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. Molecular & Cellular Proteomics 9:1063-1084, 2010.Plastids are semiautonomous organelles that are ubiquitously found in plant cells. They are derived from an endosymbiotic event and are thought to have evolved from an ancient photosynthetic prokaryote related to present-day cyanobacteria. Following endosymbiosis, the plastid genome has been reduced to ϳ100 genes, mainly coding for housekeeping functions (translation and transcription of the plastid genome), proteins required for primary photosynthetic reactions, and a few, yet poorly characterized, gene products (1). The most conspicuous plastid type is the chloroplast, found in leaves and carrying out photosynthesis as its main function. Photosynthesis is an integrated biological process involving the coordinated functioning of ...

The development of chloroplasts and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting envelope membranes. To provide the most exhaustive view of the protein repertoire of chloroplast envelope membranes, we analyzed this membrane system using proteomics. To this purpose, we first developed a procedure to prepare highly purified envelope membranes from Arabidopsis chloroplasts. We then extracted envelope proteins using different methods, i.e. chloroform/methanol extraction and alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to least hydrophobic ones. Liquid chromatography tandem mass spectrometry analyses were then performed on each envelope membrane subfraction, leading to the identification of more than 100 proteins. About Plastids are semiautonomous organelles that present a wide structural diversity and contain unique biosynthetic pathways. They are strongly dependent on proteins that are nuclear encoded, translated in the cytoplasm, and imported into this organelle. A pair of membranes called the envelope surrounds all plastids. As the vast majority of plastid proteins are nuclear encoded, the plastid envelope contains a protein import machinery. Translocation at the envelope membranes is directed by a general import machinery composed of the outer-membrane Toc complex and the inner-membrane Tic complex (for reviews, see Refs. 1-4).Located at the interface between the stroma and the cytosol, the envelope is also the site of various transports and exchanges of ions and metabolites required for the integration of the plastid metabolism within the plant cell. Few envelope transporters have been identified and characterized at the molecular level: the triose-phosphate/phosphate translocator, an ADP/ATP translocator, several substrate-specific outer membrane channels, and two dicarboxylate translocators (for a review, see Ref. 5). Recently, a putative hexose transporter was also identified (6). More recently we described a proteomic approach that allowed the identification of several putative transporters of the chloroplast envelope (7).A unique biochemical machinery is also present in envelope membranes. The chloroplast envelope is the site of specific biosynthetic functions i.e. synthesis of plastid membrane components (glycerolipids, pigments, prenylquinones), chlorophyll breakdown, synthesis of lipid-derived signaling molecules (fatty acid hydroperoxydes, growth regulators, or chlorophyll precursors), and participates in the coordination of the expression of nuclear and plastid genes (for a review, see Ref. 8). So far, and as for other plastid envelope components, few proteins catalyzing these biosynthetic functions have been identified and characterized at the molecular level.Subcellular proteomic studies are essential to get access to protein location in relation with their function (for a review, see Ref. 9). Plant proteomics exemplifies perfectly this functional dimensi...

In Arabidopsis, monogalactosyldiacylglycerol (MGDG) is synthesized by a multigenic family of MGDG synthases consisting of two types of enzymes differing in their N-terminal portion: type A (atMGD1) and type B (atMGD2 and atMGD3). The present paper compares type B isoforms with the enzymes of type A that are known to sit in the inner membrane of plastid envelope. The occurrence of types A and B in 16:3 and 18:3 plants shows that both types are not specialized isoforms for the prokaryotic and eukaryotic glycerolipid biosynthetic pathways. Type A atMGD1 gene is abundantly expressed in green tissues and along plant development and encodes the most active enzyme. Its mature polypeptide is immunodetected in the envelope of chloroplasts from Arabidopsis leaves after cleavage of its transit peptide. atMGD1 is therefore likely devoted to the massive production of MGDG required to expand the inner envelope membrane and build up the thylakoids network. Transient expression of green fluorescent protein fusions in Arabidopsis leaves and in vitro import experiments show that type B precursors are targeted to plastids, owing to a different mechanism. Noncanonical addressing peptides, whose processing could not be assessed, are involved in the targeting of type B precursors, possibly to the outer envelope membrane where they might contribute to membrane expansion. Expression of type B enzymes was higher in nongreen tissues, i.e., in inflorescence (atMGD2) and roots (atMGD3), where they conceivably influence the eukaryotic structure prominence in MGDG. In addition, their expression of type B enzymes is enhanced under phosphate deprivation.G alactolipids are a major class of higher plant glycerolipids because they are unique to plastid membranes from which they represent up to 80% of the total lipids (1). They contain one or two galactose molecules attached to the sn-3 position of a glycerol backbone, respectively monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). In 16:3 plants, § two distinct pathways lead to the prokaryotic and eukaryotic sn-1,2-diacylglycerol (DAG) molecules, the substrates used to generate MGDG (1). The last step for MGDG biosynthesis is catalyzed by a UDP-galactose:sn-1,2-DAG 3-␤-galactosyltransferase or MGDG synthase activity.MGDG synthase activity was localized in the inner envelope membrane in spinach (a 16:3 plant) (3), whereas it was detected in the outer envelope membrane from pea (a 18:3 plant) (4). Further investigations in MGDG synthase localization were obviously limited by the lack of characterized polypeptides associated with the galactosylation activity. MGDG synthase encoding cDNAs were cloned in cucumber (5) and spinach (6).The encoded enzyme from spinach (soMGD1) could synthesize both prokaryotic and eukaryotic MGDG molecular species, and its processed form was imported in chloroplasts and immunodetected in the inner envelope membrane (6).In Arabidopsis, at least two classes of MGDG synthase homologues can be distinguished according to the length of the N-terminal por...

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies. Molecular & Cellular Proteomics 3: 675-691, 2004.

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In many soils plants have to grow in a shortage of phosphate, leading to development of phosphate-saving mechanisms. At the cellular level, these mechanisms include conversion of phospholipids into glycolipids, mainly digalactosyldiacylglycerol (DGDG). The lipid changes are not restricted to plastid membranes where DGDG is synthesized and resides under normal conditions. In plant cells deprived of phosphate, mitochondria contain a high concentration of DGDG, whereas mitochondria have no glycolipids in control cells. Mitochondria do not synthesize this pool of DGDG, which structure is shown to be characteristic of a DGD type enzyme present in plastid envelope. The transfer of DGDG between plastid and mitochondria is investigated and detected between mitochondria-closely associated envelope vesicles and mitochondria. This transfer does not apparently involve the endomembrane system and would rather be dependent upon contacts between plastids and mitochondria. Contacts sites are favored at early stages of phosphate deprivation when DGDG cell content is just starting to respond to phosphate deprivation.

A two-membrane system, or envelope, surrounds plastids. Because of the integration of chloroplast metabolism within the plant cell, the envelope is the site of many specific transport activities. However, only a few proteins involved in the processes of transport across the chloroplast envelope have been identified already at the molecular level. To discover new envelope transporters, we developed a subcellular proteomic approach, which is aimed to identify the most hydrophobic envelope proteins. This strategy combined the use of highly purified and characterized membrane fractions, extraction of the hydrophobic proteins with organic solvents, SDS͞PAGE separation, and tandem mass spectrometry analysis. To process the large amount of MS͞MS data, a BLAST-based program was developed for searching in protein, expressed sequence tag, and genomic plant databases. Among the 54 identified proteins, 27 were new envelope proteins, with most of them bearing multiple ␣-helical transmembrane regions and being very likely envelope transporters. The present proteomic study also allowed us to identify common features among the known and newly identified putative envelope inner membrane transporters. These features were used to mine the complete Arabidopsis genome and allowed us to establish a virtual plastid envelope integral protein database. Altogether, both proteomic and in silico approaches identified more than 50 candidates for the as yet previously uncharacterized plastid envelope transporters. The predictable function of some of these proteins opens up areas of investigation that may lead to a better understanding of the chloroplast metabolism. The present subcellular proteomic approach is amenable to the analysis of the hydrophobic core of other intracellular membrane systems. P lastids, and especially chloroplasts, conduct vital biosynthetic functions, and many reactions are located exclusively within these unique organelles. A two-membrane system, the envelope, surrounds all plastid types and separates the plastid stroma from the cytosol. As a consequence, the envelope is involved in the controlled exchange of a variety of ions and metabolites between these two subcellular compartments (1).Chloroplasts import cytoplasmically synthesized precursor proteins from the cytosol. Translocation of precursor proteins across the envelope is achieved by the joint action of Toc and Tic translocons located at the outer and inner envelope membranes, respectively, of the chloroplast envelope (2, 3). Chloroplasts also take up intermediates of various metabolic pathways such as dicarboxylic acids, acetate, and phosphoenolpyruvate. Chloroplasts also have been demonstrated to import inorganic ions like K ϩ , Na (4, 5). As the sole site of biosynthesis of most amino acids (with the exception of sulfur-containing amino acids; refs. 6 and 7), chloroplasts must export these compounds for protein synthesis in the cytosolic and mitochondrial compartments. Finally, because of metabolism compartmentation, several other organic or inorganic comp...

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.