Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves

<p>Abstract</p> <p>Background</p> <p>The N-terminal proline-rich domain (Zera) of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles.</p> <p>Results</p> <p>We analysed the proteome of PBs induced in <it>Nicotiana benthamiana </it>leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed). Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer.</p> <p>Conclusions</p> <p>This study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of recombinant proteins in molecular farming applications.</p

Targeted Proteomics Allows Quantification of Ethylene Receptors and Reveals SlETR3 Accumulation in Never-Ripe Tomatoes

Ethylene regulates fruit ripening and several plant functions (germination, plant growth, plant-microbe interactions). Protein quantification of ethylene receptors (ETRs) is essential to study their functions, but is impaired by low resolution tools such as antibodies that are mostly nonspecific, or the lack of sensitivity of shotgun proteomic approaches. We developed a targeted proteomic method, to quantify low-abundance proteins such as ETRs, and coupled this to mRNAs analyses, in two tomato lines: Wild Type (WT) and Never-Ripe (NR) which is insensitive to ethylene because of a gain-of-function mutation in ETR3. We obtained mRNA and protein abundance profiles for each ETR over the fruit development period. Despite a limiting number of replicates, we propose Pearson correlations between mRNA and protein profiles as interesting indicators to discriminate the two genotypes: such correlations are mostly positive in the WT and are affected by the NR mutation. The influence of putative post-transcriptional and post-translational changes are discussed. In NR fruits, the observed accumulation of the mutated ETR3 protein between ripening stages (Mature Green and Breaker + 8 days) may be a cause of NR tomatoes to stay orange. The label-free quantitative proteomics analysis of membrane proteins, concomitant to Parallel Reaction Monitoring analysis, may be a resource to study changes over tomato fruit development. These results could lead to studies about ETR subfunctions and interconnections over fruit development. Variations of RNA-protein correlations may open new fields of research in ETR regulation. Finally, similar approaches may be developed to study ETRs in whole plant development and plant-microorganism interactions

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma

International audienceIron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention

Effects of elevated endogenous cytokinin levels on the Arabidopsis thalianaArabidopsis\ thaliana proteome

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Effects of elevated endogenous cytokinin levels on the Arabidopsis thalianaArabidopsis\ thaliana proteome

International audienc

Different impact of staining procedures using visible stains and fluorescent dyes for large-scale investigation of proteomes by MALDI-TOF mass spectrometry

Replicate 2-D gels were stained with four visible or fluorescent dyes using published procedures, and 48 co-detected spots were selected for contrasting values in abundance, M r and pI. Success rate of identification and sequence coverage were affected in a dye-dependent manner by the three parameters. Frequency of missed cleavages and recovery of sulfur-containing peptides also depended on the dye. Finally, the dataset was used to predict the number of proteins identifiable when integrating the differential contribution of each parameter. Sypro Ruby appeared to combine several favorable features: no dependence of the identification rate upon the physicochemical properties of proteins, no impact on frequency of missed cleavages, and a higher predicted identification rate

Identification des protéines par MALDI-TOF après électrophorèse 2D : Performance et compatibilité comparaison du bleu colloïdal (CCB), nitrate d'argent (SN), SpyroTM^{TM} Ruby (SR) and Deep PurpleTM^{TM} (DP)

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Identification des protéines par MALDI-TOF après électrophorèse 2D : Performance et compatibilité comparaison du bleu colloïdal (CCB), nitrate d'argent (SN), SpyroTM^{TM} Ruby (SR) and Deep PurpleTM^{TM} (DP)

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The FW2.2/CNR protein regulates cell-to-cell communication in tomato by modulating callose deposition at plasmodesmata

The FW2.2 gene is the founding member of the CELL NUMBER REGULATOR (CNR) gene family. More than 20 years ago, FW2.2 was the first cloned gene underlying a Quantitative Trait Locus (QTL) governing fruit size/weight in tomato. However, despite this discovery, the molecular mechanisms by which FW2.2 acts as a negative regulator of cell divisions during fruit growth remain undeciphered. In the present study, we confirm that FW2.2 is a transmembrane spanning protein, whose both N-and C-terminal ends are facing the apoplast. We unexpectedly found that FW2.2 is located at plasmodesmata (PD). FW2.2 participates in the spatiotemporal regulation of callose deposition at PD via an interaction with Callose Synthases, which suggests a regulatory role in cell-to-cell communication by modulating PD transport capacity and trafficking of signaling molecules during fruit development