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 Reconstitution of the Mia40-Erv1 Oxidative Folding Pathway for the Small Tim Proteins

2009 ◽  
Vol 20 (15) ◽  
pp. 3481-3490 ◽  
Author(s):  
Heather L. Tienson ◽  
Deepa V. Dabir ◽  
Sonya E. Neal ◽  
Rachel Loo ◽  
Samuel A. Hasson ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Folding Pathway ◽  
Intermembrane Space ◽  
Oxidative Folding ◽  
Midpoint Potential ◽  
Redox Active ◽  
Disulfide Linkages ◽  
Mitochondrial Intermembrane Space ◽  
Tim Proteins

Mia40 and Erv1 execute a disulfide relay to import the small Tim proteins into the mitochondrial intermembrane space. Here, we have reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate and determined the midpoint potentials of Mia40 and Tim13. Specifically, Mia40 served as a direct oxidant of Tim13, and Erv1 was required to reoxidize Mia40. During oxidation, four electrons were transferred from Tim13 with the insertion of two disulfide bonds in succession. The extent of Tim13 oxidation was directly dependent on Mia40 concentration and independent of Erv1 concentration. Characterization of the midpoint potentials showed that electrons flowed from Tim13 with a more negative midpoint potential of −310 mV via Mia40 with an intermediate midpoint potential of −290 mV to the C130-C133 pair of Erv1 with a positive midpoint potential of −150 mV. Intermediary complexes between Tim13-Mia40 and Mia40-Erv1 were trapped. Last, mutating C133 of the catalytic C130-C133 pair or C30 of the shuttle C30-C33 pair in Erv1 abolished oxidation of Tim13, whereas mutating the cysteines in the redox-active CPC motif, but not the structural disulfide linkages of the CX9C motif of Mia40, prevented Tim13 oxidation. Thus, we demonstrate that Mia40, Erv1, and oxygen are the minimal machinery for Tim13 oxidation.

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.

scholarly journals Μηχανισμός λειτουργίας και μοριακών αλληλεπιδράσεων της σουλφυδριλοξειδάσης Erv1 στο μιτοχόνδριο του σακχαρομύκητα

2013 ◽  
Author(s):  
Εμμανουέλα Καλλέργη
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intermembrane Space ◽  
In Organello ◽  
Mitochondrial Intermembrane Space

Τα μιτοχόνδρια αποτελούν σημαντικά οργανίδια των ευκαρυωτικών κυττάρων καθώς παίζουν σημαντικό ρόλο σε πολλές κυτταρικές διαδικασίες όπως στην αναπνοή, στην παραγωγή ΑΤΡ και στην απόπτωση. Η βιογένεση των μιτοχονδρίων εξαρτάται από την εισαγωγή πρωτεϊνών στο μιτοχόνδριο που πραγματοποιείται μέσω διαφορετικών μονοπατιών εισόδου. Πρόσφατα, το μονοπάτι MIA (Mitochondrial Intermembrane space import and Assembly) έχει περιγραφεί ως ένα σύστημα οξείδωσης δισουλφιδίων στον οργανισμό Saccharomyces cerevisiae που δίνει δισουλφιδικούς δεσμούς σε μια ποικιλία διαφορετικών πρωτεϊνών στο διαμεμβρανικό χώρο των μιτοχονδρίων (IMS). Η λειτουργία αυτού του μονοπατιού εξαρτάται από δύο πρωτεΐνες: την σουλφυδριλοξειδάση Erv1/ALR και την οξειδορεδουκτάση Mia40, που μαζί οδηγούν την είσοδο πρόδρομων πρωτεϊνικών μορίων τα οποία φέρουν συντηρημένες κυστεΐνες, στο IMS μέσω της οξειδωτικής τους αναδίπλωσης.Σε αυτή τη διδακτορική διατριβή, μελετάμε την διττή αλληλεπίδραση μεταξύ των Mia40-Erv1 με βιοχημικές, in organello, in vitro και in vivo προσεγγίσεις, η οποία συμβαίνει σε δύο στάδια: (α) η Erv1, αναγνωρίζεται και οξειδώνεται απο τη Mia40, ως υπόστρωμα του MIA μονοπατιού (Στάδιο Α) και (β) η αναδιπλωμένη και λειτουργική Erv1 οξειδώνει το ενεργό κέντρο της Mia40 (Στάδιο Β). Μελετώντας την είσοδο και την ωρίμανση της Erv1 (Στάδιο Α) χαρακτηρίσαμε την ελάχιστη περιοχή στο καρβοξυτελικό της άκρο που απαιτείται για την αναγνώριση και την οξείδωσή της από τη Mia40 πριν από την μεταγενέστερη πρόσδεση ενός μορίου FAD ανά μονομερές. Απο την άλλη πλευρά, μελετώντας το ρόλο της Erv1 στην επανοξείδωση της Mia40 (Στάδιο Β) βρήκαμε ότι συγκεκριμένα υδρόφοβα αμινοξικά κατάλοιπα καθοδικά του CRSC μοτίβου κυστεϊνών στο αμινοτελικό άκρο της Erv1, απαιτούνται για την ανακύκλωση της Mia40, αλλά όχι για την εισαγωγή της στο μιτοχόνδριο. Επιπλέον, τα αποτελέσματα μας έδειξαν ότι το σε μεγάλο βαθμό αδόμητο κομμάτι των πρώτων 72 αμινοξέων της Erv1 (N72) εμφανίζεται στο κυτοσόλιο να παίζει ρόλο στη στόχευση της πρωτεΐνης στα μιτοχόνδρια, πέρα απο το ρόλο του στην επανοξείδωση της Mia40. Αυτός ο εξαρτώμενος απο το υποκυτταρικό διαμέρισμα οξειδοαναγωγικός έλεγχος του αμινοτελικού κομματιού της Erv1 γεννά πρόσθετα ερωτήματα σχετικά με την αλληλεπίδρασή του με την εξωτερική μεμβράνη των μιτοχονδρίων καθώς και με σαπερόνες του κυτοσολίου. Τα παραπάνω αποτελέσματα μας δίνουν περισσότερη πληροφορία στο πεδίο της εισόδου πρωτεϊνών στο μιτοχόνδριο προκειμένου να μελετήσουμε σε μεγαλύτερη λεπτομέρεια την αλληλεπίδραση Mia40-Erv1, η οποία είναι ζωτικής σημασίας για τη βιογένεση της Erv1, τη λειτουργία του ΜΙΑ μονοπατιού και επομένως για τη βιογένεση των μιτοχονδρίων και τη βιωσιμότητα των κυττάρων.

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.

scholarly journals Influence of N-terminal sequence variation on the sorting of major adenylate kinase to the mitochondrial intermembrane space in yeast

1998 ◽  
Vol 329 (2) ◽  
pp. 359-367 ◽  
Author(s):  
Wolfhard BANDLOW ◽  
Gertrud STROBEL ◽  
Roland SCHRICKER
Keyword(s):  
Adenylate Kinase ◽  
Matrix Protein ◽  
Helical Structure ◽  
Terminal Region ◽  
Intermembrane Space ◽  
Homologous Segment ◽  
Mutant Proteins ◽  
N Terminus ◽  
Mitochondrial Intermembrane Space

Major adenylate kinase (Aky2p) from yeast has no cleavable presequence and occurs in identical form in the mitochondrial intermembrane space (6-8%) and in the cytoplasm (approx. 90%). To identify the signal(s) on Aky2p that might be required for mitochondrial import, the N-terminal region was examined. The N-terminus of Aky2p can guide at least two cytoplasmic passengers, dihydrofolate reductase from mouse and UMP kinase (Ura6p) from yeast, to the intermembrane space in vivo, showing that the N-terminus harbours import information. In contrast, deletion of the eight N-terminal amino acid residues or the introduction of two compensating frameshifts into this segment does not abolish translocation into the organelle's intermembrane space. Thus internal targeting and sorting information must be present in Aky2p as well. Neither a pronounced amphiphilic α-helical moment nor positive charges in the N-terminal region is a necessary prerequisite for Aky2p to reach the intermembrane space. Even a surplus of negative charges in mutant N-termini does not impede basal import into the correct submitochondrial compartment. The potential to form an amphipathic α-helical structure of five to eight residues close to the N-terminus significantly improves import efficiency, whereas extension of this amphipathic structure, e.g. by replacing it with the homologous segment of Aky3p, a mitochondrial matrix protein from yeast, leads to misdirection of the chimaera to the matrix compartment. This shows that the topogenic N-terminal signal of Aky3p is dominant over the presumptive internal intermembrane space-targeting signal of Aky2p and argues that the sorting of wild-type Aky2p to the intermembrane space is not due to the presence in the protein of a specific sorting sequence for the intermembrane space, but rather is the consequence of being imported but not being sorted to the inner compartment. Some Aky2 mutant proteins are susceptible to proteolysis in the cytoplasm, indicating incorrect folding. They are nevertheless efficiently rescued by uptake into mitochondria, suggesting a negative correlation between folding velocity (or folding stability) and efficiency of import.

scholarly journals A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments

2007 ◽  
Vol 179 (7) ◽  
pp. 1355-1363 ◽  
Author(s):  
Hidenori Otera ◽  
Yohsuke Taira ◽  
Chika Horie ◽  
Yurina Suzuki ◽  
Hiroyuki Suzuki ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Benzodiazepine Receptor ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Transmembrane Segments ◽  
Import Receptors ◽  
Mitochondrial Intermembrane Space

The central channel Tom40 of the preprotein translocase of outer membrane (TOM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. In this study, we analyze the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs) and functions in cholesterol import into the inner membrane. Analyses of in vitro and in vivo import into TOM component–depleted mitochondria reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the C-tail–anchored proteins, and (3) requires factors of the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, the MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway. These findings reveal a previously unidentified pathway of the membrane integration of MOM proteins with multiple TMSs.

scholarly journals Mia40 Protein Serves as an Electron Sink in the Mia40-Erv1 Import Pathway

2015 ◽  
Vol 290 (34) ◽  
pp. 20804-20814 ◽  
Author(s):  
Sonya E. Neal ◽  
Deepa V. Dabir ◽  
Heather L. Tienson ◽  
Darryl M. Horn ◽  
Kathrin Glaeser ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Disulfide Bonds ◽  
Intermembrane Space ◽  
Isolated Mitochondria ◽  
Redox Active ◽  
In Organello ◽  
Mitochondrial Intermembrane Space ◽  
Electron Sink ◽  
Reduced State

A redox-regulated import pathway consisting of Mia40 and Erv1 mediates the import of cysteine-rich proteins into the mitochondrial intermembrane space. Mia40 is the oxidoreductase that inserts two disulfide bonds into the substrate simultaneously. However, Mia40 has one redox-active cysteine pair, resulting in ambiguity about how Mia40 accepts numerous electrons during substrate oxidation. In this study, we have addressed the oxidation of Tim13 in vitro and in organello. Reductants such as glutathione and ascorbate inhibited both the oxidation of the substrate Tim13 in vitro and the import of Tim13 and Cmc1 into isolated mitochondria. In addition, a ternary complex consisting of Erv1, Mia40, and substrate, linked by disulfide bonds, was not detected in vitro. Instead, Mia40 accepted six electrons from substrates, and this fully reduced Mia40 was sensitive to protease, indicative of conformational changes in the structure. Mia40 in mitochondria from the erv1–101 mutant was also trapped in a completely reduced state, demonstrating that Mia40 can accept up to six electrons as substrates are imported. Therefore, these studies support that Mia40 functions as an electron sink to facilitate the insertion of two disulfide bonds into substrates.

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

2015 ◽  
Vol 26 (2) ◽  
pp. 195-204 ◽  
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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