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Walter Neupert - One of the best experts on this subject based on the ideXlab platform.
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Import of small Tim proteins into the mitochondrial Intermembrane Space
The EMBO Journal, 2003Co-Authors: Thomas Lutz, Walter Neupert, Johannes M HerrmannAbstract:Proteins of the Intermembrane Space (IMS) of mitochondria are typically synthesized without presequences. Little is known about their topogenesis. We used Tim13, a member of the ‘small Tim protein’ family, as model protein to investigate the mechanism of translocation into the IMS. Tim13 contains four conserved cysteine residues that bind a zinc ion as cofactor. Import of Tim13 did not depend on the membrane potential or ATP hydrolysis. Upon import into mitochondria Tim13 adopted a stably folded conformation in the IMS. Mutagenesis of the cysteine residues or pretreatment with metal chelators interfered with folding of Tim13 in vitro and impaired its import into mitochondria. Upon depletion of metal ions or modification of cysteine residues, imported Tim13 diffused back out of the IMS. We propose an import pathway in which (1) Tim13 can pass through the TOM complex into and out of the IMS in an unfolded conformation, and (2) cofactor acquisition stabilizes folding on the trans side of the outer membrane and traps Tim13 in the IMS, and drives unidirectional movement of the protein across the outer membrane of mitochondria.
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Sorting of Proteins to the Mitochondrial Intermembrane Space
Protein Targeting to Mitochondria, 1996Co-Authors: Rosemary A. Stuart, Heike Fölsch, Albrecht Gruhler, Walter NeupertAbstract:Publisher Summary This chapter discusses the conservative sorting of Intermembrane Space (IMS) targeted proteins. Experimental evidence exists to support the conservative sorting hypothesis. This model proposes that IMS-targeted proteins embark on the general mitochondrial import pathway crossing the outer membrane (OM) and inner membrane (IM), where they emerge into the matrix. Upon exposure to the matrix, the sorting signal is recognized by a putative signal binding protein. The preprotein then embarks on a translocation event back across the IM, a process postulated to resemble the export of proteins in bacteria across the periplasmic membrane. Initiation of this export step is thought to occur prior to completion ofthe import of carboxy-terminal portions of the preproteins through the import machinery. Thus, it is highly plausible that, if of sufficient length, such IMS-targeted proteins can span both the OM and IM import machineries while being translocated across the IM during its export step. IM spanning intermediates have been identified and have been shown to be looping through the matrix by two independent approaches.
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biogenesis of mitochondrial heme lyases in yeast import and folding in the Intermembrane Space
Journal of Biological Chemistry, 1995Co-Authors: Harald Steiner, Walter Neupert, Alfred Zollner, Albert Haid, Roland LillAbstract:Abstract Heme lyases are components of the mitochondrial Intermembrane Space facilitating the covalent attachment of heme to the apoforms of c-type cytochromes. The precursors of heme lyases are synthesized in the cytosol without the typical N-terminal mitochondrial targeting signal. Here, we have analyzed the mode of import and folding of the two heme lyases of the yeast Saccharomyces cerevisiae, namely of cytochrome c heme lyase and of cytochrome c heme lyase. For transport into mitochondria, both proteins use the general protein import machinery of the outer membrane. Import occurred independently of a membrane potential, Δ, across the inner membrane and ATP in the matrix Space, suggesting that the inner membrane is not required for transport along this direct sorting pathway. The presence of a large folded domain in heme lyases was utilized to study their folding in the Intermembrane Space. Formation of this domain occurred at the same rate as import, indicating that heme lyases fold either during or immediately after their transfer across the membrane. Folding was not affected by depletion of ATP and Δ or by inhibitors of peptidylprolyl cis-trans isomerases, i.e. it does not involve homologs of known folding factors (like Hsp60 and Hsp70). The energy derived from folding cannot be regarded as a major driving force for import, since the folded domain could be imported into mitochondria with the same efficiency as the intact protein. We conclude that protein folding in the Intermembrane Space obeys principles different from those established for other subcellular compartments.
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Sorting of cytochrome b2 to the Intermembrane Space of mitochondria. Kinetic analysis of intermediates demonstrates passage through the matrix.
The Journal of biological chemistry, 1995Co-Authors: Hideyu Ono, Bernard Guiard, Rosemary A. Stuart, Albrecht Gruhler, E. Schwarz, Walter NeupertAbstract:Abstract Precytochrome b2 is targeted to the mitochondrial Intermembrane Space by a dual targeting sequence comprising 80 amino acids. A kinetic analysis of intramitochondrial sorting was performed. The intermediate-size form accumulated transiently in the matrix. When import was performed in the presence of metal chelators to prevent the first processing by the matrix processing peptidase, >40% of the imported precursor was localized in the matrix. A deletion of 13 amino acids in the Intermembrane Space sorting sequence caused partial inhibition of the first processing, and a transient accumulation of the precursor form in the matrix was also observed. The decrease in this matrix-localized precursor form paralleled an increase in the mature-size form in the Intermembrane Space. A point mutation in the mitochondrial targeting sequence (N-terminal to the sorting sequence) resulted in missorting to the matrix Space. Furthermore, a chimeric protein consisting of the initial 85 residues of cytochrome b2 fused to dihydrofolate reductase was partially targeted to the matrix at 15°C, but not at 25°C. Together, the results presented here indicate that cytochrome b2 passes through the matrix on its sorting pathway to the Intermembrane Space.
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the requirement of matrix atp for the import of precursor proteins into the mitochondrial matrix and Intermembrane Space
FEBS Journal, 1994Co-Authors: Rosemary A. Stuart, Bernard Guiard, Albrecht Gruhler, Ida J Van Der Klei, Hans Koll, Walter NeupertAbstract:The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted. Import and processing of precytochrome b, (pb,), a precursor carrying a bipartite presequence, into the Intermembrane Space was also strongly dependent on matrix ATP. Preproteins, consisting of 220 or more residues of pb, fused to dihydrofolate reductase, showed the same requirement for matrix ATP, whereas the import of shorter fusion proteins (up to 167 residues of pb,) was largely independent of matrix ATP. For those Intermembrane-Space-targeted proteins that did need matrix ATP, the dependence could be relieved either by unfolding these proteins prior to import or by introducing a deletion into the mature portion of the protein thereby impairing the tight folding of the cytochrome b, domain. These results suggest the following: (a) The import of matrix-targeted preproteins, in addition to a membrane potential AY, requires matrix ATP [most likely to facilitate reversible binding of mitochondrial heat-shock protein 70 (mt-Hsp70) to incoming precursors], for two steps, securing the presequence on the matrix side of the inner membrane and for the completion of translocation; (b) in the case of Intermembrane-Space-targeted precursors with bipartite signals, the function of ATP/mt-Hsp70 is not obligatory, as components of the Intermembrane-Space-sorting pathway may substitute for ATP/mt-Hsp70 function (however, if a tightly folded domain is present in the precursor, ATPlmt-Hsp70 is indispensable) ; (c) unfolding on the mitochondrial surface of tightly folded segments of preproteins is facilitated by matrix-ATP/mt-Hsp70.
Agnieszka Chacinska - One of the best experts on this subject based on the ideXlab platform.
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Retro-translocation of mitochondrial Intermembrane Space proteins
Proceedings of the National Academy of Sciences, 2015Co-Authors: Piotr Bragoszewski, Agnieszka Gornicka, Michal Wasilewski, Paulina Sakowska, Lena Böttinger, Jian Qiu, Nils Wiedemann, Agnieszka ChacinskaAbstract:The content of mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the Intermembrane Space by the mitochondrial Intermembrane Space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the mitochondrial proteome surveillance.
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A discrete pathway for the transfer of Intermembrane Space proteins across the outer membrane of mitochondria
Molecular biology of the cell, 2014Co-Authors: Agnieszka Gornicka, Piotr Bragoszewski, Lena-sophie Wenz, Peter Rehling, Piotr Chroscicki, Christian Schulz, Agnieszka ChacinskaAbstract:Mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria with the help of protein translocases. For the majority of precursor proteins, the role of the translocase of the outer membrane (TOM) and mechanisms of their transport across the outer mitochondrial membrane are well recognized. However, little is known about the mode of membrane translocation for proteins that are targeted to the Intermembrane Space via the redox-driven mitochondrial Intermembrane Space import and assembly (MIA) pathway. On the basis of the results obtained from an in organello competition import assay, we hypothesized that MIA-dependent precursor proteins use an alternative pathway to cross the outer mitochondrial membrane. Here we demonstrate that this alternative pathway involves the protein channel formed by Tom40. We sought a translocation intermediate by expressing tagged versions of MIA-dependent proteins in vivo. We identified a transient interaction between our model substrates and Tom40. Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22. Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.
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The Ubiquitin-Proteasome System Regulates Mitochondrial Intermembrane Space Proteins
Molecular and Cellular Biology, 2013Co-Authors: Piotr Bragoszewski, Agnieszka Gornicka, Malgorzata E. Sztolsztener, Agnieszka ChacinskaAbstract:Mitochondrial precursor proteins are synthesized in the cytosol and subsequently imported into mitochondria. The import of mitochondrial Intermembrane Space proteins is coupled with their oxidative folding and governed by the mitochondrial Intermembrane Space import and assembly (MIA) pathway. The cytosolic steps that precede mitochondrial import are not well understood. We identified a role for the ubiquitin-proteasome system in the biogenesis of Intermembrane Space proteins. Interestingly, the function of the ubiquitin-proteasome system is not restricted to conditions of mitochondrial protein import failure. The ubiquitin-proteasome system persistently removes a fraction of Intermembrane Space proteins under physiological conditions, acting as a negative regulator in the biogenesis of this class of proteins. Thus, the ubiquitin-proteasome system plays an important role in determining the levels of proteins targeted to the Intermembrane Space of mitochondria.
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Intermembrane Space Proteome of Yeast Mitochondria
Molecular & cellular proteomics : MCP, 2012Co-Authors: F.-nora Vögtle, Agnieszka Chacinska, Carolin Gerbeth, Julia M. Burkhart, Sanjana Rao, Jens Hinrichs, Jean-claude Martinou, Albert Sickmann, René P. Zahedi, Chris MeisingerAbstract:The Intermembrane Space (IMS) represents the smallest subcompartment of mitochondria. Nevertheless, it plays important roles in the transport and modification of proteins, lipids, and metal ions and in the regulation and assembly of the respiratory chain complexes. Moreover, it is involved in many redox processes and coordinates key steps in programmed cell death. A comprehensive profiling of IMS proteins has not been performed so far. We have established a method that uses the proapoptotic protein Bax to release IMS proteins from isolated mitochondria, and we profiled the protein composition of this compartment. Using stable isotope-labeled mitochondria from Saccharomyces cerevisiae, we were able to measure specific Bax-dependent protein release and distinguish between quantitatively released IMS proteins and the background efflux of matrix proteins. From the known 31 soluble IMS proteins, 29 proteins were reproducibly identified, corresponding to a coverage of >90%. In addition, we found 20 novel Intermembrane Space proteins, out of which 10 had not been localized to mitochondria before. Many of these novel IMS proteins have unknown functions or have been reported to play a role in redox regulation. We confirmed IMS localization for 15 proteins using in organello import, protease accessibility upon osmotic swelling, and Bax-release assays. Moreover, we identified two novel mitochondrial proteins, Ymr244c-a (Coa6) and Ybl107c (Mic23), as substrates of the MIA import pathway that have unusual cysteine motifs and found the protein phosphatase Ptc5 to be a novel substrate of the inner membrane protease (IMP). For Coa6 we discovered a role as a novel assembly factor of the cytochrome c oxidase complex. We present here the first and comprehensive proteome of IMS proteins of yeast mitochondria with 51 proteins in total. The IMS proteome will serve as a valuable source for further studies on the role of the IMS in cell life and death.
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Identification of the Signal Directing Tim9 and Tim10 into the Intermembrane Space of Mitochondria
Molecular Biology of the Cell, 2009Co-Authors: Dusanka Milenkovic, Diana Stojanovski, Judith M. Müller, Agnes Schulze-specking, Thomas Ramming, Lena-sophie Wenz, Natalia Gebert, Sabine Rospert, Agnieszka ChacinskaAbstract:The Intermembrane Space of mitochondria contains the specific mitochondrial Intermembrane Space assembly (MIA) machinery that operates in the biogenesis pathway of precursor proteins destined to this compartment. The Mia40 component of the MIA pathway functions as a receptor and binds incoming precursors, forming an essential early intermediate in the biogenesis of Intermembrane Space proteins. The elements that are crucial for the association of the Intermembrane Space precursors with Mia40 have not been determined. In this study, we found that a region within the Tim9 and Tim10 precursors, consisting of only nine amino acid residues, functions as a signal for the engagement of substrate proteins with the Mia40 receptor. Furthermore, the signal contains sufficient information to facilitate the transfer of proteins across the outer membrane to the Intermembrane Space. Thus, here we have identified the mitochondrial Intermembrane Space sorting signal required for delivery of proteins to the mitochondrial Intermembrane Space.
Gottfried Schatz - One of the best experts on this subject based on the ideXlab platform.
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different import pathways through the mitochondrial Intermembrane Space for inner membrane proteins
The EMBO Journal, 1999Co-Authors: Danielle Leuenberger, Gottfried Schatz, Nikolaus A Bally, Carla M KoehlerAbstract:Earlier work on the protein import system of yeast mitochondria has identified two soluble 70 kDa protein complexes in the Intermembrane Space. One complex contains the essential proteins Tim9p and Tim10p and mediates transport of cytosolically‐made metabolite carrier proteins from the outer to the inner membrane. The other complex contains the non‐essential proteins Tim8p and Tim13p as well as loosely associated Tim9p; its function was unclear, but it interacted structurally or functionally with the Tim9p–Tim10p complex. We now show that the two 70 kDa complexes each mediate the import of a different subset of integral inner membrane proteins and that they can transfer these proteins to one of three different membrane insertion sites: the TIM22 complex, the TIM23 complex or an as yet uncharacterized insertion site. Yeast mitochondria thus use multiple pathways for escorting hydrophobic inner membrane proteins across the aqueous Intermembrane Space.
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How membrane proteins travel across the mitochondrial Intermembrane Space
Trends in Biochemical Sciences, 1999Co-Authors: Carla M Koehler, Sabeeha S. Merchant, Gottfried SchatzAbstract:A newly discovered family of small proteins in the yeast mitochondrial Intermembrane Space mediates import of hydrophobic proteins from the cytoplasm into the inner membrane. Loss of one of these chaperone-like proteins from human mitochondria results in a disease that causes deafness, muscle weakness and blindness.
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import of mitochondrial carriers mediated by essential proteins of the Intermembrane Space
Science, 1998Co-Authors: Carla M Koehler, Kostas Tokatlidis, E. Jarosch, Rudolf J. Schweyen, K Schmid, Gottfried SchatzAbstract:In order to reach the inner membrane of the mitochondrion, multispanning carrier proteins must cross the aqueous Intermembrane Space. Two essential proteins of that Space, Tim10p and Tim12p, were shown to mediate import of multispanning carriers into the inner membrane. Both proteins formed a complex with the inner membrane protein Tim22p. Tim10p readily dissociated from the complex and was required to transport carrier precursors across the outer membrane; Tim12p was firmly bound to Tim22p and mediated the insertion of carriers into the inner membrane. Neither protein was required for protein import into the other mitochondrial compartments. Both proteins may function as Intermembrane Space chaperones for the highly insoluble carrier proteins.
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fusion proteins containing the cytochrome b2 presequence are sorted to the mitochondrial Intermembrane Space independently of hsp60
Journal of Biological Chemistry, 1994Co-Authors: Sabine Rospert, Gottfried Schatz, S Muller, Benjamin S. GlickAbstract:Abstract hsp60 is a chaperonin located in the mitochondrial matrix. It has been suggested that hsp60 participates in two processes: protein folding in the matrix, and the sorting of imported proteins to the Intermembrane Space. We analyzed hsp60 function by allowing isolated mitochondria to import two model precursor proteins and then measuring the binding of these proteins to the chaperonin. Of the methods that we tested for monitoring the association of imported proteins with hsp60, only co-immunoprecipitation with specific anti-hsp60 antibodies proved to be reliable. A chimeric matrix-targeted precursor, consisting of a mitochondrial presequence fused to a chloroplast-encoded protein, bound stably to hsp60 after import. In contrast, there was no detectable binding to hsp60 with a fusion protein that was targeted to the Intermembrane Space by the bipartite cytochrome b2 presequence. Analysis of a translocation intermediate demonstrated that the cytochrome b2 presequence arrests import through the inner membrane, with the result that the attached passenger protein is never exposed to hsp60.
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import of cytochrome b2 to the mitochondrial Intermembrane Space the tightly folded heme binding domain makes import dependent upon matrix atp
Protein Science, 1993Co-Authors: Benjamin S. Glick, Clemens Wachter, Graeme A Reid, Gottfried SchatzAbstract:Cytochrome b2 is synthesized as a precursor in the cytoplasm and imported to the Intermembrane Space of yeast mitochondria. We show here that the precursor contains a tightly folded heme-binding domain and that translocation of this domain across the outer membrane requires ATP. Surprisingly, it is ATP in the mitochondrial matrix rather than external ATP that drives import of the heme-binding domain. When the folded structure of the heme-binding domain is disrupted by mutation or by urea denaturation, import and correct processing take place in ATP-depleted mitochondria. These results indicate that (1) cytochrome b2 reaches the Intermembrane Space without completely crossing the inner membrane, and (2) some precursors fold outside the mitochondria but remain translocation-competent, and import of these precursors in vitro does not require ATP-dependent cytosolic chaperone proteins.
Enrique Cadenas - One of the best experts on this subject based on the ideXlab platform.
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Redox activation of mitochondrial Intermembrane Space Cu,Zn-superoxide dismutase.
Biochemical Journal, 2005Co-Authors: Pedro Iñarrea, Hadi Moini, Daniel Rettori, Derick Han, Jesús I. Martínez, Inés Bayona García, Erika Fernandez-vizarra, María Iturralde, Enrique CadenasAbstract:The localization of Cu,Zn-superoxide dismutase in the mitochondrial Intermembrane Space suggests a functional relationship with superoxide anion (O2•−) released into this compartment. The present study was aimed at examining the functionality of Cu,Zn-superoxide dismutase and elucidating the molecular basis for its activation in the Intermembrane Space. Intact rat liver mitochondria neither scavenged nor dismutated externally generated O2•−, unless the mitochondrial outer membrane was disrupted selectively by digitonin. The activation of the Intermembrane Space Cu,Zn-superoxide dismutase following the disruption of mitochondrial outer membrane was largely inhibited by bacitracin, an inhibitor of protein disulphide-isomerase. Thiol alkylating agents, such as N-methylmaleimide or iodoacetamide, decreased Intermembrane Space Cu,Zn-superoxide dismutase activation during, but not after, disruption of the outer membrane. This inhibitory effect was overcome by exposing mitochondria to low micromolar concentrations of H2O2 before disruption of the outer membrane in the presence of the alkylating agents. Moreover, H2O2 treatment alone enabled intact mitochondria to scavenge externally generated O2•−. These findings suggest that Intermembrane Space Cu,Zn-superoxide dismutase is inactive in intact mitochondria and that an oxidative modification of its critical thiol groups is necessary for its activation.
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mitochondrial superoxide anion production and release into Intermembrane Space
Methods in Enzymology, 2002Co-Authors: Derick Han, Fernando Antunes, Francesca Daneri, Enrique CadenasAbstract:The topological distribution of ubiquinone in the mitochondrial respiratory chain suggests that both ubiquinone pools may participate in O2.- production and, hence, are vectorially released into the matrix and Intermembrane Space. Mitoplasts, obtained by either digitonin or hypotonic KCl treatment, are a suitable experimental model for measuring O2.- in the Intermembrane Space. The use of membrane-impermeable spin-broadening agents strengthens the notion that part of the O2.- generated by the respiratory chain may be released into the Intermembrane Space. This, together with the putative occurrence of a Cu, Zn-superoxide dismutase in this compartment may account for part of H2O2 released by mitochondria and contributing to a cytosolic steady-state level of this species in cytosol.
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mitochondrial respiratory chain dependent generation of superoxide anion and its release into the Intermembrane Space
Biochemical Journal, 2001Co-Authors: Derick Han, Everett Williams, Enrique CadenasAbstract:It has been generally accepted that superoxide anion generated by the mitochondrial respiratory transport chain are vectorially released into the mitochondrial matrix, where they are converted to hydrogen peroxide through the catalytic action of Mn-superoxide dismutase. Release of superoxide anion into the Intermembrane Space is a controversial topic, partly unresolved by the reaction of superoxide anion with cytochrome c, which faces the Intermembrane Space and is present in this compartment at a high concentration. This study was aimed at assessing the topological site(s) of release of superoxide anion during respiratory chain activity. To address this issue, mitoplasts were prepared from isolated mitochondria by digitonin treatment to remove portions of the outer membrane along with portions of cytochrome c. EPR analysis in conjunction with spin traps of antimycin-supplemented mitoplasts revealed the formation of a spin adduct of superoxide anion. The EPR signal was (i) abrogated by superoxide dismutase, (ii) decreased competitively by exogenous ferricytochrome c and (iii) broadened by the membrane-impermeable spin-broadening agent chromium trioxalate. These results confirm the production and release of superoxide anion towards the cytosolic side of the inner mitochondrial membrane. In addition, co-treatment of mitoplasts with myxothiazol and antimycin A, resulting in an inhibition of the oxidation of ubiquinol to ubisemiquinone, abolished the EPR signal, thus suggesting that ubisemiquinone autoxidation at the outer site of the complex-III ubiquinone pool is a pathway for superoxide anion formation and subsequent release into the Intermembrane Space. The generation of superoxide anion towards the Intermembrane Space requires consideration of the mitochondrial steady-state values for superoxide anion and hydrogen peroxide, the decay pathways of these oxidants in this compartment and the implications of these processes for cytosolic events.
Nikolaus Pfanner - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial biogenesis switching the sorting pathway of the Intermembrane Space receptor mia40
Journal of Biological Chemistry, 2008Co-Authors: Agnieszka Chacinska, Judith M. Müller, Bernard Guiard, Agnes Schulzespecking, Kipros Gabriel, Stephan Kutik, Nikolaus PfannerAbstract:Mitochondrial precursor proteins are directed into the Intermembrane Space via two different routes, the presequence pathway and the redox-dependent MIA pathway. The pathways were assumed to be independent and transport different proteins. We report that the Intermembrane Space receptor Mia40 can switch between both pathways. In fungi, Mia40 is synthesized as large protein with an N-terminal presequence, whereas in metazoans and plants, Mia40 consists only of the conserved C-terminal domain. Human MIA40 and the C-terminal domain of yeast Mia40 (termed Mia40core) rescued the viability of Mia40-deficient yeast independently of the presence of a presequence. Purified Mia40core was imported into mitochondria via the MIA pathway. With cells expressing both full-length Mia40 and Mia40core, we demonstrate that yeast Mia40 contains dual targeting information, directing the large precursor onto the presequence pathway and the smaller Mia40core onto the MIA pathway, raising interesting implications for the evolution of mitochondrial protein sorting.
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The MIA system for protein import into the mitochondrial Intermembrane Space.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2008Co-Authors: Diana Stojanovski, Judith M. Müller, Dusanka Milenkovic, Bernard Guiard, Nikolaus Pfanner, Agnieszka ChacinskaAbstract:Abstract When thinking of the mitochondrial Intermembrane Space we envisage a small compartment that is bordered by the mitochondrial outer and inner membranes. Despite this somewhat simplified perception the Intermembrane Space has remained a central focus in mitochondrial biology. This compartment accommodates many proteinaceous factors that play critical roles in mitochondrial and cellular metabolism, including the regulation of programmed cell death and energy conversion. The mechanism by which Intermembrane Space proteins are transported into the organelle and folded remained largely unknown until recently. In pursuit of the answer to this question a novel machinery, the Mitochondrial Intermembrane Space Assembly machinery, exploiting a unique regulated thiol–disulfide exchange mechanism has been revealed. This exciting discovery has not only put in place novel concepts for the biogenesis of Intermembrane Space precursors but also raises important implications on the mechanisms involved in the generation and transfer of disulfide bonds.
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Precursor oxidation by Mia40 and Erv1 promotes vectorial transport of proteins into the mitochondrial Intermembrane Space.
Molecular Biology of the Cell, 2008Co-Authors: Judith M. Müller, Dusanka Milenkovic, Bernard Guiard, Nikolaus Pfanner, Agnieszka ChacinskaAbstract:The mitochondrial Intermembrane Space contains chaperone complexes that guide hydrophobic precursor proteins through this aqueous compartment. The chaperones consist of hetero-oligomeric complexes of small Tim proteins with conserved cysteine residues. The precursors of small Tim proteins are synthesized in the cytosol. Import of the precursors requires the essential Intermembrane Space proteins Mia40 and Erv1 that were proposed to form a relay for disulfide formation in the precursor proteins. However, experimental evidence for a role of Mia40 and Erv1 in the oxidation of Intermembrane Space precursors has been lacking. We have established a system to directly monitor the oxidation of precursors during import into mitochondria and dissected distinct steps of the import process. Reduced precursors bind to Mia40 during translocation into mitochondria. Both Mia40 and Erv1 are required for formation of oxidized monomers of the precursors that subsequently assemble into oligomeric complexes. Whereas the reduced precursors can diffuse back into the cytosol, the oxidized precursors are retained in the Intermembrane Space. Thus, oxidation driven by Mia40 and Erv1 determines vectorial transport of the precursors into the mitochondrial Intermembrane Space.
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Chaperoning through the Mitochondrial Intermembrane Space
Molecular Cell, 2006Co-Authors: Nils Wiedemann, Nikolaus Pfanner, Agnieszka ChacinskaAbstract:The first high-resolution structure of a mitochondrial translocase complex, the Tim9-Tim10 chaperone, is reported by Webb et al. (2006) in a recent issue of Molecular Cell, providing important insight in the transport of hydrophobic proteins through the aqueous Intermembrane Space and the mechanisms of protein assembly.
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biogenesis of the protein import channel tom40 of the mitochondrial outer membrane Intermembrane Space components are involved in an early stage of the assembly pathway
Journal of Biological Chemistry, 2004Co-Authors: Nils Wiedemann, Bernard Guiard, Kaye N Truscott, Sylvia Pfannschmidt, Chris Meisinger, Nikolaus PfannerAbstract:Abstract Tom40 forms the central channel of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The precursor of Tom40 is encoded in the nucleus, synthesized in the cytosol, and imported into mitochondria via a multi-step assembly pathway that involves the mature TOM complex and the sorting and assembly machinery of the outer membrane (SAM complex). We report that opening of the mitochondrial Intermembrane Space by swelling blocks the assembly pathway of the β-barrel protein Tom40. Mitochondria with defects in small Tim proteins of the Intermembrane Space are impaired in the Tom40 assembly pathway. Swelling as well as defects in the small Tim proteins inhibit an early stage of the Tom40 import pathway that is needed for formation of a Tom40-SAM intermediate. We propose that the biogenesis pathway of β-barrel proteins of the outer mitochondrial membrane not only requires TOM and SAM components, but also involves components of the Intermembrane Space.