Conserved motifs reveal details of ancestry and structure in the small tim chaperones of the mitochondrial intermembrane space

The mitochondrial inner and outer membranes are composed of a variety of integral membrane proteins, assembled into the membranes posttranslationally. The small translocase of the inner mitochondrial membranes (TIMs) are a group of ∼10 kDa proteins that function as chaperones to ferry the imported proteins across the mitochondrial intermembrane space to the outer and inner membranes. In yeast, there are 5 small TIM proteins: Tim8, Tim9, Tim10, Tim12, and Tim13, with equivalent proteins reported in humans. Using hidden Markov models, we find that many eukaryotes have proteins equivalent to the Tim8 and Tim13 and the Tim9 and Tim10 subunits. Some eukaryotes provide "snapshots" of evolution, with a single protein showing the features of both Tim8 and Tim13, suggesting that a single progenitor gene has given rise to each of the small TIMs through duplication and modification. We show that no "Tim12" family of proteins exist, but rather that variant forms of the cognate small TIMs have been recently duplicated and modified to provide new functions: the yeast Tim12 is a modified form of Tim10, whereas in humans and some protists variant forms of Tim9, Tim8, and Tim13 are found instead. Sequence motif analysis reveals acidic residues conserved in the Tim10 substrate-binding tentacles, whereas more hydrophobic residues are found in the equivalent substrate-binding region of Tim13. The substrate-binding region of Tim10 and Tim13 represent structurally independent domains: when the acidic domain from Tim10 is attached to Tim13, the Tim8–Tim13¹⁰ complex becomes essential and the Tim9–Tim10 complex becomes dispensable. The conserved features in the Tim10 and Tim13 subunits provide distinct binding surfaces to accommodate the broad range of substrate proteins delivered to the mitochondrial inner and outer membranes

Mitochondrial tRNA import in the parasitic protozoon "Trypanosoma brucei" and its consequences on mitochondrial translation

Le parasite protozoaire Trypanosoma brucei est l’agent pathogène responsable de la maladie du sommeil chez l’homme. En plus de son importance dans le domaine de la lutte contre les maladies tropicales, T. brucei est également un excellent modèle pour la recherche fondamentale car il présente beaucoup de caractéristiques qui lui sont propres. Par exemple, aucun ARN de transfert (ARNt) n’est codé dans le génome mitochondrial. Pour cette raison, les ARNts nécessaires au processus de traduction mitochondriale sont codés dans le noyau, puis importés depuis le cytosol [1]. Ainsi, mis à part l’initiateur ARNtMet et l’ARNtSec, tous les ARNts du trypanosome fonctionnent à la fois dans le cytosol et dans la mitochondrie. Une conséquence importante de l’import mitochondrial des ARNts réside dans le fait que les ARNts utilisés dans la mitochondrie de T. brucei ont une origine évolutionnaire eucaryote. Cependant la mitochondrie provient d’un ancêtre bactérien. C’est pourquoi nous avons voulu étudier comment le système de traduction mitochondriale, qui est de type bactérien, s’est adapté aux ARNts de type eucaryote durant l’évolution. Etant donné que l’initiateur ARNtMet du trypanosome est localisé exclusivement dans le cytosol, le seul ARNtMet présent dans la mitochondrie de T. brucei est l’élongateur ARNtMet qui est importé depuis le cytosol. Dans les bactéries et les organelles, la formylation spécifique de l’initiateur methionyl-ARNtMet catalisée par la methionyl- ARNt formyltransferase (MTF) est nécessaire au processus d’initiation de la traduction. L’initiateur formylmethionyl-ARNtMet résultant de cette réaction se lie alors avec le facteur d’initiation 2 de type bactérien (IF2), ce qui favorise l’intéraction de l’ARNt avec le ribosome. Etonnamment, dans la mitochondrie de T. brucei, une partie de l’élongateur ARNtMet importé est formylée par un orthologue de MTF dont la taille est spécialement grande [2]. Durant ce travail de thèse, nous avons identifié IF2 chez T. brucei et démontré que cette protéine est nécessaire à la croissance normale du parasite. De plus, nous avons montré qu’IF2 ne reconnaît l’élongateur methionyl-ARNtMet que si ce dernier est formylé. Ainsi, en complément de précédentes études [1, 2], ce travail met l’accent sur le double rôle de l’élongateur cytosolique ARNtMet en tant qu’initiateur et élongateur dans un système de traduction mitochondriale (Résultats A). Pour être fonctionnel pendant la traduction, chaque ARNt doit être attaché à son acide aminé. Le processus d’attachement, appelé aminoacylation, est catalisé par des aminoacyl-ARNt synthétases. Etant donné que chez T. brucei les ARNts cytosoliques et importés proviennent du même ensemble de gènes nucléaires, on s’attend à ce qu’ils soient aminoacylés par les mêmes enzymes. Le fait que la plupart des aminoacyl-ARNt synthétases du trypanosome sont représentées par des gènes uniques supporte cette hypothèse. Cependant, le génome du trypanosome contient deux différents gènes codant pour deux tryptophanyl-tRNA synthétases de type eucaryote. Nous montrons dans ce travail que ces deux enzymes sont essentielles pour une croissance normale. De plus nous démontrons que la nécessité d’une deuxième tryptophanyl-tRNA synthétase chez T. brucei est due à l’édition intra-mitochondriale de l’ARNtTrp (tRNA editing) requise pour le ré-assignement du codon UGA en tryptophane (Résultats B). Dans la partie C des résultats, certains composants impliqués dans l’insertion de la sérine et de la sélénocystéine dans les protéines ont été caractérisés. Dans le contexte de l’import mitochondrial des ARNts nous avons montré que l’ARNtSec est le second ARNt localisé exclusivement dans le cytosol du trypanosome. Dans le but d’examiner le rapport entre l’import mitochondrial des ARNts et des protéines chez T. brucei, nous avons utilisé la technique de l’ARN interférence (RNAi) en vue de réduire sensiblement l’expression des homologues de Tim17-22 et Tim8-13. Ces protéines sont connues pour faire partie de l’appareil de translocation des protéines mitochondriales chez d’autres organismes. Les effets morphologiques causés par leur ablation sont présentés dans la partie D des résultats.The protozoon parasite Trypanosoma brucei is the causing agent of human sleeping sickness. Besides its clinical importance T. brucei is also an excellent model for basic research since it has many unique features. The mitochondrion of T. brucei, for example, lacks tRNA genes. The tRNAs required for mitochondrial translation are therefore encoded in the nucleus and imported from the cytosol [1]. Thus, except for the initiator tRNAMet and tRNASec, all trypanosomal tRNAs function in both the cytosol and the mitochondrion. An important consequence of mitochondrial tRNA import is that the imported tRNAs are of eukaryotic evolutionary origin. The mitochondrion however derives from a bacterial ancestor. Thus, we wanted to investigate how the bacterial-type translation system of the mitochondrion has adapted to eukaryotic-type tRNAs during evolution. Due to the exclusive cytosolic localization of the trypanosomal initiator tRNAMet, the only tRNAMet present in the mitochondrion of T. brucei is the imported eukaryotic elongator tRNAMet. In bacteria and organelles, the translation initiation process requires the specific formylation of the initiator methionyl-tRNAMet by the methionyl-tRNA formyltransferase (MTF). The subsequent binding of the resulting initiator formylmethionyl-tRNAMet to the bacterial-type initiation factor 2 (IF2) promotes the interaction of the tRNA with the ribosome. In the mitochondrion of T. brucei a fraction of the imported elongator methionyl-tRNAMet is unexpectedly formylated by an extraordinary large MTF orthologue [2]. In the present work we identified the trypanosomal IF2 and we demonstrated that it is required for normal growth of the parasite. Furthermore, we showed that it recognizes the formylmethionylated imported elongator tRNAMet, but not its unformylated counterpart. Hence, together with previous studies [1, 2], this work emphasizes the dual use of a cytosolic elongator tRNAMet as initiator and elongator in a mitochondrial translation system (Results A). In order to be used in translation each tRNA needs to be attached to its cognate amino acid. The process of attachment is called aminoacylation and is catalyzed by aminoacyl-tRNA synthetases. Since cytosolic and imported tRNAs of T. brucei derive from the same set of nuclear genes they are expected to be aminoacylated by the same enzymes. In agreement with this hypothesis, most trypanosomal aminoacyl-tRNA synthetases are represented by single genes. Interestingly however, the T. brucei genome contains two different genes for eukaryotic tryptophanyl-tRNA synthetases. We show in this work that both of these enzymes are essential for normal growth. Furthermore we demonstrate that the unexpected use of a second tryptophanyl-tRNA synthetase in T. brucei is caused by a mitochondria-specific editing event of the tRNATrp which is required for the mitochondrial reassignment of the UGA codon to tryptophan (Results B). In the part C of the results section some components involved in the insertion of serine and selenocysteine into trypanosomal proteins were characterized. In the context of the mitochondrial tRNA import it was shown that tRNASec is the second cytosol-specific tRNA in T. brucei. In order to understand the connection between the mitochondrial tRNA import and protein translocation in T. brucei we used the RNA interference (RNAi) strategy to knock down the expression of the Tim17-22 and Tim8-13 homologues. These proteins are known to be components of the mitochondrial protein translocation machinery in other organisms. Morphological effects caused by their depletion are presented in the part D of the results

Hydroxyl radical-induced photochemical formation of dicarboxylic acids from unsaturated fatty acid (oleic acid) in aqueous solution

In this study, we assess under laboratory controlled conditions the direct and hydroxyl radical (OH)-induced photochemical production of low molecular weight (LMW) dicarboxylic acids and related compounds (C2–C9) (DCAs) from oleic acid (cis-9-octadecenoic, Δ9C18) in aqueous solution. Nitrate (NO3−)-amended and unamended oleate solutions were irradiated under ultraviolet-B radiation (UV-B, 313 nm) for 5 h, with NO3− being the source of OH. The OH-induced photochemical production of DCAs (C2di–C9di) (170 ± 26 nM h−1) was much higher than that induced by the direct effect of UV-B (33 ± 22 nM h−1), accounting for approximately 85% of the total (direct + OH-induced) photochemical production of DCAs (C2di–C9di) (198 ± 15 nM h−1). Azelaic acid (C9di) was the dominant photoproduct (comprising 63 and 44% of DCAs in the direct and OH-induced photochemical production, respectively) followed by C8di, C7di and C6di, whereas shorter chain compounds (C2di–C5di) were minor produced species. Using our estimate of OH photoproduction (P-OH in nM h−1), the production of C9di from 50 μM of oleic acid was evaluated at 45 nM (nM OH)−1

In vivo study in Trypanosoma brucei links mitochondrial transfer RNA import to mitochondrial protein import

Trypanosoma brucei imports all mitochondrial transfer RNAs (tRNAs) from the cytosol. By using cell lines that allow independent tetracycline-inducible RNA interference and isopropyl-β-D-thiogalactopyranoside-inducible expression of a tagged tRNA, we show that ablation of Tim17 and mitochondrial heat-shock protein 70, components of the inner-membrane protein translocation machinery, strongly inhibits import of newly synthesized tRNAs. These findings, together with previous results in yeast and plants, suggest that the requirement for mitochondrial protein-import factors might be a conserved feature of mitochondrial tRNA import in all systems

Trypanosoma seryl-tRNA synthetase Is a metazoan-like enzyme with high affinity for tRNA^Sec

Trypanosomatids are important human pathogens that form a basal branch of eukaryotes. Their evolutionary history is still unclear as are many aspects of their molecular biology. Here we characterize essential components required for the incorporation of serine and selenocysteine into the proteome of Trypanosoma. First, the biological function of a putative Trypanosoma seryl-tRNA synthetase was characterized in vivo. Secondly, the molecular recognition by Trypanosoma seryl-tRNA synthetase of its cognate tRNAs was dissected in vitro. The cellular distribution of tRNASec was studied, and the catalytic constants of its aminoacylation were determined. These were found to be markedly different from those reported in other organisms, indicating that this reaction is particularly efficient in trypanosomatids. Our functional data were analyzed in the context of a new phylogenetic analysis of eukaryotic seryl-tRNA synthetases that includes Trypanosoma and Leishmania sequences. Our results show that trypanosomatid seryl-tRNA synthetases are functionally and evolutionarily more closely related to their metazoan homologous enzymes than to other eukaryotic enzymes. This conclusion is supported by sequence synapomorphies that clearly connect metazoan and trypanosomatid seryl-tRNA synthetases