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Subcellular and subplastidial proteomics to study intracellular and intraplastidial trafficking of proteins
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International audience. Recent advances in the proteomic field have allowed high throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused either on the whole chloroplast fractions or on independent suplastidial fractions. However, these previous studies raised the question of the accurate localization of many proteins that were identified in different suplastidial compartments. We recently went a step further into the knowledge of A. thaliana chloroplast proteins with regards to their accurate localization within the chloroplast. To achieve this goal, we first obtained highly pure subfractions of envelope, stroma and thylakoids and evaluated their cross-contaminations using biochemical methods. We then performed a comprehensive analysis of the Arabidopsis thaliana chloroplast proteome starting from the whole chloroplast and its three main compartments. Then, we assessed the partitioning of each identified protein in the three above-cited compartments using a semi-quantitative proteomic approach and also performed a curated information on envelope proteins (Ferro et al., 2010). An in depth investigation of the proteins identified within the purified envelope fraction allowed new insights over this subplastidial compartment to be revealed (Joyard et al., 2009; Joyard et al., 2010).During the course of this project, we also generated an important tool to investigate the dynamics of the chloroplast proteome at the scale of the whole organelle. This yet unique tool is the first database based on the accurate mass and time tags (AMT) strategy dedicated to plants: the chloroplast AMT database AT-CHLORO (http://www.grenoble.prabi.fr/at_chloro/). This proteomic database was generated in such a way that it can be used for quantitative studies (e.g. comparisons of mutants, impact of adverse growth conditions...). This AMT-based strategy is now used to characterize mechanisms that regulate protein trafficking between the cytosol and the chloroplast, to identify chloroplast proteins that are subjected to these regulatory mechanisms before being imported or during their import into the chloroplast and to understand the physiological significance of these mechanisms.Selected recent papersJoyard J, Ferro M, Masselon C, Seigneurin-Berny D, Salvi D, Garin J, Rolland N (2009) Chloroplast proteomics and the compartmentation of plastidial isoprenoid biosynthetic pathways. Mol Plant. 2: 1154-1180.Joyard J, Ferro M, Masselon C, Seigneurin-Berny D, Salvi D, Garin J, Rolland N (2010) Chloroplast proteomics highlights the subcellular compartmentation of lipid metabolism. Prog Lipid Res. 49: 128-158.Ferro M, Brugière S, Salvi D, Seigneurin-Berny D, Court M, Moyet L, Ramus C, Miras S, Mellal M, Le Gall S, Kieffer-Jaquinod S, Bruley C, Garin J, Joyard J, Masselon C, Rolland N (2010) AT_CHLORO: A comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins. Mol Cell Proteomics. 9: 1063-1084.Agrawal GK, Bourguignon J, Rolland N, Ephritikhine G, Ferro M, Jaquinod M, Kitsios G, Chardot T, Chakraborty N., Jolivet P, Doonan JH & Rakwal R (2011) Plant organelle proteomics: altruistic collaboration for proper cell function. Mass Spectrom Reviews. In press. PMID: 2103843Joshi H, Hirsch-Hoffmann M, Baerenfaller K, Gruissem W, Baginsky S, Schmidt R, Shulze WX, Sun Q, van Wijk KJ, Egelhofer V, Wienkoop S, Weckwerth W, Bruley C, Rolland N, Toyoda T, Nakagami H, Jones AME, Briggs SP, Castleden I, Tanz SK, Millar AH and Heazlewood JL (2011) MASCP Gator: An aggregation portal for the visualization of Arabidopsis proteomics data. Plant Physiol. 155(1) In press. PMID: 21075962
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