Lipid Trafficking at Membrane Contact Sites During Plant Development and Stress Response

The biogenesis of cellular membranes involves an important traffic of lipids from their site of synthesis to their final destination. Lipid transfer can be mediated by vesicular or non-vesicular pathways. The non-vesicular pathway requires the close apposition of two membranes to form a functional platform, called membrane contact sites (MCSs), where lipids are exchanged. These last decades, MCSs have been observed between virtually all organelles and a role in lipid transfer has been demonstrated for some of them. In plants, the lipid composition of membranes is highly dynamic and can be drastically modified in response to environmental changes. This highlights the importance of understanding the mechanisms involved in the regulation of membrane lipid homeostasis in plants. This review summarizes our current knowledge about the non-vesicular transport of lipids at MCSs in plants and its regulation during stress

PlantRNA, a database for tRNAs of photosynthetic eukaryotes.

International audiencePlantRNA database (http://plantrna.ibmp.cnrs.fr/) compiles transfer RNA (tRNA) gene sequences retrieved from fully annotated plant nuclear, plastidial and mitochondrial genomes. The set of annotated tRNA gene sequences has been manually curated for maximum quality and confidence. The novelty of this database resides in the inclusion of biological information relevant to the function of all the tRNAs entered in the library. This includes 5'- and 3'-flanking sequences, A and B box sequences, region of transcription initiation and poly(T) transcription termination stretches, tRNA intron sequences, aminoacyl-tRNA synthetases and enzymes responsible for tRNA maturation and modification. Finally, data on mitochondrial import of nuclear-encoded tRNAs as well as the bibliome for the respective tRNAs and tRNA-binding proteins are also included. The current annotation concerns complete genomes from 11 organisms: five flowering plants (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Medicago truncatula and Brachypodium distachyon), a moss (Physcomitrella patens), two green algae (Chlamydomonas reinhardtii and Ostreococcus tauri), one glaucophyte (Cyanophora paradoxa), one brown alga (Ectocarpus siliculosus) and a pennate diatom (Phaeodactylum tricornutum). The database will be regularly updated and implemented with new plant genome annotations so as to provide extensive information on tRNA biology to the research community

Cytosolic mRNA targeting to plant mitochondria

La biogénèse des mitochondries est un processus qui implique l’importation de plus de 98% des protéines constitutives de ces organites. Les signaux protéiques impliqués dans l’importation de ces protéines dans les mitochondries sont relativement bien caractérisés. Il y a une dizaine d’année, il a été montré chez la levure et les mammifères que d’autres signaux, présents au niveau des ARNm étaient également impliqués dans l’adressage et l’importation des protéines dans les mitochondries. Ce processus d’adressage d’ARNm à la surface des mitochondries s’est montré fondamental pour la biogénèse et la fonction des mitochondries chez la levure. Au cours de cette thèse, nous avons démontré que des ARNm étaient adressés à la surface des mitochondries chez trois espèces végétales. Par la combinaison d’approches in vitro et in vivo, nous avons également identifié des éléments cis permettant l’adressage d’ARNm à la surface des mitochondries à partir d’un messager candidat : AtVDAC3. Ces éléments cis sont localisés dans une séquence de 142 nt présente dans la région 3’UTR du messager AtVDAC3. Le rôle de ce processus chez les plantes est actuellement en cours d’étude.Mitochondria biogenesis requires the import of more than 98 % of their constitutive proteins. Proteic signals involved in mitochondrial protein import are well known today. Ten years ago, it was shown in yeast and mammals that targeting signals are also present at the level of mRNAs. Cytosolic mRNA targeting to mitochondria is an extended process concerning half of the mRNAs encoded mitochondrial proteins in yeast. Furthermore, this process is fundamental for yeast mitochondria biogenesis and functions. During this PhD, we showed that some mRNAs are also targeting to mitochondria in three different plant species. These results highlighted the conservation of this process during evolution. By using in vivo and in vitro experimental strategies, we also identified a mitochondrial cis-targeting element in one candidate mRNA: AtVDAC3. This cis-element is 142 nt long and is located in the 3’UTR of the AtVDAC3 mRNA. We are now investigating the roles of this process in mitochondria biogenesis and functions in plants

Cytosolic mRNA targeting to plant mitochondria

La biogénèse des mitochondries est un processus qui implique l’importation de plus de 98% des protéines constitutives de ces organites. Les signaux protéiques impliqués dans l’importation de ces protéines dans les mitochondries sont relativement bien caractérisés. Il y a une dizaine d’année, il a été montré chez la levure et les mammifères que d’autres signaux, présents au niveau des ARNm étaient également impliqués dans l’adressage et l’importation des protéines dans les mitochondries. Ce processus d’adressage d’ARNm à la surface des mitochondries s’est montré fondamental pour la biogénèse et la fonction des mitochondries chez la levure. Au cours de cette thèse, nous avons démontré que des ARNm étaient adressés à la surface des mitochondries chez trois espèces végétales. Par la combinaison d’approches in vitro et in vivo, nous avons également identifié des éléments cis permettant l’adressage d’ARNm à la surface des mitochondries à partir d’un messager candidat : AtVDAC3. Ces éléments cis sont localisés dans une séquence de 142 nt présente dans la région 3’UTR du messager AtVDAC3. Le rôle de ce processus chez les plantes est actuellement en cours d’étude.Mitochondria biogenesis requires the import of more than 98 % of their constitutive proteins. Proteic signals involved in mitochondrial protein import are well known today. Ten years ago, it was shown in yeast and mammals that targeting signals are also present at the level of mRNAs. Cytosolic mRNA targeting to mitochondria is an extended process concerning half of the mRNAs encoded mitochondrial proteins in yeast. Furthermore, this process is fundamental for yeast mitochondria biogenesis and functions. During this PhD, we showed that some mRNAs are also targeting to mitochondria in three different plant species. These results highlighted the conservation of this process during evolution. By using in vivo and in vitro experimental strategies, we also identified a mitochondrial cis-targeting element in one candidate mRNA: AtVDAC3. This cis-element is 142 nt long and is located in the 3’UTR of the AtVDAC3 mRNA. We are now investigating the roles of this process in mitochondria biogenesis and functions in plants

Analysis of the MTL Supercomplex at Contact Sites Between Mitochondria and Plastids

International audiencePlastids are organelles playing fundamental roles in different cellular processes such as energy metabolism or lipid biosynthesis. To fulfill their biogenesis and their function in the cell, plastids have to communicate with other cellular compartments. This communication can be mediated by the establishment of direct contact sites between plastids envelop and other organelles. These contacts are dynamic structures that are modified in response to stress. As example, during phosphate (Pi) starvation, the number of contact sites between plastids and mitochondria significantly increases. In this situation, these contacts play an important role in the transfer of galactoglycerolipids from plastids to mitochondria. Recently, Pi starvation stress was used to identify key proteins involved in the traffic of galactoglycerolipids from plastids to mitochondria in Arabidopsis thaliana. A mitochondrial lipoprotein complex called MTL (mitochondrial transmembrane lipoprotein complex) was identified. This complex contains mitochondrial proteins but also proteins located in the plastid envelope, suggesting its presence at the plastid-mitochondria junction. This chapter describes the protocol to isolate the MTL complex by clear-native polyacrylamide gel electrophoresis (CN-PAGE) from the mitochondrial fraction of Arabidopsis cell cultures and the methods to study different features of this complex

Mitochondrial membrane biogenesis: A new pathway for lipid transport mediated by PERK/E-Syt1 complex

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Non-vesicular glycerolipids transport in plant cells

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Adressage des ARNm cytosoliques à la surface des mitochondries végétales

La biogénèse des mitochondries est un processus qui implique l importation de plus de 98% des protéines constitutives de ces organites. Les signaux protéiques impliqués dans l importation de ces protéines dans les mitochondries sont relativement bien caractérisés. Il y a une dizaine d année, il a été montré chez la levure et les mammifères que d autres signaux, présents au niveau des ARNm étaient également impliqués dans l adressage et l importation des protéines dans les mitochondries. Ce processus d adressage d ARNm à la surface des mitochondries s est montré fondamental pour la biogénèse et la fonction des mitochondries chez la levure. Au cours de cette thèse, nous avons démontré que des ARNm étaient adressés à la surface des mitochondries chez trois espèces végétales. Par la combinaison d approches in vitro et in vivo, nous avons également identifié des éléments cis permettant l adressage d ARNm à la surface des mitochondries à partir d un messager candidat : AtVDAC3. Ces éléments cis sont localisés dans une séquence de 142 nt présente dans la région 3 UTR du messager AtVDAC3. Le rôle de ce processus chez les plantes est actuellement en cours d étude.Mitochondria biogenesis requires the import of more than 98 % of their constitutive proteins. Proteic signals involved in mitochondrial protein import are well known today. Ten years ago, it was shown in yeast and mammals that targeting signals are also present at the level of mRNAs. Cytosolic mRNA targeting to mitochondria is an extended process concerning half of the mRNAs encoded mitochondrial proteins in yeast. Furthermore, this process is fundamental for yeast mitochondria biogenesis and functions. During this PhD, we showed that some mRNAs are also targeting to mitochondria in three different plant species. These results highlighted the conservation of this process during evolution. By using in vivo and in vitro experimental strategies, we also identified a mitochondrial cis-targeting element in one candidate mRNA: AtVDAC3. This cis-element is 142 nt long and is located in the 3 UTR of the AtVDAC3 mRNA. We are now investigating the roles of this process in mitochondria biogenesis and functions in plants.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

Measurement of Lipid Transport in Mitochondria by the MTL Complex

International audienceMembrane biogenesis requires an extensive traffic of lipids between different cell compartments. Two main pathways, the vesicular and non-vesicular pathways, are involved in such a process. Whereas the mechanisms involved in vesicular trafficking are well understood, fewer is known about non-vesicular lipid trafficking, particularly in plants. This pathway involves the direct exchange of lipids at membrane contact sites (MCSs) between organelles. In plants, an extensive traffic of the chloroplast-synthesized digalactosyldiacylglycerol (DGDG) to mitochondria occurs during phosphate starvation. This lipid exchange occurs by non-vesicular trafficking pathways at MCSs between mitochondria and plastids. By a biochemical approach, a mitochondrial lipoprotein super-complex called MTL (Mitochondrial Transmembrane Lipoprotein complex) involved in mitochondria lipid trafficking has been identified in Arabidopsis thaliana. This protocol describes the method to isolate the MTL complex and to study the implication of a component of this complex (AtMic60) in mitochondria lipid trafficking

Isolation of mitochondria for lipid analysis

International audienceDiverse classes of lipids are found in cell membranes, the major ones being glycerolipids, sphingolipids, and sterols. In eukaryotic cells, each organelle has a specific lipid composition, which defines its identity and regulates its biogenesis and function. For example, glycerolipids are present in all membranes, whereas sphingolipids and sterols are mostly enriched in the plasma membrane. In addition to phosphoglycerolipids, plants also contain galactoglycerolipids, a family of glycerolipids present mainly in chloroplasts and playing an important role in photosynthesis. During phosphate starvation, galactoglycerolipids are also found in large amounts in other organelles, illustrating the dynamic nature of membrane lipid composition. Thus, it is important to determine the lipid composition of each organelle, as analyses performed on total cells do not represent the specific changes occurring at the organelle level. This task requires the optimization of standard protocols to isolate organelles with high yield and low contamination by other cellular fractions. In this chapter, we describe a protocol to isolate mitochondria from Arabidopsis thaliana cell cultures to perform lipidomic analysis