In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database

An extensive analysis of the Arabidopsis thaliana peripheral and integral thylakoid membrane proteome was performed by sequential extractions with salt, detergent, and organic solvents, followed by multidimensional protein separation steps (reverse-phase HPLC and one- and two-dimensional electrophoresis gels), different enzymatic and nonenzymatic protein cleavage techniques, mass spectrometry, and bioinformatics. Altogether, 154 proteins were identified, of which 76 (49%) were alpha-helical integral membrane proteins. Twenty-seven new proteins without known function but with predicted chloroplast transit peptides were identified, of which 17 (63%) are integral membrane proteins. These new proteins, likely important in thylakoid biogenesis, include two rubredoxins, a potential metallochaperone, and a new DnaJ-like protein. The data were integrated with our analysis of the lumenal-enriched proteome. We identified 83 out of 100 known proteins of the thylakoid localized photosynthetic apparatus, including several new paralogues and some 20 proteins involved in protein insertion, assembly, folding, or proteolysis. An additional 16 proteins are involved in translation, demonstrating that the thylakoid membrane surface is an important site for protein synthesis. The high coverage of the photosynthetic apparatus and the identification of known hydrophobic proteins with low expression levels, such as cpSecE, Ohp1, and Ohp2, indicate an excellent dynamic resolution of the analysis. The sequential extraction process proved very helpful to validate transmembrane prediction. Our data also were cross-correlated to chloroplast subproteome analyses by other laboratories. All data are deposited in a new curated plastid proteome database (PPDB) with multiple search functions (http://cbsusrv01.tc.cornell.edu/users/ppdb/). This PPDB will serve as an expandable resource for the plant community.     

Combining experimental and predicted datasets for determination of the subcellular location of proteins in Arabidopsis

Substantial experimental datasets defining the subcellular location of Arabidopsis (Arabidopsis thaliana) proteins have been reported in the literature in the form of organelle proteomes built from mass spectrometry data (approximately 2,500 proteins). Subcellular location for specific proteins has also been published based on imaging of chimeric fluorescent fusion proteins in intact cells (approximately 900 proteins). Further, the more diverse history of biochemical determination of subcellular location is stored in the entries of the Swiss-Prot database for the products of many Arabidopsis genes (approximately 1,800 proteins). Combined with the range of bioinformatic targeting prediction tools and comparative genomic analysis, these experimental datasets provide a powerful basis for defining the final location of proteins within the wide variety of subcellular structures present inside Arabidopsis cells. We have analyzed these published experimental and prediction data to answer a range of substantial questions facing researchers about the veracity of these approaches to determining protein location and their interrelatedness. We have merged these data to form the subcellular location database for Arabidopsis proteins (SUBA), providing an integrated understanding of protein location, encompassing the plastid, mitochondrion, peroxisome, nucleus, plasma membrane, endoplasmic reticulum, vacuole, Golgi, cytoskeleton structures, and cytosol (www.suba.bcs.uwa.edu.au). This includes data on more than 4,400 nonredundant Arabidopsis protein sequences. We also provide researchers with an online resource that may be used to query protein sets or protein families and determine whether predicted or experimental location data exist; to analyze the nature of contamination between published proteome sets; and/or for building theoretical subcellular proteomes in Arabidopsis using the latest experimental data.     

Non-canonical transit peptide for import into the chloroplast

The large majority of plastid proteins are nuclear-encoded and, thus, must be imported within these organelles. Unlike most of the outer envelope proteins, targeting of proteins to all other plastid compartments (inner envelope membrane, stroma, and thylakoid) is strictly dependent on the presence of a cleavable transit sequence in the precursor N-terminal region. In this paper, we describe the identification of a new envelope protein component (ceQORH) and demonstrate that its subcellular localization is limited to the inner membrane of the chloroplast envelope. Immunopurification, microsequencing of the natural envelope protein and cloning of the corresponding full-length cDNA demonstrated that this protein is not processed in the N-terminal region during its targeting to the inner envelope membrane. Transient expression experiments in plant cells were performed with truncated forms of the ceQORH protein fused to the green fluorescent protein. These experiments suggest that neither the N-terminal nor the C-terminal are essential for chloroplastic localization of the ceQORH protein. These observations are discussed in the frame of the endosymbiotic theory of chloroplast evolution and suggest that a domain of the ceQORH bacterial ancestor may have evolved so as to exclude the general requirement of an N-terminal plastid transit sequence.     

A small ATPase protein of Arabidopsis, TGD3, involved in chloroplast lipid import

Polar lipid trafficking is essential in eukaryotic cells as membranes of lipid assembly are often distinct from final destination membranes. A striking example is the biogenesis of the photosynthetic membranes (thylakoids) in plastids of plants. Lipid biosynthetic enzymes at the endoplasmic reticulum and the inner and outer plastid envelope membranes are involved. This compartmentalization requires extensive lipid trafficking. Mutants of Arabidopsis are available that are disrupted in the incorporation of endoplasmic reticulum-derived lipid precursors into thylakoid lipids. Two proteins affected in two of these mutants, trigalactosyldiacylglycerol 1 (TGD1) and TGD2, encode the permease and substrate binding component, respectively, of a proposed lipid translocator at the inner chloroplast envelope membrane. Here we describe a third protein of Arabidopsis, TGD3, a small ATPase proposed to be part of this translocator. As in the tgd1 and tgd2 mutants, triacylglycerols and trigalactolipids accumulate in a tgd3 mutant carrying a T-DNA insertion just 5' of the TGD3 coding region. The TGD3 protein shows basal ATPase activity and is localized inside the chloroplast beyond the inner chloroplast envelope membrane. Proteins orthologous to TGD1, -2, and -3 are predicted to be present in Gram- bacteria, and the respective genes are organized in operons suggesting a common biochemical role for the gene products. Based on the current analysis, it is hypothesized that TGD3 is the missing ATPase component of a lipid transporter involving TGD1 and TGD2 required for the biosynthesis of ER-derived thylakoid lipids in Arabidopsis.     

Genome-wide identification of the subcellular localization of the Escherichia coli B proteome using experimental and computational methods

Escherichia coli K-12 and B strains have most widely been employed for scientific studies as well as industrial applications. Recently, the complete genome sequences of two representative descendants of E. coli B strains, REL606 and BL21(DE3), have been determined. Here, we report the subproteome reference maps of E. coli B REL606 by analyzing cytoplasmic, periplasmic, inner and outer membrane, and extracellular proteomes based on the genome information using experimental and computational approaches. Among the total of 3487 spots, 651 proteins including 410 non-redundant proteins were identified and characterized by 2-DE and LC-MS/MS; they include 440 cytoplasmic, 45 periplasmic, 50 inner membrane, 61 outer membrane, and 55 extracellular proteins. In addition, subcellular localizations of all 4205 ORFs of E. coli B were predicted by combined computational prediction methods. The subcellular localizations of 1812 (43.09%) proteins of currently unknown function were newly assigned. The results of computational prediction were also compared with the experimental results, showing that overall precision and recall were 92.16 and 92.16%, respectively. This work represents the most comprehensive analyses of the subproteomes of E. coli B, and will be useful as a reference for proteome profiling studies under various conditions. The complete proteome data are available online (http://ecolib.kaist.ac.kr).     

Plastids contain a second sec translocase system with essential functions

Proteins that are synthesized on cytoplasmic ribosomes but function within plastids must be imported and then targeted to one of six plastid locations. Although multiple systems that target proteins to the thylakoid membranes or thylakoid lumen have been identified, a system that can direct the integration of inner envelope membrane proteins from the stroma has not been previously described. Genetics and localization studies were used to show that plastids contain two different Sec systems with distinct functions. Loss-of-function mutations in components of the previously described thylakoid-localized Sec system, designated as SCY1 (At2g18710), SECA1 (At4g01800), and SECE1 (At4g14870) in Arabidopsis (Arabidopsis thaliana), result in albino seedlings and sucrose-dependent heterotrophic growth. Loss-of-function mutations in components of the second Sec system, designated as SCY2 (At2g31530) and SECA2 (At1g21650) in Arabidopsis, result in arrest at the globular stage and embryo lethality. Promoter-swap experiments provided evidence that SCY1 and SCY2 are functionally nonredundant and perform different roles in the cell. Finally, chloroplast import and fractionation assays and immunogold localization of SCY2-green fluorescent protein fusion proteins in root tissues indicated that SCY2 is part of an envelope-localized Sec system. Our data suggest that SCY2 and SECA2 function in Sec-mediated integration and translocation processes at the inner envelope membrane.     

New functions of the thylakoid membrane proteome of Arabidopsis thaliana revealed by a simple, fast, and versatile fractionation strategy

Identification of membrane proteomes remains challenging. Here, we present a simple, fast, and scalable off-line procedure based on three-phase partitioning with butanol to fractionate membrane proteomes in combination with both in-gel and in-solution digestions and mass spectrometry. This should help to further accelerate the field of membrane proteomics. Using this new strategy, we analyzed the salt-stripped thylakoid membrane of chloroplasts of Arabidopsis thaliana. 242 proteins were identified, at least 40% of which are integral membrane proteins. The functions of 86 proteins are unknown; these include proteins with TPR, PPR, rhodanese, and DnaJ domains. These proteins were combined with all known thylakoid proteins and chloroplast (associated) envelope proteins, collected from primary literature, resulting in 714 non-redundant proteins. They were assigned to functional categories using a classification developed for MapMan (Thimm, O., Blasing, O., Gibon, Y., Nagel, A., Meyer, S., Kruger, P., Selbig, J., Muller, L. A., Rhee, S. Y., and Stitt, M. (2004) Plant J. 37, 914-939), updated with information from primary literature. The analysis elucidated the likely location of many membrane proteins, including 190 proteins of unknown function, holding the key to better understanding the two membrane systems. The three-phase partitioning procedure added a new level of dynamic resolution to the known thylakoid proteome. An automated strategy was developed to track possible ambiguous identifications to more than one gene model or family member. Mass spectrometry search results, ambiguities, and functional classifications can be searched via the Plastid Proteome Database.     

Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the predominant lipids in thylakoid membranes and indispensable for photosynthesis. Among the three isoforms that catalyze MGDG synthesis in Arabidopsis thaliana, MGD1 is responsible for most galactolipid synthesis in chloroplasts, whereas MGD2 and MGD3 are required for DGDG accumulation during phosphate (Pi) starvation. A null mutant of Arabidopsis MGD1 (mgd1‐2), which lacks both galactolipids and shows a severe defect in chloroplast biogenesis under nutrient‐sufficient conditions, accumulated large amounts of DGDG, with a strong induction of MGD2/3 expression, during Pi starvation. In plastids of Pi‐starved mgd1‐2 leaves, biogenesis of thylakoid‐like internal membranes, occasionally associated with invagination of the inner envelope, was observed, together with chlorophyll accumulation. Moreover, the mutant accumulated photosynthetic membrane proteins upon Pi starvation, indicating a compensation for MGD1 deficiency by Pi stress‐induced galactolipid biosynthesis. However, photosynthetic activity in the mutant was still abolished, and light‐harvesting/photosystem core complexes were improperly formed, suggesting a requirement for MGDG for proper assembly of these complexes. During Pi starvation, distribution of plastid nucleoids changed concomitantly with internal membrane biogenesis in the mgd1‐2 mutant. Moreover, the reduced expression of nuclear‐ and plastid‐encoded photosynthetic genes observed in the mgd1‐2 mutant under Pi‐sufficient conditions was restored after Pi starvation. In contrast, Pi starvation had no such positive effects in mutants lacking chlorophyll biosynthesis. These observations demonstrate that galactolipid biosynthesis and subsequent membrane biogenesis inside the plastid strongly influence nucleoid distribution and the expression of both plastid‐ and nuclear‐encoded photosynthetic genes, independently of photosynthesis.     

The role of the transmembrane domain in determining the targeting of membrane proteins to either the inner envelope or thylakoid membrane

Chloroplastic membrane proteins can be targeted to any of three distinct membrane systems, i.e., the outer envelope membrane (OEM), inner envelope membrane (IEM), and thylakoid membrane. This complex structure of chloroplasts adds significantly to the challenge of studying protein targeting to various membrane sub-compartments within a chloroplast. In this investigation, we examined the role played by the transmembrane domain (TMD) in directing membrane proteins to either the IEM or thylakoid membrane. Using the IEM protein, Arc6 (Accumulation and Replication of Chloroplasts 6), we exchanged the stop-transfer TMD of Arc6 with various TMDs derived from different IEM and thylakoid membrane proteins and monitored the subcellular localization of these Arc6-hybrid proteins. We showed that when the Arc6 TMD was replaced with a TMD derived from various thylakoid membrane proteins, these Arc6(thylTMD) hybrid proteins could be directed to the thylakoid membrane rather than to the IEM. Conversely, when the TMD of the thylakoid membrane proteins, STN8 (State Transition protein kinase 8) or Plsp1 (Plastidic type I signal peptidase 1), was replaced with the stop-transfer TMD of Arc6, STN8 and Plsp1 were halted at the IEM. From our investigation, we conclude that the TMD plays a critical role in targeting integral membrane proteins to either the IEM or thylakoid membrane.     

The functional network of the Arabidopsis plastoglobule proteome based on quantitative proteomics and genome-wide coexpression analysis

Plastoglobules (PGs) in chloroplasts are thylakoid-associated monolayer lipoprotein particles containing prenyl and neutral lipids and several dozen proteins mostly with unknown functions. An integrated view of the role of the PG is lacking. Here, we better define the PG proteome and provide a conceptual framework for further studies. The PG proteome from Arabidopsis (Arabidopsis thaliana) leaf chloroplasts was determined by mass spectrometry of isolated PGs and quantitative comparison with the proteomes of unfractionated leaves, thylakoids, and stroma. Scanning electron microscopy showed the purity and size distribution of the isolated PGs. Compared with previous PG proteome analyses, we excluded several proteins and identified six new PG proteins, including an M48 metallopeptidase and two Absence of bc1 complex (ABC1) atypical kinases, confirmed by immunoblotting. This refined PG proteome consisted of 30 proteins, including six ABC1 kinases and seven fibrillins together comprising more than 70% of the PG protein mass. Other fibrillins were located predominantly in the stroma or thylakoid and not in PGs; we discovered that this partitioning can be predicted by their isoelectric point and hydrophobicity. A genome-wide coexpression network for the PG genes was then constructed from mRNA expression data. This revealed a modular network with four distinct modules that each contained at least one ABC1K and/or fibrillin gene. Each module showed clear enrichment in specific functions, including chlorophyll degradation/senescence, isoprenoid biosynthesis, plastid proteolysis, and redox regulators and phosphoregulators of electron flow. We propose a new testable model for the PGs, in which sets of genes are associated with specific PG functions.     

The Sec2 translocase of the chloroplast inner envelope contains a unique and dedicated SECE2 component

Biogenesis of chloroplasts involves a series of protein trafficking events. Nuclear‐encoded proteins are imported into the organelle, and then trafficked to various chloroplast locations by systems that are directly homologous to bacterial systems. Although the thylakoid‐based systems have been studied extensively, much less is known about the systems that reside and function in the inner envelope membrane. One such system, the Sec2 system, is homologous to both the thylakoid‐based Sec1 system and bacterial Sec systems, and may mediate both integration and translocation across the inner envelope. At a minimum, this system is expected to include three components, but only two, SCY2 and SECA2, have been identified in Arabidopsis. Bioinformatics and protein modeling were used to identify the protein encoded by At4g38490 as a candidate for the missing component (SECE2). Cellular localization, biochemistry, protein interaction assays in yeast, and co‐immunoprecipitation experiments were used to establish that this protein is an integral membrane protein of the inner envelope, and specifically interacts with the SCY2 component in vivo. Sequence analyses indicated that SECE2 proteins are found in a variety of plants, and differ from the thylakoid SECE1 proteins in a stroma‐exposed helical domain, which may contribute to their specificity. Finally, a genetic analysis indicated that SECE2 plays an essential role in plant growth and development.     

Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-specific proteomic approach to identify rare intrinsic membrane proteins

Identification of rare hydrophobic membrane proteins is a major biological problem that is limited by the specific biochemical approaches required to extract these proteins from membranes and purify them. This is especially true for membranes, such as plastid envelope membranes, that have a high lipid content, present a wide variety of specific functions and therefore contain a large number of unique, but minor, proteins. We have optimized a procedure, based on the differential solubilization of membrane proteins in chloroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrophilic proteins were excluded. In addition to previously characterized chloroplast envelope proteins, such as the phosphate/triose phosphate translocator, we have identified new proteins that were shown to contain putative transmembrane α-helices. Moreover, using different chloroform/methanol mixtures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. All the proteins identified were genuine chloroplast envelope proteins, most of them being localized within the inner membrane. Our procedure enables direct mapping (by classical SDS-PAGE) and identification of hydrophobic membrane proteins, whatever their isoelectric point was, that are minor components of specific subcellular compartments. Thus, it complements other techniques that give access to peripheral membrane proteins. If applied to various cell membranes, it is anticipated that it can expedite the identification of hydrophobic proteins involved in transport systems for ions or organic solutes, or it may act as signal receptors or to control metabolic processes and vesicle trafficking.     

The membrane proteome of stroma thylakoids from Arabidopsis thaliana studied by successive in‐solution and in‐gel digestion

From individual localization and large‐scale proteomic studies, we know that stroma‐exposed thylakoid membranes harbor part of the machinery performing the light‐dependent photosynthetic reactions. The minor components of the stroma thylakoid proteome, regulating and maintaining the photosynthetic machinery, are in the process of being unraveled. In this study, we developed in‐solution and in‐gel proteolytic digestion methods, and used them to identify minor membrane proteins, e.g. transporters, in stroma thylakoids prepared from Arabidopsis thaliana (L.) Heynh Columbia‐0 leaves. In‐solution digestion with chymotrypsin yielded the largest number of peptides, but in combination with methanol extraction resulted in identification of the largest number of membrane proteins. Although less efficient in extracting peptides, in‐gel digestion with trypsin and chymotrypsin led to identification of additional proteins. We identified a total of 58 proteins including 44 membrane proteins. Almost half are known thylakoid proteins with roles in photosynthetic light reactions, proteolysis and import. The other half, including many transporters, are not known as chloroplast proteins, because they have been either curated (manually assigned) to other cellular compartments or not curated at all at the plastid protein databases. Transporters include ATP‐binding cassette (ABC) proteins, transporters for K⁺ and other cations. Other proteins either have a role in processes probably linked to photosynthesis, namely translation, metabolism, stress and signaling or are contaminants. Our results indicate that all these proteins are present in stroma thylakoids; however, individual studies are required to validate their location and putative roles. This study also provides strategies complementary to traditional methods for identification of membrane proteins from other cellular compartments.     

A role for lipid trafficking in chloroplast biogenesis

Chloroplasts are the defining plant organelle carrying out photosynthesis. Photosynthetic complexes are embedded into the thylakoid membrane which forms an intricate system of membrane lamellae and cisternae. The chloroplast boundary consists of two envelope membranes controlling the exchange of metabolites between the plastid and the extraplastidic compartments of the cell. The plastid internal matrix (stroma) is the primary location for fatty acid biosynthesis in plants. Fatty acids can be assembled into glycerolipids at the envelope membranes of plastids or they can be exported and assembled into lipids at the endoplasmic reticulum (ER) to provide building blocks for extraplastidic membranes. Some of these glycerolipids, assembled at the ER, return to the plastid where they are remodeled into the plastid typical glycerolipids. As a result of this cooperation of different subcellular membrane systems, a rich complement of lipid trafficking phenomena contributes to the biogenesis of chloroplasts. Considerable progress has been made in recent years towards a better mechanistic understanding of lipid transport across plastid envelopes. Lipid transporters of bacteria and plants have been discovered and their study begins to provide detailed mechanistic insights into lipid trafficking phenomena relevant to chloroplast biogenesis.     

A second thylakoid membrane-localized Alb3/OxaI/YidC homologue is involved in proper chloroplast biogenesis in Arabidopsis thaliana

The integral membrane proteins Alb3, OxaI, and YidC belong to an evolutionary conserved protein family mediating protein insertion into the thylakoid membrane of chloroplasts, the inner membrane of mitochondria, and bacteria, respectively. Whereas OxaI and YidC are involved in the insertion of a wide range of membrane proteins, the function of Alb3 seems to be limited to the insertion of a subset of the light-harvesting chlorophyll-binding proteins. In this study, we identified a second chloroplast homologue of the Alb3/OxaI/YidC family, named Alb4. Alb4 is almost identical to the Alb3/OxaI/YidC domain of the previously described 110-kDa inner envelope protein Artemis. We show that Alb4 is expressed as a separate 55-kDa protein and that Artemis was identified mistakenly. Alb4 is located in the thylakoid membrane of Arabidopsis thaliana chloroplasts. Analysis of an Arabidopsis mutant (Salk_136199) and RNA interference lines with a reduced level of Alb4 revealed chloroplasts with an altered ultrastructure. Mutant plastids are larger and more spherical in appearance, and the grana stacks within the mutant lines are less appressed than in the wild-type chloroplasts. These data indicate that Alb4 is required for proper chloroplast biogenesis.     

Role of signal peptides in targeting of proteins in cyanobacteria.

Proteins of cyanobacteria may be transported across one of two membrane systems: the typical eubacterial cell envelope (consisting of an inner membrane, periplasmic space, and an outer membrane) and the photosynthetic thylakoids. To investigate the role of signal peptides in targeting in cyanobacteria, Synechococcus sp. strain PCC 7942 was transformed with vectors carrying the chloramphenicol acetyltransferase reporter gene fused to coding sequences for one of four different signal peptides. These included signal peptides of two proteins of periplasmic space origin (one from Escherichia coli and the other from Synechococcus sp. strain PCC 7942) and two other signal peptides of proteins located in the thylakoid lumen (one from a cyanobacterium and the other from a higher plant). The location of the gene fusion products expressed in Synechococcus sp. strain PCC 7942 was determined by a chloramphenicol acetyltransferase enzyme-linked immunosorbent assay of subcellular fractions. The distribution pattern for gene fusions with periplasmic signal peptides was different from that of gene fusions with thylakoid lumen signal peptides. Primary sequence analysis revealed conserved features in the thylakoid lumen signal peptides that were absent from the periplasmic signal peptides. These results suggest the importance of the signal peptide in protein targeting in cyanobacteria and point to the presence of signal peptide features conserved between chloroplasts and cyanobacteria for targeting of proteins to the thylakoid lumen.     

Isoelectric points of membrane surfaces of three spinach chloroplast classes determined by cross-partition

The isoelectric points of the membranes surrounding three classes of spinach chloroplasts have been determined by partition at different pH values in aqueous two-phase systems where the electrical potential differences at the interface are opposite (cross-partition). Class I chloroplasts, intact chloroplasts, have an isoelectric point at pH 3.8–4.1 and class II chloroplasts, broken chloroplasts or intact thylakoid membranes, have an isoelectric point at pH 4.7–4.9. The third class of particles, class III ‘chloroplasts’, that contain one or more chloroplasts, mitochondria, peroxisomes and some cytoplasm all surrounded by a membrane, probably the plasma membrane, have an isoelectric point at pH 3.4–4.0. The partition technique used presumably yields the isoelectric point of the surface of the membranes exposed to the phase system by the three classes of chloroplasts, i.e., the outer envelope membrane, the thylakoid membrane and the plasma membrane, respectively. The isoelectric points obtained with this technique are suggested to reflect protein to charged-lipid differences in the composition of the membranes.     

Proteomics of chloroplast envelope membranes

Proteomics is a very powerful approach to link the information contained in sequenced genomes, like Arabidopsis, to the functional knowledge provided by studies of plant cell compartments, such as chloroplast envelope membranes. This review summarizes the present state of proteomic analyses of highly purified spinach and Arabidopsis envelope membranes. Methods targeted towards the hydrophobic core of the envelope allow identifying new proteins, and especially new transport systems. Common features were identified among the known and newly identified putative envelope inner membrane transporters and were used to mine the complete Arabidopsis genome to establish a virtual plastid envelope integral protein database. Arabidopsis envelope membrane proteins were extracted using different methods, that is, chloroform/methanol extraction, alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to the less hydrophobic ones. Mass spectrometry analyses lead to the identification of more than 100 proteins. More than 50% of the identified proteins have functions known or very likely to be associated with the chloroplast envelope. These proteins are (a) involved in ion and metabolite transport, (b) components of the protein import machinery and (c) involved in chloroplast lipid metabolism. Some soluble proteins, like proteases, proteins involved in carbon metabolism or in responses to oxidative stress, were associated with envelope membranes. Almost one third of the newly identified proteins have no known function. The present stage of the work demonstrates that a combination of different proteomics approaches together with bioinformatics and the use of different biological models indeed provide a better understanding of chloroplast envelope biochemical machinery at the molecular level.