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

Sonya E. Neal; Deepa V. Dabir; Heather L. Tienson; Darryl M. Horn; Kathrin Glaeser; Rachel R. Ogozalek Loo; Antoni Barrientos; Carla M. Koehler

doi:10.1074/jbc.m115.669440

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

Journal of Biological Chemistry ◽

10.1074/jbc.m115.669440 ◽

2015 ◽

Vol 290 (34) ◽

pp. 20804-20814 ◽

Cited By ~ 9

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e08-10-1062 ◽

2009 ◽

Vol 20 (15) ◽

pp. 3481-3490 ◽

Cited By ~ 52

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.

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

10.12681/eadd/33116 ◽

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.

Oxidative folding competes with mitochondrial import of the small Tim proteins

Biochemical Journal ◽

10.1042/bj20071476 ◽

2008 ◽

Vol 411 (1) ◽

pp. 115-122 ◽

Cited By ~ 22

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e12-05-0358 ◽

2012 ◽

Vol 23 (20) ◽

pp. 3957-3969 ◽

Cited By ~ 36

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.

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.

Transfer of disulfide bonds in biogenesis of mitochondrial intermembrane space proteins

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/j.bbabio.2010.04.324 ◽

2010 ◽

Vol 1797 ◽

pp. 107

Author(s):

Judith M. Müller ◽

Dusanka Milenkovic ◽

Diana Stojanovski ◽

Lena Böttinger ◽

Thomas Ramming ◽

...

Keyword(s):

Disulfide Bonds ◽

Intermembrane Space ◽

Mitochondrial Intermembrane Space

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.

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.

Uptake, Trapping, and Release of Organometallic Cations in Redox-Active Cationic Hosts

10.26434/chemrxiv.14778147.v1 ◽

2021 ◽

Author(s):

Iram F. Mansoor ◽

Kaitlyn Dutton ◽

Daniel A. Rothschild ◽

Richard C. Remsing ◽

Mark C. Lipke

Keyword(s):

Conformational Changes ◽

Electrostatic Interactions ◽

Dft Studies ◽

Redox Active ◽

Metal Organic ◽

Kinetic Effects ◽

Organometallic Cations ◽

Cation Complexes ◽

Uptake And Release ◽

Reduced State

The host-guest chemistry of metal-organic nanocages is typically driven by thermodynamically favorable interactions with their guests, such that uptake and release of guests can be controlled by switching affinity on/off. Herein, we achieve this effect by reducing porphyrin-walled cationic nanoprisms 1a12+ and 1b12+ to zwitterionic states that rapidly uptake organometallic cations Cp*2Co+ or Cp2Co+. Cp*2Co+ binds strongly (Ka = 1.3 x 103 M−1) in the neutral state 1a0 of host 1a12+, which has its three porphyrin walls doubly reduced and its six (bipy)Pt2+ linkers singly reduced. The less-reduced states of the host 1a3+ and 1a9+ also bind Cp*2Co+, though with lower affinities. The smaller Cp2Co+ cation binds strongly (Ka = 1.7 x 103 M-1) in the 3 e− reduced state 1b9+ of (tmeda)Pt2+ linked host 1b12+. Upon reoxidation of the hosts with Ag+, the guests become trapped to provide unprecedented metastable cation-in-cation complexes Cp*2Co+@1a12+ and Cp2Co+@1b12+ that persist for >1 month. Thus, dramatic kinetic effects reveal a way to confine the guests in thermodynamically unfavorable environments. Experimental and DFT studies indicate that PF6− anions kinetically stabilize Cp*2Co+@1a12+ through electrostatic interactions and by influencing conformational changes of the host that open and close its apertures. However, when Cp*2Co+@1a12+ was prepared using ferrocenium (Fc+) instead of Ag+ to reoxidize the host, dissociation was accelerated >200-fold even though neither Fc+ nor Fc have any competing affinity for 1a12+. This finding shows that metastable host-guest complexes can respond to subtler stimuli than are required to induce guest release from thermodynamically favorable complexes.

Erv1 of Arabidopsis thaliana can directly oxidize mitochondrial intermembrane space proteins in the absence of redox-active Mia40

BMC Biology ◽

10.1186/s12915-017-0445-8 ◽

2017 ◽

Vol 15 (1) ◽

Cited By ~ 15

Author(s):

Valentina Peleh ◽

Flavien Zannini ◽

Sandra Backes ◽

Nicolas Rouhier ◽

Johannes M. Herrmann

Keyword(s):

Arabidopsis Thaliana ◽

Intermembrane Space ◽

Redox Active ◽

Mitochondrial Intermembrane Space