfhydryl oxidase Erv1 to Mia40 and from Mia40 to substrate proteins. However, the substrates of the MIA typically contain two disulfide bonds. It was unclear what the mechanisms are that ensure that proteins are released from Mia40 in a fully oxidized form. In this work, we dissect the stage of the oxidative folding relay, in which Mia40 binds to its substrate. We identify dynamics of the Mia40–substrate intermediate complex. Our experiments performed in a native environment, both in organello and in vivo, show that Erv1 directly participates in Mia40–substrate complex dynamics by forming a ternary complex. Thus Mia40 in cooperation with Erv1 promotes the formation of two disulfide bonds in the substrate protein, ensuring the efficiency of oxidative folding in the intermembrane space of mitochondria.

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