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.

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 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.

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 Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

2007 ◽  
Vol 6 (12) ◽  
pp. 2214-2221 ◽  
Author(s):  
Lois M. Douglas ◽  
Li Li ◽  
Yang Yang ◽  
A. M. Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biofilm Formation ◽  
In Vitro System ◽  
Glycosylphosphatidylinositol Anchor ◽  
Fungal Adhesion ◽  
Very High ◽  
Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

scholarly journals The Highly Conserved tRNAHis Guanylyltransferase Thg1p Interacts with the Origin Recognition Complex and Is Required for the G2/M Phase Transition in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 4 (4) ◽  
pp. 832-835 ◽  
Author(s):  
Terri S. Rice ◽  
Min Ding ◽  
David S. Pederson ◽  
Nicholas H. Heintz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Origin Recognition Complex ◽  
Nuclear Division ◽  
Permissive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
M Phase ◽  
And Migration ◽  
Origin Recognition

ABSTRACT Here we show that the Saccharomyces cerevisiae tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex in vivo and in vitro and that overexpression of hemagglutinin-Thg1p selectively impedes growth of orc2-1(Ts) cells at the permissive temperature. Studies with conditional mutants indicate that Thg1p couples nuclear division and migration to cell budding and cytokinesis in yeast.

scholarly journals Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo.

1986 ◽  
Vol 6 (7) ◽  
pp. 2663-2673 ◽  
Author(s):  
M C Strobel ◽  
J Abelson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trna Gene ◽  
Nuclear Extract ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Specific Sequence ◽  
Made In

The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.

scholarly journals Binding and Partial Denaturing of G-quartet DNA by Cdc13p ofSaccharomyces cerevisiae

2001 ◽  
Vol 276 (50) ◽  
pp. 47671-47674 ◽  
Author(s):  
Yi-Chien Lin ◽  
Jing-Wen Shih ◽  
Chia-Ling Hsu ◽  
Jing-Jer Lin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intermolecular Association ◽  
Telomere Association ◽  
Dna Quadruplex ◽  
Telomere Replication ◽  
Dna Quadruplexes

The protein Cdc13p binds telomeresin vivoand is essential for the maintenance of the telomeres ofSaccharomyces cerevisiae. In addition, Cdc13p is known to bind single-stranded TG1–3DNAin vitro. Here we have shown that Cdc13p also binds DNA quadruplex, G-quartet, formed by TG1–3DNA. Moreover, the binding of Cdc13p causes a partial denaturing of the G-quartet DNA. Formation of DNA quadruplexes may involve the intermolecular association of TG1–3DNA and inhibit the extension of telomeres by telomerase. Thus, our finding suggests that Cdc13p may disrupt telomere association and facilitate telomere replication.

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