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.

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

eLife ◽  
2016 ◽  
Vol 5 ◽  
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.

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 Structure of Yeast Sulfhydryl Oxidase Erv1 Reveals Electron Transfer of the Disulfide Relay System in the Mitochondrial Intermembrane Space

2012 ◽  
Vol 287 (42) ◽  
pp. 34961-34969 ◽  
Author(s):  
Peng-Chao Guo ◽  
Jin-Di Ma ◽  
Yong-Liang Jiang ◽  
Shu-Jie Wang ◽  
Zhang-Zhi Bao ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Electron Donor ◽  
Full Length ◽  
Intermembrane Space ◽  
Amphipathic Helix ◽  
Sulfhydryl Oxidase ◽  
Relay System ◽  
Helix Bundle ◽  
Mitochondrial Intermembrane Space ◽  
Four Helix Bundle

The disulfide relay system in the mitochondrial intermembrane space drives the import of proteins with twin CX9C or twin CX3C motifs by an oxidative folding mechanism. This process requires disulfide bond transfer from oxidized Mia40 to a substrate protein. Reduced Mia40 is reoxidized/regenerated by the FAD-linked sulfhydryl oxidase Erv1 (EC 1.8.3.2). Full-length Erv1 consists of a flexible N-terminal shuttle domain (NTD) and a conserved C-terminal core domain (CTD). Here, we present crystal structures at 2.0 Å resolution of the CTD and at 3.0 Å resolution of a C30S/C133S double mutant of full-length Erv1 (Erv1FL). Similar to previous homologous structures, the CTD exists as a homodimer, with each subunit consisting of a conserved four-helix bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. The structure of Erv1FL enabled us to identify, for the first time, the three-dimensional structure of the Erv1NTD, which is an amphipathic helix flanked by two flexible loops. This structure also represents an intermediate state of electron transfer from the NTD to the CTD of another subunit. Comparative structural analysis revealed that the four-helix bundle of the CTD forms a wide platform for the electron donor NTD. Moreover, computational simulation combined with multiple-sequence alignment suggested that the amphipathic helix close to the shuttle redox enter is critical for the recognition of Mia40, the upstream electron donor. These findings provide structural insights into electron transfer from Mia40 via the shuttle domain of one subunit of Erv1 to the CTD of another Erv1 subunit.

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