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.

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 Functional characterization of an N-terminally-truncated mitochondrial porin expressed in Neurospora crassa

2017 ◽  
Vol 63 (8) ◽  
pp. 730-738 ◽  
Author(s):  
Sabbir R. Shuvo ◽  
Uliana Kovaltchouk ◽  
Abdullah Zubaer ◽  
Ayush Kumar ◽  
William A.T. Summers ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Outer Membrane ◽  
Functional Characterization ◽  
Helical Structure ◽  
Terminal Segment ◽  
Wild Type ◽  
Mitochondrial Porin ◽  
N Terminus

Mitochondrial porin, which forms voltage-dependent anion-selective channels (VDAC) in the outer membrane, can be folded into a 19-β-stranded barrel. The N terminus of the protein is external to the barrel and contains α-helical structure. Targeted modifications of the N-terminal region have been assessed in artificial membranes, leading to different models for gating in vitro. However, the in vivo requirements for gating and the N-terminal segment of porin are less well-understood. Using Neurospora crassa porin as a model, the effects of a partial deletion of the N-terminal segment were investigated. The protein, ΔN2-12porin, is assembled into the outer membrane, albeit at lower levels than the wild-type protein. The resulting strain displays electron transport chain deficiencies, concomitant expression of alternative oxidase, and decreased growth rates. Nonetheless, its mitochondrial genome does not contain any significant mutations. Most of the genes that are expressed in high levels in porin-less N. crassa are expressed at levels similar to that of wild type or are slightly increased in ΔN2-12porin strains. Thus, although the N-terminal segment of VDAC is required for complete function in vivo, low levels of a protein lacking part of the N terminus are able to rescue some of the defects associated with the absence of porin.

scholarly journals In Vivo Structure-Function Analysis and Redox Interactomes of Leishmania tarentolae Erv

2021 ◽  
Author(s):  
Gino L. Turra ◽  
Linda Liedgens ◽  
Frederik Sommer ◽  
Luzia Schneider ◽  
David Zimmer ◽  
...  
Keyword(s):  
Protein Import ◽  
Function Analysis ◽  
Mitochondrial Protein ◽  
Intermembrane Space ◽  
Mitochondrial Protein Import ◽  
Redox Biology ◽  
Oxidative Protein Folding ◽  
Mitochondrial Intermembrane Space ◽  
New Research

The discovery of the redox proteins Mia40/CHCHD4 and Erv1/ALR, as well as the elucidation of their relevance for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals, founded a new research topic in redox biology and mitochondrial protein import. The lack of Mia40/CHCHD4 in protist lineages raises fundamental and controversial questions regarding the conservation and evolution of this essential pathway.

scholarly journals The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Heike Rampelt ◽  
Iva Sucec ◽  
Beate Bersch ◽  
Patrick Horten ◽  
Inge Perschil ◽  
...  
Keyword(s):  
Intermembrane Space ◽  
Inner Mitochondrial Membrane ◽  
Inner Membrane ◽  
Mitochondrial Carrier ◽  
Transmembrane Segments ◽  
Mitochondrial Inner Membrane ◽  
N Terminus ◽  
The Matrix ◽  
Mitochondrial Carrier Family ◽  
Mitochondrial Intermembrane Space

Abstract Background The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway. Results Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins. Conclusions The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins.

scholarly journals A New Function in Translocation for the Mitochondrial i-AAA Protease Yme1: Import of Polynucleotide Phosphorylase into the Intermembrane Space

2006 ◽  
Vol 26 (22) ◽  
pp. 8488-8497 ◽  
Author(s):  
Robert N. Rainey ◽  
Jenny D. Glavin ◽  
Hsiao-Wen Chen ◽  
Samuel W. French ◽  
Michael A. Teitell ◽  
...  
Keyword(s):  
Protein Translocation ◽  
Rna Degradation ◽  
Mechanistic Study ◽  
Intermembrane Space ◽  
Polynucleotide Phosphorylase ◽  
Cellular Effects ◽  
The Matrix ◽  
Mitochondrial Intermembrane Space ◽  
Processing Peptidase

ABSTRACT Polynucleotide phosphorylase (PNPase) is an exoribonuclease and poly(A) polymerase postulated to function in the cytosol and mitochondrial matrix. Prior overexpression studies resulted in PNPase localization to both the cytosol and mitochondria, concurrent with cytosolic RNA degradation and pleiotropic cellular effects, including growth inhibition and apoptosis, that may not reflect a physiologic role for endogenous PNPase. We therefore conducted a mechanistic study of PNPase biogenesis in the mitochondrion. Interestingly, PNPase is localized to the intermembrane space by a novel import pathway. PNPase has a typical N-terminal targeting sequence that is cleaved by the matrix processing peptidase when PNPase engaged the TIM23 translocon at the inner membrane. The i-AAA protease Yme1 mediated translocation of PNPase into the intermembrane space but did not degrade PNPase. In a yeast strain deleted for Yme1 and expressing PNPase, nonimported PNPase accumulated in the cytosol, confirming an in vivo role for Yme1 in PNPase maturation. PNPase localization to the mitochondrial intermembrane space suggests a unique role distinct from its highly conserved function in RNA processing in chloroplasts and bacteria. Furthermore, Yme1 has a new function in protein translocation, indicating that the intermembrane space harbors diverse pathways for protein translocation.

Phosphorylation of the carboxyl-terminal region of dystrophin

1996 ◽  
Vol 74 (4) ◽  
pp. 431-437 ◽  
Author(s):  
Marek Michalak ◽  
Susan Y. Fu ◽  
Rachel E. Milner ◽  
Jody L. Busaan ◽  
Jacqueline E. Hance
Keyword(s):  
Extracellular Matrix ◽  
Muscular Dystrophy ◽  
Protein Kinases ◽  
Protein Interactions ◽  
Matrix Protein ◽  
Casein Kinase ◽  
Terminal Region ◽  
Extracellular Matrix Protein

Dystrophin is a protein product of the gene responsible for Duchenne and Becker muscular dystrophy. The protein is localized to the inner surface of sarcolemma and is associated with a group of membrane (glyco)proteins. Dystrophin links cytoskeletal actins via the dystrophin-associated protein complex to extracellular matrix protein, laminin. This structural organization implicates the role of dystrophin in stabilizing the sarcolemma of muscle fibers. Precisely how dystrophin functions is far from clear. The presence of an array of isoforms of the C-terminal region of dystrophin suggests that dystrophin may have functions other than structural. In agreement, many potential phosphorylation sites are found in the C-terminal region of dystrophin, and the C-terminal region of dystrophin is phosphorylated both in vitro and in vivo by many protein kinases, including MAP kinase, p34cdc2 kinase, CaM kinase, and casein kinase, and is dephosphorylated by calcineurin. The C-terminal domain of dystrophin is also a substrate for hierarchical phosporylation by casein kinase-2 and GSK-3. These observations, in accordance with the finding that the cysteine-rich region binds to Ca2+, Zn2+, and calmodulin, suggest an active involvement of dystrophin in transducing signals across muscle sarcolemma. Phosphorylation–dephosphorylation of the C-terminal region of dystrophin may play a role in regulating dystrophin–protein interactions and (or) transducing signal from the extracellular matrix via the dystrophin molecule to the cytoskeleton.Key words: Duchenne muscular dystrophy, protein phosphorylation, protein kinases, calcineurin, cytoskeleton.

scholarly journals Neuroprotective Role of the Reaper-Related Serine Protease HtrA2/Omi Revealed by Targeted Deletion in Mice

2004 ◽  
Vol 24 (22) ◽  
pp. 9848-9862 ◽  
Author(s):  
L. Miguel Martins ◽  
Alastair Morrison ◽  
Kristina Klupsch ◽  
Valentina Fedele ◽  
Nicoleta Moisoi ◽  
...  
Keyword(s):  
Cell Death ◽  
Serine Protease ◽  
Neurodegenerative Disorder ◽  
Binding Motif ◽  
Intermembrane Space ◽  
Targeted Deletion ◽  
Amino Terminal ◽  
Its Gene ◽  
Mitochondrial Intermembrane Space

ABSTRACT The serine protease HtrA2/Omi is released from the mitochondrial intermembrane space following apoptotic stimuli. Once in the cytosol, HtrA2/Omi has been implicated in promoting cell death by binding to inhibitor of apoptosis proteins (IAPs) via its amino-terminal Reaper-related motif, thus inducing caspase activity, and also in mediating caspase-independent death through its own protease activity. We report here the phenotype of mice entirely lacking expression of HtrA2/Omi due to targeted deletion of its gene, Prss25. These animals, or cells derived from them, show no evidence of reduced rates of cell death but on the contrary suffer loss of a population of neurons in the striatum, resulting in a neurodegenerative disorder with a parkinsonian phenotype that leads to death of the mice around 30 days after birth. The phenotype of these mice suggests that it is the protease function of this protein and not its IAP binding motif that is critical. This conclusion is reinforced by the finding that simultaneous deletion of the other major IAP binding protein, Smac/DIABLO, does not obviously alter the phenotype of HtrA2/Omi knockout mice or cells derived from them. Mammalian HtrA2/Omi is therefore likely to function in vivo in a manner similar to that of its bacterial homologues DegS and DegP, which are involved in protection against cell stress, and not like the proapoptotic Reaper family proteins in Drosophila melanogaster.

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.

Sign in / Sign up

Export Citation Format

Share Document