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Nikolaus Pfanner - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial protein import motor differential role of tim44 in the recruitment of pam17 and j complex to the presequence Translocase
Molecular Biology of the Cell, 2008Co-Authors: Agnieszka Chacinska, Nikolaus Pfanner, Peter Rehling, Bernard Guiard, Dana P Hutu, Dorothea Becker, Martin Van Der LaanAbstract:The presequence Translocase of the mitochondrial inner membrane (TIM23 complex) mediates the import of preproteins with amino-terminal presequences. To drive matrix translocation the TIM23 complex recruits the presequence Translocase-associated motor (PAM) with the matrix heat shock protein 70 (mtHsp70) as central subunit. Activity and localization of mtHsp70 are regulated by four membrane-associated cochaperones: the adaptor protein Tim44, the stimulatory J-complex Pam18/Pam16, and Pam17. It has been proposed that Tim44 serves as molecular platform to localize mtHsp70 and the J-complex at the TIM23 complex, but it is unknown how Pam17 interacts with the Translocase. We generated conditional tim44 yeast mutants and selected a mutant allele, which differentially affects the association of PAM modules with TIM23. In tim44-804 mitochondria, the interaction of the J-complex with the TIM23 complex is impaired, whereas unexpectedly the binding of Pam17 is increased. Pam17 interacts with the channel protein Tim23, revealing a new interaction site between TIM23 and PAM. Thus, the motor PAM is composed of functional modules that bind to different sites of the Translocase. We suggest that Tim44 is not simply a scaffold for binding of motor subunits but plays a differential role in the recruitment of PAM modules to the inner membrane Translocase.
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cooperation of Translocase complexes in mitochondrial protein import
Journal of Cell Biology, 2007Co-Authors: Stephan Kutik, Bernard Guiard, Nils Wiedemann, Helmut E Meyer, Nikolaus PfannerAbstract:Most mitochondrial proteins are synthesized in the cytosol and imported into one of the four mitochondrial compartments: outer membrane, intermembrane space, inner membrane, and matrix. Each compartment contains protein complexes that interact with precursor proteins and promote their transport. These Translocase complexes do not act as independent units but cooperate with each other and further membrane complexes in a dynamic manner. We propose that a regulated coupling of Translocases is important for the coordination of preprotein translocation and efficient sorting to intramitochondrial compartments.
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motor free mitochondrial presequence Translocase drives membrane integration of preproteins
Nature Cell Biology, 2007Co-Authors: Martin Van Der Laan, Bernard Guiard, Inge Perschil, Richard Wagner, Michael Meinecke, Jan Dudek, Dana P Hutu, Maria I Lind, Nikolaus PfannerAbstract:The mitochondrial inner membrane is the central energy-converting membrane of eukaryotic cells. The electrochemical proton gradient generated by the respiratory chain drives the ATP synthase. To maintain this proton-motive force, the inner membrane forms a tight barrier and strictly controls the translocation of ions. However, the major preprotein transport machinery of the inner membrane, termed the presequence Translocase, translocates polypeptide chains into or across the membrane. Different views exist of the molecular mechanism of the Translocase, in particular of the coupling with the import motor of the matrix. We have reconstituted preprotein transport into the mitochondrial inner membrane by incorporating the purified presequence Translocase into cardiolipin-containing liposomes. We show that the motor-free form of the presequence Translocase integrates preproteins into the membrane. The reconstituted presequence Translocase responds to targeting peptides and mediates voltage-driven preprotein translocation, lateral release and insertion into the lipid phase. Thus, the minimal system for preprotein integration into the mitochondrial inner membrane is the presequence Translocase, a cardiolipin-rich membrane and a membrane potential.
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Mitochondrial import and the twin-pore Translocase
Nature Reviews Molecular Cell Biology, 2004Co-Authors: Peter Rehling, Katrin Brandner, Nikolaus PfannerAbstract:The mitochondrial inner membrane is rich in multispanning integral membrane proteins, most of which mediate the vital transport of molecules between the matrix and the intermembrane space. The correct transport and membrane insertion of such proteins is essential for maintaining the correct exchange of molecules between mitochondria and the rest of the cell. Mitochondria contain several specific complexes — known as Translocases — that translocate precursor proteins. Recent analysis of the inner-membrane, twin-pore protein Translocase (TIM22 complex) allows a glimpse of the molecular mechanisms by which this machinery triggers protein insertion using the membrane potential as an external driving force.
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machinery for protein sorting and assembly in the mitochondrial outer membrane
Nature, 2003Co-Authors: Nils Wiedemann, Agnieszka Chacinska, Nikolaus Pfanner, Birgit Schonfisch, Michael T. Ryan, Vera Kozjak, Sabine Rospert, Chris MeisingerAbstract:Mitochondria contain Translocases for the transport of precursor proteins across their outer and inner membranes1,2,3,4,5. It has been assumed that the Translocases also mediate the sorting of proteins to their submitochondrial destination1,2,5,6,7,8,9,10. Here we show that the mitochondrial outer membrane contains a separate sorting and assembly machinery (SAM) that operates after the Translocase of the outer membrane (TOM). Mas37 forms a constituent of the SAM complex. The central role of the SAM complex in the sorting and assembly pathway of outer membrane proteins explains the various pleiotropic functions that have been ascribed to Mas37 (refs 4, 11–15). These results suggest that the TOM complex, which can transport all kinds of mitochondrial precursor proteins, is not sufficient for the correct integration of outer membrane proteins with a complicated topology, and instead transfers precursor proteins to the SAM complex.
Peter Rehling - One of the best experts on this subject based on the ideXlab platform.
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motor recruitment to the tim23 channel s lateral gate restricts polypeptide release into the inner membrane
Nature Communications, 2018Co-Authors: Alexander Benjamin Schendzielorz, Agnieszka Chacinska, Peter Rehling, Bernard Guiard, Piotr Bragoszewski, Nataliia Naumenko, Ridhima Gomkale, Christian SchulzAbstract:The presequence Translocase of the mitochondrial inner membrane (TIM23 complex) facilitates anterograde precursor transport into the matrix and lateral release of precursors with stop-transfer signal into the membrane (sorting). Sorting requires precursor exit from the translocation channel into the lipid phase through the lateral gate of the TIM23 complex. How the two transport modes are regulated and balanced against each other is unknown. Here we show that the import motor J-protein Pam18, which is essential for matrix import, controls lateral protein release into the lipid bilayer. Constitutively Translocase-associated Pam18 obstructs lateral precursor transport. Concomitantly, Mgr2, implicated in precursor quality control, is displaced from the Translocase. We conclude that during motor-dependent matrix protein transport, the transmembrane segment of Pam18 closes the lateral gate to promote anterograde polypeptide movement. This finding explains why a motor-free form of the Translocase facilitates the lateral movement of precursors with a stop-transfer signal. The mitochondrial TIM23-complex facilitates anterograde precursor transport across the inner membrane into the matrix and lateral release of precursors into the membrane. Here authors show that the import motor J-protein Pam18 controls lateral protein release into the lipid bilayer.
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signal recognition initiates reorganization of the presequence Translocase during protein import
The EMBO Journal, 2013Co-Authors: Oleksandr Lytovchenko, Peter Rehling, Christian Schulz, Dana P Hutu, Jonathan Melin, Markus KilischAbstract:The mitochondrial presequence Translocase interacts with presequence-containing precursors at the intermembrane space (IMS) side of the inner membrane to mediate their translocation into the matrix. Little is known as too how these matrix-targeting signals activate the Translocase in order to initiate precursor transport. Therefore, we analysed how signal recognition by the presequence Translocase initiates reorganization among Tim-proteins during import. Our analyses revealed that the presequence receptor Tim50 interacts with Tim21 in a signal-sensitive manner in a process that involves the IMS-domain of the Tim23 channel. The signal-driven release of Tim21 from Tim50 promotes recruitment of Pam17 and thus triggers formation of the motor-associated form of the TIM23 complex required for matrix transport.
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mitochondrial protein import motor differential role of tim44 in the recruitment of pam17 and j complex to the presequence Translocase
Molecular Biology of the Cell, 2008Co-Authors: Agnieszka Chacinska, Nikolaus Pfanner, Peter Rehling, Bernard Guiard, Dana P Hutu, Dorothea Becker, Martin Van Der LaanAbstract:The presequence Translocase of the mitochondrial inner membrane (TIM23 complex) mediates the import of preproteins with amino-terminal presequences. To drive matrix translocation the TIM23 complex recruits the presequence Translocase-associated motor (PAM) with the matrix heat shock protein 70 (mtHsp70) as central subunit. Activity and localization of mtHsp70 are regulated by four membrane-associated cochaperones: the adaptor protein Tim44, the stimulatory J-complex Pam18/Pam16, and Pam17. It has been proposed that Tim44 serves as molecular platform to localize mtHsp70 and the J-complex at the TIM23 complex, but it is unknown how Pam17 interacts with the Translocase. We generated conditional tim44 yeast mutants and selected a mutant allele, which differentially affects the association of PAM modules with TIM23. In tim44-804 mitochondria, the interaction of the J-complex with the TIM23 complex is impaired, whereas unexpectedly the binding of Pam17 is increased. Pam17 interacts with the channel protein Tim23, revealing a new interaction site between TIM23 and PAM. Thus, the motor PAM is composed of functional modules that bind to different sites of the Translocase. We suggest that Tim44 is not simply a scaffold for binding of motor subunits but plays a differential role in the recruitment of PAM modules to the inner membrane Translocase.
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Mitochondrial import and the twin-pore Translocase
Nature Reviews Molecular Cell Biology, 2004Co-Authors: Peter Rehling, Katrin Brandner, Nikolaus PfannerAbstract:The mitochondrial inner membrane is rich in multispanning integral membrane proteins, most of which mediate the vital transport of molecules between the matrix and the intermembrane space. The correct transport and membrane insertion of such proteins is essential for maintaining the correct exchange of molecules between mitochondria and the rest of the cell. Mitochondria contain several specific complexes — known as Translocases — that translocate precursor proteins. Recent analysis of the inner-membrane, twin-pore protein Translocase (TIM22 complex) allows a glimpse of the molecular mechanisms by which this machinery triggers protein insertion using the membrane potential as an external driving force.
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protein insertion into the mitochondrial inner membrane by a twin pore Translocase
Science, 2003Co-Authors: Peter Rehling, Katrin Brandner, Albert Sickmann, Werner Kuhlbrandt, Kirstin Model, Peter Kovermann, Helmut E Meyer, Richard Wagner, Kaye N Truscott, Nikolaus PfannerAbstract:The mitochondrial inner membrane imports numerous proteins that span it multiple times using the membrane potential Δψ as the only external energy source. We purified the protein insertion complex (TIM22 complex), a twin-pore Translocase that mediated the insertion of precursor proteins in a three-step process. After the precursor is tethered to the Translocase without losing energy from the Δψ, two energy-requiring steps were needed. First, Δψ acted on the precursor protein and promoted its docking in the Translocase complex. Then, Δψ and an internal signal peptide together induced rapid gating transitions in one pore and closing of the other pore and drove membrane insertion to completion. Thus, protein insertion was driven by the coordinated action of a twin-pore complex in two voltage-dependent steps.
Timothy M Lohman - One of the best experts on this subject based on the ideXlab platform.
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uvrd helicase activation by mutl involves rotation of its 2b subdomain
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Yerdos Ordabayev, Alexander G Kozlov, Binh Nguyen, Haifeng Jia, Timothy M LohmanAbstract:Escherichia coli UvrD is a superfamily 1 helicase/Translocase that functions in DNA repair, replication, and recombination. Although a UvrD monomer can translocate along single-stranded DNA, self-assembly or interaction with an accessory protein is needed to activate its helicase activity in vitro. Our previous studies have shown that an Escherichia coli MutL dimer can activate the UvrD monomer helicase in vitro, but the mechanism is not known. The UvrD 2B subdomain is regulatory and can exist in extreme rotational conformational states. By using single-molecule FRET approaches, we show that the 2B subdomain of a UvrD monomer bound to DNA exists in equilibrium between open and closed states, but predominantly in an open conformation. However, upon MutL binding to a UvrD monomer–DNA complex, a rotational conformational state is favored that is intermediate between the open and closed states. Parallel kinetic studies of MutL activation of the UvrD helicase and of MutL-dependent changes in the UvrD 2B subdomain show that the transition from an open to an intermediate 2B subdomain state is on the pathway to helicase activation. We further show that MutL is unable to activate the helicase activity of a chimeric UvrD containing the 2B subdomain of the structurally similar Rep helicase. Hence, MutL activation of the monomeric UvrD helicase is regulated specifically by its 2B subdomain.
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chemo mechanical pushing of proteins along single stranded dna
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Joshua E Sokoloski, Alexander G Kozlov, Roberto Galletto, Timothy M LohmanAbstract:Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA Translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3′-Cy3–labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5′ to 3′ ssDNA Translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from Translocase-induced directional (5′ to 3′) pushing of the SSB toward the 3′ ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3′ to 5′) direction, are observed with EcRep and EcUvrD (both 3′ to 5′ ssDNA Translocases). Simulations indicate that these events reflect active pushing by the Translocase. The ability of Translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.
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bacillus stearothermophilus pcra monomer is a single stranded dna Translocase but not a processive helicase in vitro
Journal of Biological Chemistry, 2007Co-Authors: Anita Niedzielamajka, Marla A Chesnik, Eric J Tomko, Timothy M LohmanAbstract:Structural studies of the Bacillus stearothermophilus PcrA protein along with biochemical studies of the single-stranded (ss) DNA translocation activity of PcrA monomers have led to the suggestion that a PcrA monomer possesses processive helicase activity in vitro. Yet definitive studies testing whether the PcrA monomer possesses processive helicase activity have not been performed. Here we show, using single turnover kinetic methods, that monomers of PcrA are able to translocate along ssDNA, in the 3' to 5' direction, rapidly and processively, whereas these same monomers display no detectable helicase activity under the same solution conditions in vitro. The PcrA monomer ssDNA translocation activity, although necessary, is not sufficient for processive helicase activity, and thus the Translocase and helicase activities of PcrA are separable. These results also suggest that the helicase activity of PcrA needs to be activated either by self-assembly or through interactions with accessory proteins. This same behavior is displayed by both the Escherichia coli Rep and UvrD monomers. Hence, all three of these SF1 enzymes are ssDNA Translocases as monomers but do not display processive helicase activity in vitro unless activated. The fact that the Translocase and helicase activities are separable suggests that each activity may be used for different functions in vivo.
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bacillus stearothermophilus pcra monomer is a single stranded dna Translocase but not a processive helicase in vitro
Journal of Biological Chemistry, 2007Co-Authors: Anita Niedzielamajka, Marla A Chesnik, Eric J Tomko, Timothy M LohmanAbstract:Structural studies of the Bacillus stearothermophilus PcrA protein along with biochemical studies of the single-stranded (ss) DNA translocation activity of PcrA monomers have led to the suggestion that a PcrA monomer possesses processive helicase activity in vitro. Yet definitive studies testing whether the PcrA monomer possesses processive helicase activity have not been performed. Here we show, using single turnover kinetic methods, that monomers of PcrA are able to translocate along ssDNA, in the 3' to 5' direction, rapidly and processively, whereas these same monomers display no detectable helicase activity under the same solution conditions in vitro. The PcrA monomer ssDNA translocation activity, although necessary, is not sufficient for processive helicase activity, and thus the Translocase and helicase activities of PcrA are separable. These results also suggest that the helicase activity of PcrA needs to be activated either by self-assembly or through interactions with accessory proteins. This same behavior is displayed by both the Escherichia coli Rep and UvrD monomers. Hence, all three of these SF1 enzymes are ssDNA Translocases as monomers but do not display processive helicase activity in vitro unless activated. The fact that the Translocase and helicase activities are separable suggests that each activity may be used for different functions in vivo.
Bettina Warscheid - One of the best experts on this subject based on the ideXlab platform.
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trnas and proteins use the same import channel for translocation across the mitochondrial outer membrane of trypanosomes
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Moritz Niemann, Silke Oeljeklaus, Bettina Warscheid, Richard Wagner, Anke Judith Harsman, Jan Mani, Christian D Peikert, Andre SchneiderAbstract:Mitochondrial tRNA import is widespread, but the mechanism by which tRNAs are imported remains largely unknown. The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes, and thus imports all tRNAs from the cytosol. Here we show that in T. brucei in vivo import of tRNAs requires four subunits of the mitochondrial outer membrane protein Translocase but not the two receptor subunits, one of which is essential for protein import. The latter shows that it is possible to uncouple mitochondrial tRNA import from protein import. Ablation of the intermembrane space domain of the Translocase subunit, archaic Translocase of the outer membrane (ATOM)14, on the other hand, while not affecting the architecture of the Translocase, impedes both protein and tRNA import. A protein import intermediate arrested in the translocation channel prevents both protein and tRNA import. In the presence of tRNA, blocking events of single-channel currents through the pore formed by recombinant ATOM40 were detected in electrophysiological recordings. These results indicate that both types of macromolecules use the same import channel across the outer membrane. However, while tRNA import depends on the core subunits of the protein import Translocase, it does not require the protein import receptors, indicating that the two processes are not mechanistically linked.
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the non canonical mitochondrial inner membrane presequence Translocase of trypanosomatids contains two essential rhomboid like proteins
Nature Communications, 2016Co-Authors: Anke Judith Harsman, Silke Oeljeklaus, Bettina Warscheid, Christoph Wenger, Jonathan L Huot, Andre SchneiderAbstract:Mitochondrial protein import is essential for all eukaryotes. Here we show that the early diverging eukaryote Trypanosoma brucei has a non-canonical inner membrane (IM) protein translocation machinery. Besides TbTim17, the single member of the Tim17/22/23 family in trypanosomes, the presequence Translocase contains nine subunits that co-purify in reciprocal immunoprecipitations and with a presequence-containing substrate that is trapped in the translocation channel. Two of the newly discovered subunits are rhomboid-like proteins, which are essential for growth and mitochondrial protein import. Rhomboid-like proteins were proposed to form the protein translocation pore of the ER-associated degradation system, suggesting that they may contribute to pore formation in the presequence Translocase of T. brucei. Pulldown of import-arrested mitochondrial carrier protein shows that the carrier Translocase shares eight subunits with the presequence Translocase. This indicates that T. brucei may have a single IM Translocase that with compositional variations mediates import of presequence-containing and carrier proteins.
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the mitochondrial adp atp carrier associates with the inner membrane presequence Translocase in a stoichiometric manner
Journal of Biological Chemistry, 2014Co-Authors: Carola S Mehnert, Bernard Guiard, Silke Oeljeklaus, Bettina Warscheid, Heike Rampelt, Michael Gebert, Sandra G Schrempp, Lioba Kochbeck, Martin Van Der LaanAbstract:The majority of mitochondrial proteins are synthesized with amino-terminal signal sequences. The presequence Translocase of the inner membrane (TIM23 complex) mediates the import of these preproteins. The essential TIM23 core complex closely cooperates with partner protein complexes like the presequence Translocase-associated import motor and the respiratory chain. The inner mitochondrial membrane also contains a large number of metabolite carriers, but their association with preprotein Translocases has been controversial. We performed a comprehensive analysis of the TIM23 interactome based on stable isotope labeling with amino acids in cell culture. Subsequent biochemical studies on identified partner proteins showed that the mitochondrial ADP/ATP carrier associates with the membrane-embedded core of the TIM23 complex in a stoichiometric manner, revealing an unexpected connection of mitochondrial protein biogenesis to metabolite transport. Our data indicate that direct TIM23-AAC coupling may support preprotein import into mitochondria when respiratory activity is low.
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role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane
Molecular Biology of the Cell, 2012Co-Authors: Maria Bohnert, Silke Oeljeklaus, Ralf M Zerbes, David A Stroud, Karina Von Der Malsburg, Lenasophie Wenz, Judith M Muller, Susanne E Horvath, Inge Perschil, Bettina WarscheidAbstract:Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the Translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both Translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport–associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins.
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mitochondrial preprotein Translocase of trypanosomatids has a bacterial origin
Current Biology, 2011Co-Authors: Mascha Pusnik, Oliver Schmidt, Silke Oeljeklaus, Bettina Warscheid, Chris Meisinger, Trevor Lithgow, Andrew J Perry, Moritz Niemann, Andre SchneiderAbstract:Summary Mitochondria are found in all eukaryotic cells and derive from a bacterial endosymbiont [1, 2]. The evolution of a protein import system was a prerequisite for the conversion of the endosymbiont into a true organelle. Tom40, the essential component of the protein Translocase of the outer membrane, is conserved in mitochondria of almost all eukaryotes but lacks bacterial orthologs [3–6]. It serves as the gateway through which all mitochondrial proteins are imported. The parasitic protozoa Trypanosoma brucei and its relatives do not have a Tom40-like protein, which raises the question of how proteins are imported by their mitochondria [7, 8]. Using a combination of bioinformatics and in vivo and in vitro studies, we have discovered that T. brucei likely employs a different import channel, termed ATOM ( a rchaic t ranslocase of the o uter m itochondrial membrane). ATOM mediates the import of nuclear-encoded proteins into mitochondria and is essential for viability of trypanosomes. It is not related to Tom40 but is instead an ortholog of a subgroup of the Omp85 protein superfamily that is involved in membrane translocation and insertion of bacterial outer membrane proteins [9]. This suggests that the protein import channel in trypanosomes is a relic of an archaic protein transport system that was operational in the ancestor of all eukaryotes.
Silke Oeljeklaus - One of the best experts on this subject based on the ideXlab platform.
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trnas and proteins use the same import channel for translocation across the mitochondrial outer membrane of trypanosomes
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Moritz Niemann, Silke Oeljeklaus, Bettina Warscheid, Richard Wagner, Anke Judith Harsman, Jan Mani, Christian D Peikert, Andre SchneiderAbstract:Mitochondrial tRNA import is widespread, but the mechanism by which tRNAs are imported remains largely unknown. The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes, and thus imports all tRNAs from the cytosol. Here we show that in T. brucei in vivo import of tRNAs requires four subunits of the mitochondrial outer membrane protein Translocase but not the two receptor subunits, one of which is essential for protein import. The latter shows that it is possible to uncouple mitochondrial tRNA import from protein import. Ablation of the intermembrane space domain of the Translocase subunit, archaic Translocase of the outer membrane (ATOM)14, on the other hand, while not affecting the architecture of the Translocase, impedes both protein and tRNA import. A protein import intermediate arrested in the translocation channel prevents both protein and tRNA import. In the presence of tRNA, blocking events of single-channel currents through the pore formed by recombinant ATOM40 were detected in electrophysiological recordings. These results indicate that both types of macromolecules use the same import channel across the outer membrane. However, while tRNA import depends on the core subunits of the protein import Translocase, it does not require the protein import receptors, indicating that the two processes are not mechanistically linked.
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the non canonical mitochondrial inner membrane presequence Translocase of trypanosomatids contains two essential rhomboid like proteins
Nature Communications, 2016Co-Authors: Anke Judith Harsman, Silke Oeljeklaus, Bettina Warscheid, Christoph Wenger, Jonathan L Huot, Andre SchneiderAbstract:Mitochondrial protein import is essential for all eukaryotes. Here we show that the early diverging eukaryote Trypanosoma brucei has a non-canonical inner membrane (IM) protein translocation machinery. Besides TbTim17, the single member of the Tim17/22/23 family in trypanosomes, the presequence Translocase contains nine subunits that co-purify in reciprocal immunoprecipitations and with a presequence-containing substrate that is trapped in the translocation channel. Two of the newly discovered subunits are rhomboid-like proteins, which are essential for growth and mitochondrial protein import. Rhomboid-like proteins were proposed to form the protein translocation pore of the ER-associated degradation system, suggesting that they may contribute to pore formation in the presequence Translocase of T. brucei. Pulldown of import-arrested mitochondrial carrier protein shows that the carrier Translocase shares eight subunits with the presequence Translocase. This indicates that T. brucei may have a single IM Translocase that with compositional variations mediates import of presequence-containing and carrier proteins.
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the mitochondrial adp atp carrier associates with the inner membrane presequence Translocase in a stoichiometric manner
Journal of Biological Chemistry, 2014Co-Authors: Carola S Mehnert, Bernard Guiard, Silke Oeljeklaus, Bettina Warscheid, Heike Rampelt, Michael Gebert, Sandra G Schrempp, Lioba Kochbeck, Martin Van Der LaanAbstract:The majority of mitochondrial proteins are synthesized with amino-terminal signal sequences. The presequence Translocase of the inner membrane (TIM23 complex) mediates the import of these preproteins. The essential TIM23 core complex closely cooperates with partner protein complexes like the presequence Translocase-associated import motor and the respiratory chain. The inner mitochondrial membrane also contains a large number of metabolite carriers, but their association with preprotein Translocases has been controversial. We performed a comprehensive analysis of the TIM23 interactome based on stable isotope labeling with amino acids in cell culture. Subsequent biochemical studies on identified partner proteins showed that the mitochondrial ADP/ATP carrier associates with the membrane-embedded core of the TIM23 complex in a stoichiometric manner, revealing an unexpected connection of mitochondrial protein biogenesis to metabolite transport. Our data indicate that direct TIM23-AAC coupling may support preprotein import into mitochondria when respiratory activity is low.
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role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane
Molecular Biology of the Cell, 2012Co-Authors: Maria Bohnert, Silke Oeljeklaus, Ralf M Zerbes, David A Stroud, Karina Von Der Malsburg, Lenasophie Wenz, Judith M Muller, Susanne E Horvath, Inge Perschil, Bettina WarscheidAbstract:Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the Translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both Translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport–associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins.
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mitochondrial preprotein Translocase of trypanosomatids has a bacterial origin
Current Biology, 2011Co-Authors: Mascha Pusnik, Oliver Schmidt, Silke Oeljeklaus, Bettina Warscheid, Chris Meisinger, Trevor Lithgow, Andrew J Perry, Moritz Niemann, Andre SchneiderAbstract:Summary Mitochondria are found in all eukaryotic cells and derive from a bacterial endosymbiont [1, 2]. The evolution of a protein import system was a prerequisite for the conversion of the endosymbiont into a true organelle. Tom40, the essential component of the protein Translocase of the outer membrane, is conserved in mitochondria of almost all eukaryotes but lacks bacterial orthologs [3–6]. It serves as the gateway through which all mitochondrial proteins are imported. The parasitic protozoa Trypanosoma brucei and its relatives do not have a Tom40-like protein, which raises the question of how proteins are imported by their mitochondria [7, 8]. Using a combination of bioinformatics and in vivo and in vitro studies, we have discovered that T. brucei likely employs a different import channel, termed ATOM ( a rchaic t ranslocase of the o uter m itochondrial membrane). ATOM mediates the import of nuclear-encoded proteins into mitochondria and is essential for viability of trypanosomes. It is not related to Tom40 but is instead an ortholog of a subgroup of the Omp85 protein superfamily that is involved in membrane translocation and insertion of bacterial outer membrane proteins [9]. This suggests that the protein import channel in trypanosomes is a relic of an archaic protein transport system that was operational in the ancestor of all eukaryotes.
