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.

Owing to the complexity of higher eukaryotic cells, characterization of a complete proteome is likely to be difficult to achieve. However, advantage can be taken of the cell compartmentalization to build organelle proteomes, which can moreover be viewed as specialized tools to study specifically the biology and "physiology" of the target organelle. Within this frame, we report here the construction of the human mitochondrial proteome, using placenta as the source tissue. Protein identification was carried out mainly by peptide mass fingerprinting, but other methods were also used (N-terminal microsequencing, blotting). The optimization steps in two-dimensional (2-D) electrophoresis needed for proteome research are discussed. However, the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome, which should be a useful tool for researchers working on human mitochondria and their deficiencies.

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp. strain 6PY1. [14 C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with M r s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in soils and sediments and are of environmental concern because of their mutagenic and/or carcinogenic effects. While low-molecular-weight PAHs (composed of two or three rings) are readily degraded by bacteria, PAHs consisting of four rings or more are recalcitrant to biodegradation and persist in the environment (6, 41). The biodegradation of lowmolecular-weight PAHs, especially naphthalene, has been extensively studied with pseudomonads, leading to a good understanding of the bacterial catabolic pathway (42). On the other hand, relatively little information is available on the metabolism of high-molecular-weight PAHs (20). A number of bacterial isolates capable of pyrene mineralization have been described. Most of them are actinomycetes and belong to the genus Mycobacterium (2, 7, 37), Rhodococcus (4, 40), or Gordonia (21). A few pyrene-degrading strains have been identified as gram-negative species, including Stenotrophomonas maltophilia, Pseudomonas fluorescens (3), Sphingomonas paucimobilis (22), and Burkholder...

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysenedegrading organism Sphingomonas sp. strain CHY-1 were investigated. [14 C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1 a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.

To expand the functional analysis of plant mitochondria, we have undertaken the building of the proteome of pea mitochondria purified from leaves (green and etiolated), roots and seeds. In the first stage, we focused our proteomic exploration on the soluble protein complement of the green leaf mitochondria. We used traditional two-dimensional polyacrylamide gel electrophoresis, in combination with size exclusion chromatography as a third dimension, to identify the major proteins and further resolve their macromolecular complexity. The two-dimensional map of soluble proteins of green leaf mitochondria revealed 433 spots (with Coomassie blue staining) and around 73% of the proteins (in mass) were identified using three different approaches: Edman degradation, matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry. Quite a lot of the polypeptides were present in multiforms which indicated the presence of isoforms or the occurrence of post-translational modifications. Among these proteins, we uncovered an abundant family that was identified as aldehyde dehydrogenases, representing approximately 7.5% of the soluble proteins. The comparative analysis of soluble mitochondrial proteomes led to the identification of a number of proteins which were specifically present in root or in seed mitochondria, thus revealing the impact of tissue differentiation at the mitochondrial level.

The lysosomal compartment of human monocytic cells has never been investigated by a proteomic approach. By a combination of one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis, protein identification by N-terminal sequencing, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting and tandem mass spectrometry (MS/MS) peptide sequence analysis, we initiated an exhaustive study of the human lyososomal proteome, which aims at establishing a 2-D reference map of human soluble lyososomal proteins. Human monocytic U937 cells were induced to secrete lysosomal soluble hydrolases by addition of NH4Cl in the culture medium. Since lysosomal soluble proteins are characterized by the presence of mannose-6-phosphate, they were purified on an affinity support bearing mannose-6-phosphate receptor. Analysis of the purified fraction led to the preliminary identification of fifteen proteins, among which twelve are well-known lysosomal hydrolases, one is assumed to be lysosomal on the basis of sequence homology to cysteine proteinases of the papain family, and two (leukocystatin and the human cellular repressor of E1A-stimulated genes) are described here for the first time as mannose-6-phosphate-containing proteins.