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 higher plants, the Ndh complex reduces plastoquinones and is involved in cyclic electron flow around photosystem I, supplying extra-ATP for photosynthesis, particularly under environmental stress conditions. Based on plastid genome sequences, the Ndh complex would contain 11 subunits (NDH-A to -K), but homologies with bacterial complex indicate the probable existence of additional subunits. To identify missing subunits, tobacco (Nicotiana tabacum) NDH-H was His tagged at its N terminus using plastid transformation. A functional Ndh subcomplex was purified by Ni 2þ affinity chromatography and its subunit composition analyzed by mass spectrometry. Five plastid encoded subunits (NDH-A, -H, -I, -J, and -K) were identified as well as three new subunits (NDH-M, -N, and -O) homologous to cyanobacterial and higher plant proteins. Arabidopsis thaliana mutants missing one of these new subunits lack a functional Ndh complex, and NDH-M and NDH-N are not detected in a tobacco transformant lacking the Ndh complex. We discuss the involvement of these three nuclear-encoded subunits in the functional integrity of the plastidial complex.

A growing body of evidence indicates that phytooxylipins play important roles in plant defense responses. However, many enzymes involved in the biosynthesis of these metabolites are still elusive. We have purified one of these enzymes, the peroxygenase (PXG), from oat microsomes and lipid droplets. It is an integral membrane protein requiring detergent for its solubilization. Proteinase K digestion showed that PXG is probably deeply buried in lipid droplets or microsomes with only about 2 kDa at the C-terminal region accessible to proteolytic digestion. Sequencing of the N terminus of the purified protein showed that PXG had no sequence similarity with either a peroxidase or a cytochrome P450 but, rather, with caleosins, i.e. calcium-binding proteins. In agreement with this finding, we demonstrated that recombinant thale cress and rice caleosins, expressed in yeast, catalyze hydroperoxide-dependent mono-oxygenation reactions that are characteristic of PXG. Calcium was also found to be crucial for peroxygenase activity, whereas phosphorylation of the protein had no impact on catalysis. Site-directed mutagenesis studies revealed that PXG catalytic activity is dependent on two highly conserved histidines, the 9 GHz EPR spectrum being consistent with a high spin pentacoordinated ferric heme.