Crystal structure of Saccharomyces cerevisiae mitochondrial GatFAB reveals a novel subunit assembly in tRNA-dependent amidotransferases

Yeast mitochondrial Gln-mtRNAGln is synthesized by the transamidation of mischarged Glu-mtRNAGln by a non-canonical heterotrimeric tRNA-dependent amidotransferase (AdT). The GatA and GatB subunits of the yeast AdT (GatFAB) are well conserved among bacteria and eukaryota, but the GatF subunit is a fungi-specific ortholog of the GatC subunit found in all other known heterotrimeric AdTs (GatCAB). Here we report the crystal structure of yeast mitochondrial GatFAB at 2.0 Å resolution. The C-terminal region of GatF encircles the GatA-GatB interface in the same manner as GatC, but the N-terminal extension domain (NTD) of GatF forms several additional hydrophobic and hydrophilic interactions with GatA. NTD-deletion mutants displayed growth defects, but retained the ability to respire. Truncation of the NTD in purified mutants reduced glutaminase and transamidase activities when glutamine was used as the ammonia donor, but increased transamidase activity relative to the full-length enzyme when the donor was ammonium chloride. Our structure-based functional analyses suggest the NTD is a trans-acting scaffolding peptide for the GatA glutaminase active site. The positive surface charge and novel fold of the GatF-GatA interface, shown in this first crystal structure of an organellar AdT, stand in contrast with the more conventional, negatively charged bacterial AdTs described previousl

The asparagine-transamidosome from Helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-tRNA synthetase safeguards the genetic code

Helicobacter pylori catalyzes Asn-tRNAAsn formation by use of the indirect pathway that involves charging of Asp onto tRNAAsn by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS), followed by conversion of the mischarged Asp into Asn by the GatCAB amidotransferase. We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate. The complex is described by gel-filtration, dynamic light scattering and kinetic measurements. Two strategies for asparaginylation are shown: (i) tRNAAsn binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNAAsn is bound by ND-AspRS which releases the Asp-tRNAAsn product much slower than the cognate Asp-tRNAAsp; this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNAAsn before its release by the ND-AspRS. These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn

Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation

The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNAGln. The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNAGlu and Glu-tRNAGln. The Glu-tRNAGln is then converted to Gln-tRNAGln by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNAGlu and tRNAGln with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNAGlu/tRNAGln discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNAGln complex reveals the structural determinants responsible for specific tRNAGln recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine

Unterwerfungsverhältnisse, autorität, besessenheit und gehorsamkeit im roman von Bernhard Schlink <i>Der Vorleser</i> (1995) und in Stephen Daldrys Verfilmung <i>The reader</i> (2008)

Der Titel meiner Abschlußarbeit lautet: Unterwerfungsverhältnisse, Autorität, Besessenheit und Gehorsamkeit im Roman Der Vorleser (1995) von Bernhard Schlink und im Film The Reader von Stephen Daldry. Der Roman umfaßt zahlreiche Themen: den Umgang der verschiedenen Generationen mit der deutschen nationalsozialistischen Vergangenheit, bzw. den Holocaust, die Verurteilung und die Haft der Angeklagten, die Prägung der ersten Liebe, das Erwachsenwerden, die Bedeutung von Lesen und Schreiben sowie den Analphabetismus. Dieser Roman wurde nämlich zum Best-Seller und in ca. dreißig Sprachen übersetzt. Der Vorleser ist jedoch kein Roman über die Shoah, sondern ein Generationsroman über das Verhältnis zwischen den deutschen Neugeborenen und den Eltern, die sich NS-Verbrechen schuldig oder mitschuldig gemacht haben

Identification et rôles des partenaires de la voie de transamidation de la mitochondrie de Saccharomyces cerevisiae dans l'adaptation à la respiration

La formation du glutaminyl (Q)-tRNAQ cytoplasmique (c) permettant l insertion de Q dans les protéines lors de la traduction ribosomale se fait généralement par aminoacylation directe de l ARNt par une Q-ARNt synthétase (QRS). Cependant les mécanismes de synthèse du QtRNAQ mitochondrial (m) requis pour le bon fonctionnement de la machinerie traductionnelle organellaire ne sont toujours pas bien caractérisés. En effet, aucune mQRS n a été retrouvée dans les génomes eucaryotiques séquencés jusqu à ce jour. Ainsi, il est impossible de prédire quelle voie génère cet aminoacyl (aa)-ARNt dans un eucaryote donné. Les eucaryotes ont à priori deux possibilités pour générer du Q-mtRNAQ: soit utiliser la voie directe via l import de cQRS, ou alors utiliser une voie ARNt-dépendante de transamidation, ce qui requiert dans ce cas la présence d une ERS non discriminante (nd) et d une amidotransférase ARNt-dépendante (AdT) dans la mitochondrie. Nous avons montré que la protéine Pet112 fait partie d une amidotransférase, mais également que la ndERS essentielle à la voie est l ERS du cytoplasme qui est capable de se relocaliser dans la mitochondrie. La double localisation de la nd-cERS est contrôlée par Arc1p, son partenaire cytoplasmique. Ces résultats représentent une avancée intéressante dans le domaine de l aminoacylation et de l import mitochondrial. En effet nous décrivons une nouvelle stratégie : l utilisation d une plateforme d ancrage cytoplasmique afin de réguler la capacité de double-localisation d un seul et même produit traductionnel, suggérant que toute protéine dans un complexe pourrait être capable d atteindre d autres compartiments une fois relâchées. Nous avons alors montré que la transcription d ARC1 est contrôlée par la voie de signalisation Snf1/4 qui induit la diminution de la quantité d Arc1p lors de l adaptation à la respiration. Cependant ses deux partenaires, la cERS et la cMRS, restent exprimées de manière stable ce qui induit une augmentation de la quantité de leur forme libre. Les cMRS et cERS libres sont alors capables d être importées dans le noyau et la mitochondrie respectivement, dans le but de synchroniser l expression des partenaires de la chaîne respiratoire (RC). En effet, les partenaires de la RC sont encodés de manière séparée dans le noyau et la mitochondrie, la cMRS promeut la transcription d une partie des gènes de la RC qui sont encodés dans le noyau, alors que la cERS augmente le taux de traduction des partenaires de la RC produits dans la mitochondrie. En prouvant qu Arc1p est un relai essentiel pour la voie Snf1/4 et l adaptation à la respiration, nous décrivons pour la première fois un mécanisme de synchronisation de la chaine respiratoire. C est ce concept qui semble être le point le plus important de mon travail, nous montrons les avantages que représente l utilisation d un complexe protéique en tant qu élément synchroniseur de l expression de gènes présents dans différents compartiments. Nous pouvons en effet penser que ce mécanisme est utilisé pour beaucoup d autres fonctions où la synchronisation des différents acteurs est essentielle.The formation of cytoplasmic (c) glutaminyl (Q)-tRNAQ allowing insertion of Q into proteins during ribosome-mediated translation proceeds via direct tRNA aminoacylation by a specific Q-tRNA synthetase (QRS). However, the synthesis of mitochondrial (m) Q-tRNAQ required for the specific organellar translation system is still matter of debate. In fact, no mQRS can be found in any eukaryotic genomes sequenced so far. Thus, it is almost impossible to predict which pathway, direct or indirect, generates this organellar aminoacyl (aa)-tRNA species in a given eukaryote. Eukaryotes have, a priori, two possibilities to generate a Q-mtRNAQ: either they use the direct pathway via the import the cQRS or they use an indirect tRNA-dependent transamidation pathway which implies the presence of a non discriminating (nd) ERS and of a tRNA-dependent amidotransferase (AdT) in the organelle. We have shown that Pet112 is a part of a yeast mitochondrial amidotransferase, but also that the necessary ndERS is the cytoplasmic form of ERS (nd-cERS) which is able to be localized both in the cytoplasm and the mitochondrion. The dual localization of the nd-cERS is controlled by Arc1p, the cytoplasmic partner of the nd-cERS. This project represents an important breakthrough in the fields of aminoacylation and mitochondrial import. We describe a new strategy: the use of a cytosolic anchoring platform, for the dual localization of a single translational product, suggesting that any protein in a complex, even if well characterized in a specific subcellular compartment, might be able to reach other compartments upon release from the complex. We then show that ARC1 transcription is controlled by the Snf1/4 pathway that decreases Arc1p upon adaptation to respiration. However, its two partners, cERS and cMRS, stay stably expressed leading to an increase of the free cMRS and cERS pools. These released forms are then imported in the nucleus and the mitochondria respectively, in order to synchronize expression of respiratory chain (RC) partners. RC partners are encoded in a split manner in the nucleus and the mitochondrion, cMRS promotes transcription of a subset of the RC genes encoded by the nucleus, whereas cERS increase the translation rate of mitochondrial-encoded partners of the RC. By proving that Arc1p is an essential relay for the Snf1/4 pathway we propose for the first time a mechanism explaining how synchronization of the RC gene expression is achieved. This represents the most important conceptual change we made, in which we show the advantages of the dynamic control of a protein complex as a strategy to synchronize gene expression of genomes located in different compartments.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

A new Speyeria from Washington

Volume: 23Start Page: 111End Page: 11

Misacylation of tRNA with methionine in Saccharomyces cerevisiae

Accurate transfer RNA (tRNA) aminoacylation by aminoacyl-tRNA synthetases controls translational fidelity. Although tRNA synthetases are generally highly accurate, recent results show that the methionyl-tRNA synthetase (MetRS) is an exception. MetRS readily misacylates non-methionyl tRNAs at frequencies of up to 10% in mammalian cells; such mismethionylation may serve a beneficial role for cells to protect their own proteins against oxidative damage. The Escherichia coli MetRS mismethionylates two E. coli tRNA species in vitro, and these two tRNAs contain identity elements for mismethionylation. Here we investigate tRNA mismethionylation in Saccharomyces cerevisiae. tRNA mismethionylation occurs at a similar extent in vivo as in mammalian cells. Both cognate and mismethionylated tRNAs have similar turnover kinetics upon cycloheximide treatment. We identify specific arginine/lysine to methionine-substituted peptides in proteomic mass spectrometry, indicating that mismethionylated tRNAs are used in translation. The yeast MetRS is part of a complex containing the anchoring protein Arc1p and the glutamyl-tRNA synthetase (GluRS). The recombinant Arc1p-MetRS-GluRS complex binds and mismethionylates many tRNA species in vitro. Our results indicate that the yeast MetRS is responsible for extensive misacylation of non-methionyl tRNAs, and mismethionylation also occurs in this evolutionary branc

Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux

Holo-tomographic microscopy (HTM) is a label-free microscopy method reporting the fine changes of a cell’s refractive indices (RIs) in three dimensions at high spatial and temporal resolution. By combining HTM with epifluorescence, we demonstrate that mammalian cellular organelles such as lipid droplets (LDs) and mitochondria show specific RI 3D patterns. To go further, we developed a computer-vision strategy using FIJI, CellProfiler3 (CP3), and custom code that allows us to use the fine images obtained by HTM in quantitative approaches. We could observe the shape and dry mass dynamics of LDs, endocytic structures, and entire cells’ division that have so far, to the best of our knowledge, been out of reach. We finally took advantage of the capacity of HTM to capture the motion of many organelles at the same time to report a multiorganelle spinning phenomenon and study its dynamic properties using pattern matching and homography analysis. This work demonstrates that HTM gives access to an uncharted field of biological dynamics and describes a unique set of simple computer-vision strategies that can be broadly used to quantify HTM images