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Plastids contain several key subcompartments. The two limiting envelope membranes (inner and outer membrane of the plastid envelope with an intermembrane space between), an aqueous phase (stroma), and an internal membrane system terms (thylakoids) formed of flat compressed vesicles (grana) and more light structures (lamellae). The thylakoid vesicles delimit another discrete soluble compartment, the thylakoid lumen. AT_CHLORO ( http://at-chloro.prabi.fr/at_chloro/ ) is a unique database supplying information about the subplastidial localization of proteins. It was created from simultaneous proteomic analyses targeted to the main subcompartments of the chloroplast from Arabidopsis thaliana (i.e., envelope, stroma, thylakoid) and to the two subdomains of thylakoid membranes (i.e., grana and stroma lamellae). AT_CHLORO assembles several complementary information (MS-based experimental data, curated functional annotations and subplastidial localization, links to other public databases and references) which give a comprehensive overview of the current knowledge about the subplastidial localization and the function of chloroplast proteins, with a specific attention given to chloroplast envelope proteins.

Photosynthesis has shaped atmospheric and ocean chemistries and probably changed the climate as well, as oxygen is released from water as part of the photosynthetic process. In photosynthetic eukaryotes, this process occurs in the chloroplast, an organelle containing the most abundant biological membrane, the thylakoids. The thylakoids of plants and some green algae are structurally inhomogeneous, consisting of two main domains: the grana, which are piles of membranes gathered by stacking forces, and the stroma-lamellae, which are unstacked thylakoids connecting the grana. The major photosynthetic complexes are unevenly distributed within these compartments because of steric and electrostatic constraints. Although proteomic analysis of thylakoids has been instrumental to define its protein components, no extensive proteomic study of subthylakoid localization of proteins in the BBY (grana) and the stroma-lamellae fractions has been achieved so far. To fill this gap, we performed a complete survey of the protein composition of these thylakoid subcompartments using thylakoid membrane fractionations. We employed semiquantitative proteomics coupled with a data analysis pipeline and manual annotation to differentiate genuine BBY and stroma-lamellae proteins from possible contaminants. About 300 thylakoid (or potentially thylakoid) proteins were shown to be enriched in either the BBY or the stroma-lamellae fractions. Overall, present findings corroborate previous observations obtained for photosynthetic proteins that used nonproteomic approaches. The originality of the present proteomic relies in the identification of photosynthetic proteins whose differential distribution in the thylakoid subcompartments might explain already observed phenomenon such as LHCII docking. Besides, from the present localization results we can suggest new molecular actors for photosynthesis-linked activities. For instance, most PsbP-like subunits being differently localized in stroma-lamellae, these proteins could be linked to the PSI-NDH complex in the context of cyclic electron flow around PSI. In addition, we could identify about a hundred new likely minor thylakoid (or chloroplast) proteins, some of them being potential regulators of the chloroplast physiology.

AT_CHLORO (www.grenoble.prabi.fr/at_chloro) is a database dedicated to sub-plastidial localization of A. thaliana chloroplast proteins. This information was infered from proteomics experiments obtained from a comprehensive study that allowed the identification of proteins from envelope, stroma, and thylakoid sub-compartments Ferro et al., 2010. In addition to current knowledge regarding sub-plastidial localization, AT_CHLORO provides experimental data that allowed curated information regarding subcellular localizations of chloroplast proteins to be given. A specific focus was given to proteins that were identified in envelope fractions and for which expert functional annotation was provided. The present mini review shows the specificities of AT_CHLORO with respect to available information, data export options and recent improvements in data representation.

Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions.

Recent advances in the proteomic field have allowed high throughput experiments to be conducted on chloroplast samples and the data are available in several databases such as the Plant Protein Database (PPDB), or the SubCellular Proteomic Database (SUBA). However, the accurate localization of many proteins that were identified in different subplastidial compartments often remains hypothetical, thus making quantitative proteomics important for going a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regard to their accurate localization within the chloroplast. Spectral counting, a semi-quantitative proteomic strategy based on accurate mass and time tags (AMT), was used to build up AT_CHLORO, a comprehensive chloroplast proteome database with curated subplastidial localization. In this review, we focus on about a hundred enzymes involved in fatty acid biosynthesis, export and metabolism (desaturation and oxylipin metabolism), in the synthesis of chloroplast-specific glycerolipids either with a eukaryotic or a prokaryotic structure. Two main chloroplast compartments play a major role in lipid biosynthesis: the initial steps of fatty acid biosynthesis take place in the stroma, then the envelope membranes concentrate most of the proteins involved in chloroplast glycerolipid metabolism.

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database (PPDB, Sun et al., 2009) presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. (2009) went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags (AMT) database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways (i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts) validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts (i.e. in envelope membranes, stroma, and/or thylakoids) that remains unclear until now.