Proteomic approach to identify novel mitochondrial proteins in Arabidopsis.

An Arabidopsis mitochondrial proteome project was started for a comprehensive investigation of mitochondrial functions in plants. Mitochondria were prepared from Arabidopsis stems and leaves or from Arabidopsis suspension cell cultures, and the purity of the generated fractions was tested by the resolution of organellar protein complexes applying two-dimensional blue-native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Arabidopsis mitochondrial proteome was analyzed by two-dimensional isoelectric focusing/ Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 650 different proteins in a pI range of pH 3 to 10 were separated on single gels. Solubilization conditions, pH gradients for isoelectric focusing, and gel staining procedures were varied, and the number of separable proteins increased to about 800. Fifty-two protein spots were identified by immunoblotting, direct protein sequencing, and mass spectrometry. The characterized proteins cooperate in various processes, such as respiration, citric acid cycle, amino acid and nucleotide metabolism, protection against O(2), mitochondrial assembly, molecular transport, and protein biosynthesis. More than 20% of the identified proteins were not described previously for plant mitochondria, indicating novel mitochondrial functions. The map of the Arabidopsis mitochondrial proteome should be useful for the analysis of knockout mutants concerning nuclear-encoded mitochondrial genes. Considerations of the total complexity of the Arabidopsis mitochondrial proteome are discussed. The data from this investigation will be made available at http://www.gartenbau.uni-hannover.de/genetik/AMPP.     

Comparative Analysis of Predicted Plastid-Targeted Proteomes of Sequenced Higher Plant Genomes

Plastids are actively involved in numerous plant processes critical to growth, development and adaptation. They play a primary role in photosynthesis, pigment and monoterpene synthesis, gravity sensing, starch and fatty acid synthesis, as well as oil, and protein storage. We applied two complementary methods to analyze the recently published apple genome (Malus × domestica) to identify putative plastid-targeted proteins, the first using TargetP and the second using a custom workflow utilizing a set of predictive programs. Apple shares roughly 40% of its 10,492 putative plastid-targeted proteins with that of the Arabidopsis (Arabidopsis thaliana) plastid-targeted proteome as identified by the Chloroplast 2010 project and ∼57% of its entire proteome with Arabidopsis. This suggests that the plastid-targeted proteomes between apple and Arabidopsis are different, and interestingly alludes to the presence of differential targeting of homologs between the two species. Co-expression analysis of 2,224 genes encoding putative plastid-targeted apple proteins suggests that they play a role in plant developmental and intermediary metabolism. Further, an inter-specific comparison of Arabidopsis, Prunus persica (Peach), Malus × domestica (Apple), Populus trichocarpa (Black cottonwood), Fragaria vesca (Woodland Strawberry), Solanum lycopersicum (Tomato) and Vitis vinifera (Grapevine) also identified a large number of novel species-specific plastid-targeted proteins. This analysis also revealed the presence of alternatively targeted homologs across species. Two separate analyses revealed that a small subset of proteins, one representing 289 protein clusters and the other 737 unique protein sequences, are conserved between seven plastid-targeted angiosperm proteomes. Majority of the novel proteins were annotated to play roles in stress response, transport, catabolic processes, and cellular component organization. Our results suggest that the current state of knowledge regarding plastid biology, preferentially based on model systems is deficient. New plant genomes are expected to enable the identification of potentially new plastid-targeted proteins that will aid in studying novel roles of plastids.     

Integrative identification of Arabidopsis mitochondrial proteome and its function exploitation through protein interaction network

Mitochondria are major players on the production of energy, and host several key reactions involved in basic metabolism and biosynthesis of essential molecules. Currently, the majority of nucleus-encoded mitochondrial proteins are unknown even for model plant Arabidopsis. We reported a computational framework for predicting Arabidopsis mitochondrial proteins based on a probabilistic model, called Naive Bayesian Network, which integrates disparate genomic data generated from eight bioinformatics tools, multiple orthologous mappings, protein domain properties and co-expression patterns using 1,027 microarray profiles. Through this approach, we predicted 2,311 candidate mitochondrial proteins with 84.67% accuracy and 2.53% FPR performances. Together with those experimental confirmed proteins, 2,585 mitochondria proteins (named CoreMitoP) were identified, we explored those proteins with unknown functions based on protein-protein interaction network (PIN) and annotated novel functions for 26.65% CoreMitoP proteins. Moreover, we found newly predicted mitochondrial proteins embedded in particular subnetworks of the PIN, mainly functioning in response to diverse environmental stresses, like salt, draught, cold, and wound etc. Candidate mitochondrial proteins involved in those physiological acitivites provide useful targets for further investigation. Assigned functions also provide comprehensive information for Arabidopsis mitochondrial proteome.     

The plant mitochondrial proteome

The plant mitochondrial proteome might contain as many as 2000-3000 different gene products, each of which might undergo post-translational modification. Recent studies using analytical methods, such as one-, two- and three-dimensional gel electrophoresis and one- and two-dimensional liquid chromatography linked on-line with tandem mass spectrometry, have identified >400 mitochondrial proteins, including subunits of mitochondrial respiratory complexes, supercomplexes, phosphorylated proteins and oxidized proteins. The results also highlight a range of new mitochondrial proteins, new mitochondrial functions and possible new mechanisms for regulating mitochondrial metabolism. More than 70 identified proteins in Arabidopsis mitochondrial samples lack similarity to any protein of known function. In some cases, unknown proteins were found to form part of protein complexes, which allows a functional context to be defined for them. There are indications that some of these proteins add novel activities to mitochondrial protein complexes in plants.     

Plant mitochondrial rhomboid, AtRBL12, has different substrate specificity from its yeast counterpart

Rhomboid proteins comprise a class of serine proteases that are conserved in all kingdoms of organisms. They contain six or seven transmembrane helices and control a wide range of cellular functions and developmental processes by intramembrane proteolysis. This paper provides experimental evidence for the existence of rhomboid proteases in plant mitochondria and chloroplasts. Among 15 putative rhomboid-like proteins in Arabidopsis thaliana, we selected five predicted as mitochondrially targeted. For these proteins we performed the GFP transient assay, and identified two homologues, AtRBL11 (At5g25752) and AtRBL12 (At1g18600) to be targeted into plastids and mitochondria, respectively. Phylogenetic analysis reveals that AtRBL12 or AtRBL11 have only one clear orthologue in plant species with completely sequenced genomes. Complementation of the yeast lacking a functional copy of mitochondrial rhomboid with AtRBL12 indicates that this plant protease, in contrast to the human orthologue, does not recognize the yeast substrates, cytochrome c peroxidase (Ccp1) or dynamin-like GTPase (Mgm1). In agreement with this, we did not observe processing of Mgm1 when labeled precursor of this protein was incubated in vitro with Arabidopsis mitochondrial extract. Our results imply that plant mitochondrial rhomboids function in a specific manner and thus differ from their yeast and mammal counterparts.     

Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes

Peroxisomes play key roles in energy metabolism, cell signaling, and plant development. A better understanding of these important functions will be achieved with a more complete definition of the peroxisome proteome. The isolation of peroxisomes and their separation from mitochondria and other major membrane systems have been significant challenges in the Arabidopsis (Arabidopsis thaliana) model system. In this study, we present new data on the Arabidopsis peroxisome proteome obtained using two new technical advances that have not previously been applied to studies of plant peroxisomes. First, we followed density gradient centrifugation with free-flow electrophoresis to improve the separation of peroxisomes from mitochondria. Second, we used quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions. We provide evidence for peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analyses of Arabidopsis peroxisomes. Chimeric green fluorescent protein constructs of 35 proteins have been used to confirm their localization in peroxisomes or to identify endoplasmic reticulum contaminants. The distribution of many of these peroxisomal proteins between soluble, membrane-associated, and integral membrane locations has also been determined. This core peroxisomal proteome from nonphotosynthetic cultured cells contains a proportion of proteins that cannot be predicted to be peroxisomal due to the lack of recognizable peroxisomal targeting sequence 1 (PTS1) or PTS2 signals. Proteins identified are likely to be components in peroxisome biogenesis, beta-oxidation for fatty acid degradation and hormone biosynthesis, photorespiration, and metabolite transport. A considerable number of the proteins found in peroxisomes have no known function, and potential roles of these proteins in peroxisomal metabolism are discussed. This is aided by a metabolic network analysis that reveals a tight integration of functions and highlights specific metabolite nodes that most probably represent entry and exit metabolites that could require transport across the peroxisomal membrane.     

Cytoplasmic regulation of the accumulation of nuclear-encoded proteins in the mitochondrial proteome of maize

Mitochondria from normal (NA)- and Texas (T)-cytoplasm maize (Zea mays L.) were purified from unpollinated ears via Percoll centrifugation. Approximately 300 mitochondrial proteins were resolved using two-dimensional (2-D) electrophoresis. The 197 most abundant proteins were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry involving overlapping pH gradients (pH 4-7 and 6-9). Database searches identified 58 genes that encode 100 of these protein spots. Functions could be predicted for 38 of the 58 genes (66%). All but one of these genes are located in the nuclear genome. Thirteen per cent of the analyzed protein spots (25 out of 197) exhibited at least a threefold difference in accumulation between the mitochondrial proteomes of NA- or T-cytoplasm maize plants that had essentially identical nuclear genomes. As most of these proteins were nuclear-encoded, these findings demonstrate that the genotype of a mitochondrion can regulate the accumulation of the nuclear-encoded fraction of its proteome. About half (27 out of 58) of the maize mitochondrial proteins identified in this study were not recovered in previous analyses of the Arabidopsis and rice mitochondrial proteomes.     

Wheat mitochondrial proteomes provide new links between antioxidant defense and plant salinity tolerance

The mitochondrial proteome and differences associated with salt tolerance have been investigated in Australian commercial varieties of wheat. Mitochondria isolated from shoots were used to generate a wheat mitochondrial reference map; 68 unique wheat mitochondrial proteins were identified from 192 gel spots using 2D PAGE and LC-MS/MS. This analysis also provided MS/MS spectra for 199 proteotypic peptides as a foundation for the development of targeted proteomics to study the respiratory apparatus in wheat. Using this reference map and 2D DIGE, we have found quantitative differences in the shoot mitochondrial proteomes of v. Wyalkatchem and v. Janz, two commercially important wheat varieties that are known from a range of experiments to differ in salinity tolerance. These proteins included Mn-superoxide dismutase (Mn-SOD), cysteine synthase, nucleotide diphosphate kinase, and the voltage dependent anion channel (VDAC). Antibodies to the mitochondrial alternative oxidase (AOX), previously linked to reduced ROS formation from the electron transport chain and salt tolerance in Arabidopsis, also showed a commensurate higher abundance in v. Wyakatchem in both control and salt-treated conditions. Together, the data presented here suggest that differences in mitochondrial ROS defense pathways in the mitochondrial proteomes of key Australian wheat varieties correlate with whole-plant salinity tolerance.     

A survey of the Arabidopsis thaliana mitochondrial phosphoproteome

Plant mitochondria play central roles in cellular energy production, metabolism and stress responses. Recent phosphoproteomic studies in mammalian and yeast mitochondria have presented evidence indicating that protein phosphorylation is a likely regulatory mechanism across a broad range of important mitochondrial processes. This study investigated protein phosphorylation in purified mitochondria from cell suspensions of the model plant Arabidopsis thaliana using affinity enrichment and proteomic tools. Eighteen putative phosphoproteins consisting of mitochondrial metabolic enzymes, HSPs, a protease and several proteins of unknown function were detected on 2‐DE separations of Arabidopsis mitochondrial proteins and affinity‐enriched phosphoproteins using the Pro‐Q Diamond phospho‐specific in‐gel dye. Comparisons with mitochondrial phosphoproteomes of yeast and mouse indicate that these three species share few validated phosphoproteins. Phosphorylation sites for seven of the eighteen mitochondrial proteins were characterized by titanium dioxide enrichment and MS/MS. In the process, 71 phosphopeptides from Arabidopsis proteins which are not present in mitochondria but found as contaminants in various types of mitochondrial preparations were also identified, indicating the low level of phosphorylation of mitochondrial components compared with other cellular components in Arabidopsis. Information gained from this study provides a better understanding of protein phosphorylation at both the subcellular and the cellular level in Arabidopsis.     

The hydrophobic proteome of mitochondrial membranes from Arabidopsis cell suspensions

The development of mitochondria and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting membranes. The aim of the present work was: (1) to enhance our understanding of the biochemical machinery of mitochondrial membranes and (2) to test the versatility of the procedure developed for the identification of the hydrophobic proteome of the chloroplast envelope [Molecular and Cellular Proteomics 2 (2003) 325-345]. A proteomic analysis was performed, to provide the most exhaustive view of the protein repertoire of these membranes. For this purpose, highly purified mitochondria were prepared from Arabidopsis cultured cells and membrane proteins were extracted. To get a more exhaustive array of membrane proteins from Arabidopsis mitochondria, from the most to the less hydrophobic ones, various extraction procedures (chloroform/methanol extraction, alkaline or saline treatments) were applied. LC-MS/MS analyses were then performed on each membrane subfraction, leading to the identification of more than 110 proteins. The identification of these proteins is discussed with respect to their mitochondrial localization, their physicochemical properties and their implications in the metabolism of mitochondria. In order to provide a new overview of the biochemical machinery of the plant mitochondria, proteins identified during this work were compared to the lists of proteins identified during previous proteomic analyses performed on plant and algae mitochondria (Arabidopsis, pea, Chlamydomonas, rice, etc.). A total of 502 proteins are listed. About 40% of the 114 proteins identified during this work were not identified during previous proteomic studies performed on mitochondria.     

Towards an analysis of the rice mitochondrial proteome

Purified rice (Oryza sativa) mitochondrial proteins have been arrayed by isoelectric focusing/polyacrylamide gel electrophoresis (PAGE), by blue-native (BN) PAGE, and by reverse-phase high-performance liquid chromatography (LC) separation (LC-mass spectrometry [MS]). From these protein arrays, we have identified a range of rice mitochondrial proteins, including hydrophilic/hydrophobic proteins (grand average of hydropathicity = -1.27 to +0.84), highly basic and acid proteins (isoelectric point = 4.0-12.5), and proteins over a large molecular mass range (6.7-252 kD), using proteomic approaches. BN PAGE provided a detailed picture of electron transport chain protein complexes. A total of 232 protein spots from isoelectric focusing/PAGE and BN PAGE separations were excised, trypsin digested, and analyzed by tandem MS (MS/MS). Using this dataset, 149 of the protein spots (the products of 91 nonredundant genes) were identified by searching translated rice open reading frames from genomic sequence and six-frame translated rice expressed sequence tags. Sequence comparison allowed us to assign functions to a subset of 85 proteins, including many of the major function categories expected for this organelle. A further six spots were matched to rice sequences for which no specific function has yet been determined. Complete digestion of mitochondrial proteins with trypsin yielded a peptide mixture that was analyzed directly by reverse-phase LC via organic solvent elution from a C-18 column (LC-MS). These data yielded 170 MS/MS spectra that matched 72 sequence entries from open reading frame and expressed sequence tag databases. Forty-five of these were obtained using LC-MS alone, whereas 28 proteins were identified by both LC-MS and gel-based separations. In total, 136 nonredundant rice proteins were identified, including a new set of 23 proteins of unknown function located in plant mitochondria. We also report the first direct identification, to our knowledge, of PPR (pentatricopeptide repeat) proteins in the plant mitochondrial proteome. This dataset provides the first extensive picture, to our knowledge, of mitochondrial functions in a model monocot plant.     

Multiple lines of evidence localize signaling, morphology, and lipid biosynthesis machinery to the mitochondrial outer membrane of Arabidopsis

The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction.     

ARAMEMNON,a novel database for Arabidopsis integral membrane proteins

A specialized database (DB) for Arabidopsis membrane proteins, ARAMEMNON, was designed that facilitates the interpretation of gene and protein sequence data by integrating features that are presently only available from individual sources. Using several publicly available prediction programs, putative integral membrane proteins were identified among the approximately 25,500 proteins in the Arabidopsis genome DBs. By averaging the predictions from seven programs, approximately 6,500 proteins were classified as transmembrane (TM) candidate proteins. Some 1,800 of these contain at least four TM spans and are possibly linked to transport functions. The ARAMEMNON DB enables direct comparison of the predictions of seven different TM span computation programs and the predictions of subcellular localization by eight signal peptide recognition programs. A special function displays the proteins related to the query and dynamically generates a protein family structure. As a first set of proteins from other organisms, all of the approximately 700 putative membrane proteins were extracted from the genome of the cyanobacterium Synechocystis sp. and incorporated in the ARAMEMNON DB. The ARAMEMNON DB is accessible at the URL http://aramemnon.botanik.uni-koeln.de.     

Analysis of the xylem sap proteome of Brassica oleracea reveals a high content in secreted proteins

Xylem plays a major role in plant development and is considered part of the apoplast. Here, we studied the proteome of Brassica oleracea cv Bartolo and compared it to the plant cell wall proteome of another Brassicaceae, the model plant Arabidopsis thaliana. B. oleracea was chosen because it is technically difficult to harvest enough A. thaliana xylem sap for proteomic analysis. We studied the whole proteome and an N-glycoproteome obtained after Concanavalin A affinity chromatography. Altogether, 189 proteins were identified by LC-MS/MS using Brassica EST and cDNA sequences. A predicted signal peptide was found in 164 proteins suggesting that most proteins of the xylem sap are secreted. Eighty-one proteins were identified in the N-glycoproteome, with 25 of them specific of this fraction, suggesting that they were concentrated during the chromatography step. All the protein families identified in this study were found in the cell wall proteomes. However, proteases and oxido-reductases were more numerous in the xylem sap proteome, whereas enzyme inhibitors were rare. The origin of xylem sap proteins is discussed. All the experimental data including the MS/MS data were made available in the WallProtDB cell wall proteomic database.     

The Arabidopsis thaliana 2-D gel mitochondrial proteome: Refining the value of reference maps for assessing protein abundance, contaminants and post-translational modifications

Mitochondria undertake the process of oxidative phosphorylation yielding ATP for plant cell maintenance and growth. The principles of isolation and fractionation of plant mitochondrial proteins have been improved over decades, and surveys of the mitochondrial proteome in a number of plants species have been performed. Over time, many quantitative analyses of changes in the plant mitochondrial proteome have been performed by 2-D gel analyses revealing the induction, degradation and modification of mitochondrial proteins in responses to mutation, stress and development. Here, we present a saturating MS analysis of 2-D gel separable protein spots from a typical purification of Arabidopsis mitochondria identifying 264 proteins, alongside an LC-MS/MS survey by non-gel methods identifying 220 proteins. This allowed us to characterise the major mitochondrial proteins that are not observed on 2-D gels, the common contaminants and the abundance of the protein machinery of key mitochondrial biochemical pathways, and consider the impact of N-terminal pre-sequence cleavage and phosphorylation as explanations of multiple protein spots and the co-ordinates of proteins on 2-D gels.