Marianne Tardif

A Proteomic Survey of Chlamydomonas reinhardtii Mitochondria Sheds New Light on the Metabolic Plasticity of the Organelle and on the Nature of the α-Proteobacterial Mitochondrial Ancestor

Mitochondria play a key role in the life and death of eukaryotic cells, yet the full spectrum of mitochondrial functions is far from being fully understood, especially in photosynthetic organisms. To advance our understanding of mitochondrial functions in a photosynthetic cell, an extensive proteomic survey of Percoll-purified mitochondria from the metabolically versatile, hydrogen-producing green alga Chlamydomonas reinhardtii was performed. Different fractions of purified mitochondria from Chlamydomonas cells grown under aerobic conditions were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry after protein separation on sodium dodecyl sulfate polyacrylamide gel electrophoresis or on blue-native polyacrylamide gel electrophoresis. Of the 496 nonredundant proteins identified, 149 are known or predicted to reside in other cellular compartments and were thus excluded from the molecular and evolutionary analyses of the Chlamydomonas proteome. The mitochondrial proteome of the photosynthetic alga reveals important lineage-specific differences with other mitochondrial proteomes, reflecting the high metabolic diversity of the organelle. Some mitochondrial metabolic pathways in Chlamydomonas appear to combine typical mitochondrial enzymes and bacterial-type ones, whereas others are unknown among mitochondriate eukaryotes. The comparison of the Chlamydomonas proteins to their identifiable homologs predicted from 354 sequenced genomes indicated that Arabidopsis is the most closely related nonalgal eukaryote. Furthermore, this phylogenomic analysis shows that free-living alpha-proteobacteria from the metabolically versatile orders Rhizobiales and Rhodobacterales better reflect the gene content of the ancestor of the chlorophyte mitochondria than parasitic alpha-proteobacteria with reduced and specialized genomes.

PredAlgo, a new subcellular localization prediction tool dedicated to green algae

The unicellular green alga Chlamydomonas reinhardtii is a prime model for deciphering processes occurring in the intracellular compartments of the photosynthetic cell. Organelle-specific proteomic studies have started to delineate its various subproteomes, but sequence-based prediction software is necessary to assign proteins subcellular localizations at whole genome scale. Unfortunately, existing tools are oriented toward land plants and tend to mispredict the localization of nuclear-encoded algal proteins, predicting many chloroplast proteins as mitochondrion targeted. We thus developed a new tool called PredAlgo that predicts intracellular localization of those proteins to one of three intracellular compartments in green algae: the mitochondrion, the chloroplast, and the secretory pathway. At its core, a neural network, trained using carefully curated sets of C. reinhardtii proteins, divides the N-terminal sequence into overlapping 19-residue windows and scores the probability that they belong to a cleavable targeting sequence for one of the aforementioned organelles. A targeting prediction is then deduced for the protein, and a likely cleavage site is predicted based on the shape of the scoring function along the N-terminal sequence. When assessed on an independent benchmarking set of C. reinhardtii sequences, PredAlgo showed a highly improved discrimination capacity between chloroplast- and mitochondrion-localized proteins. Its predictions matched well the results of chloroplast proteomics studies. When tested on other green algae, it gave good results with Chlorophyceae and Trebouxiophyceae but tended to underpredict mitochondrial proteins in Prasinophyceae. Approximately 18% of the nuclear-encoded C. reinhardtii proteome was predicted to be targeted to the chloroplast and 15% to the mitochondrion.

AtMic60 Is Involved in Plant Mitochondria Lipid Trafficking and Is Part of a Large Complex

The mitochondrion is an organelle originating from an endosymbiotic event and playing a role in several fundamental processes such as energy production, metabolite syntheses, and programmed cell death. This organelle is delineated by two membranes whose synthesis requires an extensive exchange of phospholipids with other cellular organelles such as endoplasmic reticulum (ER) and vacuolar membranes in yeast. These transfers of phospholipids are thought to occur by a non-vesicular pathway at contact sites between two closely apposed membranes. In plants, little is known about the biogenesis of mitochondrial membranes. Contact sites between ER and mitochondria are suspected to play a similar role in phospholipid trafficking as in yeast, but this has never been demonstrated. In contrast, it has been shown that plastids are able to transfer lipids to mitochondria during phosphate starvation. However, the proteins involved in such transfer are still unknown. Here, we identified in Arabidopsis thaliana a large lipid-enriched complex called the mitochondrial transmembrane lipoprotein (MTL) complex. The MTL complex contains proteins located in the two mitochondrial membranes and conserved in all eukaryotic cells, such as the TOM complex and AtMic60, a component of the MICOS complex. We demonstrate that AtMic60 contributes to the export of phosphatidylethanolamine from mitochondria and the import of galactoglycerolipids from plastids during phosphate starvation. Furthermore, AtMic60 promotes lipid desorption from membranes, likely as an initial step for lipid transfer, and binds to Tom40, suggesting that AtMic60 could regulate the tethering between the inner and outer membranes of mitochondria.

Characterization of Chloroplastic Fructose 1,6-Bisphosphate Aldolases as Lysine-methylated Proteins in Plants.

Background: The protein-lysine methyltransferase LSMT from pea methylates the large subunit of Rubisco. Results: In Arabidopsis, Rubisco is not methylated, and the physiological substrates of the LSMT-like enzyme are chloroplastic aldolases. Conclusion: LSMT homologs from plants display different substrate specificities, with targets involved in carbon metabolism. Significance: The study identifies chloroplastic aldolases as new lysine-methylated proteins. In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO2 fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO2 through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts.

Uncovering the Protein Lysine and Arginine Methylation Network in Arabidopsis Chloroplasts

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology.

Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii

Saturating light induces oil storage in Chlamydomonas reinhardtii, and the newly formed oil accumulates in lipid droplets distinct in protein and lipid compositions from those induced by nitrogen starvation. Enriching algal biomass in energy density is an important goal in algal biotechnology. Nitrogen (N) starvation is considered the most potent trigger of oil accumulation in microalgae and has been thoroughly investigated. However, N starvation causes the slow down and eventually the arrest of biomass growth. In this study, we show that exposing a Chlamydomonas reinhardtii culture to saturating light (SL) under a nonlimiting CO2 concentration in turbidostatic photobioreactors induces a sustained accumulation of lipid droplets (LDs) without compromising growth, which results in much higher oil productivity than N starvation. We also show that the polar membrane lipid fraction of SL-induced LDs is rich in plastidial lipids (approximately 70%), in contrast to N starvation-induced LDs, which contain approximately 60% lipids of endoplasmic reticulum origin. Proteomic analysis of LDs isolated from SL-exposed cells identified more than 200 proteins, including known proteins of lipid metabolism, as well as 74 proteins uniquely present in SL-induced LDs. LDs induced by SL and N depletion thus differ in protein and lipid contents. Taken together, lipidomic and proteomic data thus show that a large part of the sustained oil accumulation occurring under SL is likely due to the formation of plastidial LDs. We discuss our data in relation to the different metabolic routes used by microalgae to accumulate oil reserves depending on cultivation conditions. Finally, we propose a model in which oil accumulation is governed by an imbalance between photosynthesis and growth, which can be achieved by impairing growth or by boosting photosynthetic carbon fixation, with the latter resulting in higher oil productivity.

Dual Targeting of the Protein Methyltransferase PrmA Contributes to Both Chloroplastic and Mitochondrial Ribosomal Protein L11 Methylation in Arabidopsis

Methylation of ribosomal proteins has long been described in prokaryotes and eukaryotes, but our knowledge about the enzymes responsible for these modifications in plants is scarce. The bacterial protein methyltransferase PrmA catalyzes the trimethylation of ribosomal protein L11 (RPL11) at three distinct sites. The role of these modifications is still unknown. Here, we show that PrmA from Arabidopsis thaliana (AtPrmA) is dually targeted to chloroplasts and mitochondria. Mass spectrometry and enzymatic assays indicated that the enzyme methylates RPL11 in plasto- and mitoribosomes in vivo. We determined that the Arabidopsis and Escherichia coli PrmA enzymes share similar product specificity, making trimethylated residues, but, despite an evolutionary relationship, display a difference in substrate site specificity. In contrast to the bacterial enzyme that trimethylates the ε-amino group of two lysine residues and the N-terminal α-amino group, AtPrmA methylates only one lysine in the MAFCK(D/E)(F/Y)NA motif of plastidial and mitochondrial RPL11. The plant enzyme possibly methylates the N-terminus of plastidial RPL11, whereas mitochondrial RPL11 is N-α-acetylated by an unknown acetyltransferase. Lastly, we found that an Arabidopsis prma-null mutant is viable in standard environmental conditions and no molecular defect could be associated with a lack of RPL11 methylation in leaf chloroplasts or mitochondria. However, the conservation of PrmA during the evolution of photosynthetic eukaryotes together with the location of methylated residues at the binding site of translation factors to ribosomes suggests that RPL11 methylation in plant organelles could be involved, in combination with other post-translational modifications, in optimizing ribosome function.

Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants

Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity.

ChloroKB: A Web Application for the Integration of Knowledge Related to Chloroplast Metabolic Network

ChloroKB, a Web-based application that enables the user to explore the chloroplast metabolic network in Arabidopsis. Higher plants, as autotrophic organisms, are effective sources of molecules. They hold great promise for metabolic engineering, but the behavior of plant metabolism at the network level is still incompletely described. Although structural models (stoichiometry matrices) and pathway databases are extremely useful, they cannot describe the complexity of the metabolic context, and new tools are required to visually represent integrated biocurated knowledge for use by both humans and computers. Here, we describe ChloroKB, a Web application (http://chlorokb.fr/) for visual exploration and analysis of the Arabidopsis (Arabidopsis thaliana) metabolic network in the chloroplast and related cellular pathways. The network was manually reconstructed through extensive biocuration to provide transparent traceability of experimental data. Proteins and metabolites were placed in their biological context (spatial distribution within cells, connectivity in the network, participation in supramolecular complexes, and regulatory interactions) using CellDesigner software. The network contains 1,147 reviewed proteins (559 localized exclusively in plastids, 68 in at least one additional compartment, and 520 outside the plastid), 122 proteins awaiting biochemical/genetic characterization, and 228 proteins for which genes have not yet been identified. The visual presentation is intuitive and browsing is fluid, providing instant access to the graphical representation of integrated processes and to a wealth of refined qualitative and quantitative data. ChloroKB will be a significant support for structural and quantitative kinetic modeling, for biological reasoning, when comparing novel data with established knowledge, for computer analyses, and for educational purposes. ChloroKB will be enhanced by continuous updates following contributions from plant researchers.