TbLOK1/ATOM19 is a novel subunit of the noncanonical mitochondrial outer membrane protein translocase of Trypanosoma brucei

TbLOK1 has previously been characterized as a trypanosomatid-specific mitochondrial outer membrane protein whose ablation caused a collapse of the mitochondrial network, disruption of the membrane potential and loss of mitochondrial DNA. Here we show that ablation of TbLOK1 primarily abolishes mitochondrial protein import, both in vivo and in vitro. Co-immunprecipitations together with blue native gel analysis demonstrate that TbLOK1 is a stable and stoichiometric component of the archaic protein translocase of the outer membrane (ATOM), the highly diverged functional analogue of the TOM complex in other organisms. Furthermore, we show that TbLOK1 together with the other ATOM subunits forms a complex functional network where ablation of individual subunits either causes degradation of a specific set of other subunits or their exclusion from the ATOM complex. In summary these results establish that TbLOK1 is an essential novel subunit of the ATOM complex and thus that its primary molecular function is linked to mitochondrial protein import across the outer membrane. The previously described phenotypes can all be explained as consequences of the lack of mitochondrial protein import. We therefore suggest that in line with the nomenclature of the ATOM complex subunits, TbLOK1 should be renamed to ATOM19

The non-canonical mitochondrial inner membrane presequence translocase of trypanosomatids contains two essential rhomboid-like proteins

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

Helicobacter pylori VacA Toxin/Subunit p34: Targeting of an Anion Channel to the Inner Mitochondrial Membrane

The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% β-strands, similar to pore-forming β-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal

Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis

The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (delta psi m). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of delta psi m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce delta psi m loss and apoptosis, demonstrating that dissipation of delta psi m is a requirement for cell death caused by neisserial infection

The TIM translocase in T. brucei contains Rhomboid-like proteins that are essential for mitochondrial protein import

The parasitic protozoon Trypanosoma brucei is often considered as one of the earliest branching eukaryotes that have mitochondria capable of oxidative phosphorylation. Its protein import systems are therefore of great interest. Recently, it was shown that the outer mitochondrial membrane protein translocase is of similar complexity yet different composition than in other eukaryotes (1). In the inner membrane however, only a single orthologue of the pore forming Tim17/22/23 protein family was identified and termed TbTim17. Based on this finding it has been suggested that, instead of separate TIM22 and TIM23 complexes as in other eukaryotes, trypanosomes may have a single multifunctional translocase of the inner mitochondrial membrane (TIM) of reduced complexity. To elucidate the composition of the trypanosomal TIM complex we performed co-immunoprecipitations (CoIP) of epitope-tagged TbTim17 in combination with SILAC-based quantitative mass spectrometry. This led to the identification of 22 highly enriched TbTim17-interacting proteins. We tagged two of the top-scoring proteins for reciprocal CoIP analyses and recovered a set of ten proteins that are highly enriched in all three CoIPs. These proteins are excellent candidates for core subunits of the trypanosomal TIM complex. Eight of them were present in the previously determined inner membrane proteome and four show homology to small Tim chaperones. Three candidates, a novel trypanosomatid-specific 42 kDa protein, termed Tim42, and two putative orthologues of probably inactive rhomboid proteases were chosen for further analysis. All three proteins are essential in both life cycle stages and in a cell line that can grow in the absence of mitochondrial DNA. Additionally, their ablation by RNAi results in a strong protein import defect both in vivo and in vitro. Blue native PAGE reveals that Tim42, like TbTim17 is present in a high molecular weight complex. Moreover, ablation of either Tim42 or TbTim17 leads to a destabilization of the complex containing the other protein, suggesting a tight interaction of the two proteins. In summary our study shows that unlike anticipated trypanosomes have a highly complex TIM translocase that has extensively been redesigned. We have characterized three novel TIM subunits that have never been associated with mitochondrial protein import before. Two of them belong to the rhomboid protease family, a member of which recently has been implicated in the ERAD translocation system. Our study provides insight into mitochondrial evolution over large phylogenetic distances and suggests an exciting analogy between protein translocation systems of mitochondria and the ER

Probing protein import machineries of different organisms with the lipid bilayer technique: Functional comparison and phylogenetic insights

Im Rahmen dieser Arbeit wurde die Konservierung elektrophysiologischer Charakteristika von Proteintranslokasen aus verschiedenen Organismen untersucht. Die Sec61/Sec61p Komplexe aus rauen Mikrosomen von Canis familiaris und Saccharomyces cerevisiae bilden ionenpermeable Poren mit einer hohen Leitfähigkeit in planaren Lipidmembranen. In Säugerzellen werden diese durch einen Calcium-Calmodulin (Ca2+-CaM)-vermittelten negativen Feedback-Mechanismus reguliert. Im Rahmen dieser Arbeit konnte die Spezifität der zugrundeliegenden Interaktion von Ca2+-CaM mit der α-Untereinheit des Sec61 Komplexes belegt werden. Es wurde gezeigt, dass der Calmodulin Antagonist Ophiobolin A in der Lage ist, die Inhibition der Ionenpermeation durch Sec61 aufzuheben. Des Weiteren wurde anhand elektrophysiologischer Messungen nachgewiesen, dass dieser Ca2+-CaM-vermittelte Regulationsmechanismus in der Hefe S. cerevisiae nicht vorhanden ist. Dies wird auf eine kritische Variation in der Primärstruktur des Hefe-Proteins zurückgeführt, welche die Bindung von Calmodulin an den N-Terminus von Sec61p verhindert. Der bakterielle SecYEG Komplex aus E. coli konnte erfolgreich in Proteoliposomen rekonstituiert werden. Die Funktionalität des Translokons wurde in in vitro proOmpA Importexperimenten nachgewiesen. Mittels dieser Proteoliposomen sollten SecYEG Poren in den planaren Bilayer integriert werden. Sowohl für den inaktiven als auch für den durch die Anwesenheit von Substraten oder Bindepartnern aktivierten Komplex konnten keine ionenpermeablen Poren in der Membran nachgewiesen werden. Dies lässt darauf schließen, dass im Gegensatz zu den homologen Komplexen in Eukaryoten, der bakterielle Sec Komplex intrinsisch die Permeabilitätsbarriere für Ionen aufrechterhält. Die vorliegenden Ergebnisse legen nahe, dass weder die Ausbildung ionenpermeabler Poren, noch deren Regulation zwischen den Sec Komplexen von Bakterien, Hefen und Säugern vollständig konserviert ist. In einem zweiten Teilprojekt wurden auf der Suche nach der zentralen Proteinimportpore in der äußeren Mitochondrienmembran von Trypanosoma brucei zwei mögliche Kandidaten, TbSam50 und ATOM, anhand elektrophysiologischer Untersuchungen verglichen. Beide waren in der Lage in planaren Bilayern ionenpermeable Poren auszubilden. Die elektrophysiologischen Grundcharakteristika dieser Poren, wie der hohe Leitwert und die Selektivität für Kationen sowie die beobachtete Interaktion mit mitochondrialen Präsequenzen, stimmen gut mit einer potentiellen Funktion als Proteinimportpore überein. Eine detaillierte Untersuchung der Einzelkanaleigenschaften zeigte, dass TbSam50 beträcht-liche Ähnlichkeiten zu homologen Proteinen in Hefen und menschlichen Zellen aufweist. Somit bestätigen die hier präsentierten Ergebnisse die Identifikation von TbSam50 als Kern der trypanosomalen Assemblierungsmaschinerie für β-barrel Proteine in der äußeren Mitochondrienmembran. Besonderheiten in der Beeinflussung der Kanaleigenschaften durch mitochondriale Präpeptide, insbesondere die erhöhte Verweildauer des Kanals im geschlossenen Zustand, weisen darauf hin, dass TbSam50 keine duale Funktion als β-barrel Insertase und Proteintranslokase besitzt. Hingegen lieferte die elektrophysiologische Charakterisierung von ATOM Hinweise, welche die Identifikation dieses Proteins als porenbildende Untereinheit des mitochondrialen Proteinimportapparates in T. brucei bestätigen. Darüber hinaus zeigten Vergleiche der elektrophysiologischen Charakteristika, insbesondere des Schaltverhaltens und der Anzahl porenbildender Untereinheiten pro aktiver Einheit im artifiziellen Bilayer, dass ATOM stärkere Ähnlichkeiten zu Proteintranslokasen bakterieller Abstammung aufweist, als zu Tom40, der generellen Importpore der Eukaryoten. Dies unterstützt das auf Sequenzvergleichen basierende Model, dass ATOM ein evolutives Relikt repräsentiert, anhand dessen die Entwicklung der mitochondrialen Proteinimportmaschinerie aus einer bakteriellen, Omp85-artigen Protein-exportpore abgeleitet werden kann

TimX, a novel player in protein import across the inner mitochondrial membrane of T. brucei

Mitochondrial protein import is an essential function of the unique mitochondrion in T. brucei as roughly 1000 different nuclear encoded proteins need to be correctly localized to their mitochondrial subcompartment. For this reason the responsible import machinery is expected to be similarly complex as in other Eukaryotes. This was recently demonstrated for the translocation machinery in the outer mitochondrial membrane. In contrast, the composition of the inner membrane import machinery and the exact molecular pathway(s) taken by various substrates are still ill-defined. To elucidate this further, we performed a pulldown analysis of epitope tagged TbTim17 in combination with quantitative mass spectrometry. By this we identified novel components of the mitochondrial import machinery in trypanosomes. One of these, TimX, is an essential mitochondrial membrane protein of 42 kDa that is unique to kinetoplastids. This protein migrates on Blue Native PAGE in a high molecular weight complex similar to TbTim17. Ablation of either of the two proteins leads to a destabilization of the complex containing the other protein. Furthermore, its involvement in protein import could be demonstrated by in vivo and in vitro protein import assays. This corroborates that TimX together with TbTim17 forms a protein import complex in the inner mitochondrial membrane. As TbTim17 the TimX protein was subjected to pulldown analysis in combination with quantitative mass spectrometry. The overlap of candidates defined by these two sets of IPs likely defines further components of the inner membrane translocase which are presently being analyzed. In summary our study on novel components of the trypanosome mitochondrial protein import system gives us fascinating new insights into evolution of the mitochondrion

TimX, a novel player in protein import across the inner mitochondrial membrane of T. brucei

Mitochondrial protein import is an essential function of the unique mitochondrion in T. brucei as roughly 1000 different nuclear encoded proteins need to be correctly localized to their mitochondrial subcompartment. For this reason the responsible import machinery is expected to be similarly complex as in other Eukaryotes. This was recently demonstrated for the translocation machinery in the outer mitochondrial membrane. In contrast, the composition of the inner membrane import machinery and the exact molecular pathway(s) taken by various substrates are still ill-defined. To elucidate this further, we performed a pulldown analysis of epitope tagged TbTim17 in combination with quantitative mass spectrometry. By this we identified novel components of the mitochondrial import machinery in trypanosomes. One of these, TimX, is an essential mitochondrial membrane protein of 42 kDa that is unique to kinetoplastids. This protein migrates on Blue Native PAGE in a high molecular weight complex similar to TbTim17. Ablation of either of the two proteins leads to a destabilization of the complex containing the other protein. Furthermore, its involvement in protein import could be demonstrated by in vivo and in vitro protein import assays. This corroborates that TimX together with TbTim17 forms a protein import complex in the inner mitochondrial membrane. As TbTim17 the TimX protein was subjected to pulldown analysis in combination with quantitative mass spectrometry. The overlap of candidates defined by these two sets of IPs likely defines further components of the inner membrane translocase which are presently being analyzed. In summary our study on novel components of the trypanosome mitochondrial protein import system gives us fascinating new insights into evolution of the mitochondrion