Nicholas O'Toole

A Predicted Interactome for Arabidopsis

The complex cellular functions of an organism frequently rely on physical interactions between proteins. A map of all protein-protein interactions, an interactome, is thus an invaluable tool. We present an interactome for Arabidopsis (Arabidopsis thaliana) predicted from interacting orthologs in yeast (Saccharomyces cerevisiae), nematode worm (Caenorhabditis elegans), fruitfly (Drosophila melanogaster), and human (Homo sapiens). As an internal quality control, a confidence value was generated based on the amount of supporting evidence for each interaction. A total of 1,159 high confidence, 5,913 medium confidence, and 12,907 low confidence interactions were identified for 3,617 conserved Arabidopsis proteins. There was significant coexpression of genes whose proteins were predicted to interact, even among low confidence interactions. Interacting proteins were also significantly more likely to be found within the same subcellular location, and significantly less likely to be found in conflicting localizations than randomly paired proteins. A notable exception was that proteins located in the Golgi were more likely to interact with Golgi, vacuolar, or endoplasmic reticulum sorted proteins, indicating possible docking or trafficking interactions. These predictions can aid researchers by extending known complexes and pathways with candidate proteins. In addition we have predicted interactions for many previously unknown proteins in known pathways and complexes. We present this interactome, and an online Web interface the Arabidopsis Interactions Viewer, as a first step toward understanding global signaling in Arabidopsis, and to whet the appetite for those who are awaiting results from high-throughput experimental approaches.

Genome-Wide Analysis of mRNA Decay Rates and Their Determinants in Arabidopsis thaliana

To gain a global view of mRNA decay in Arabidopsis thaliana, suspension cell cultures were treated with a transcriptional inhibitor, and microarrays were used to measure transcript abundance over time. The deduced mRNA half-lives varied widely, from minutes to >24 h. Three features of the transcript displayed a correlation with decay rates: (1) genes possessing at least one intron produce mRNA transcripts significantly more stable than those of intronless genes, and this was not related to overall length, sequence composition, or number of introns; (2) various sequence elements in the 3′ untranslated region are enriched among short- and long-lived transcripts, and their multiple occurrence suggests combinatorial control of transcript decay; and (3) transcripts that are microRNA targets generally have short half-lives. The decay rate of transcripts correlated with subcellular localization and function of the encoded proteins. Analysis of transcript decay rates for genes encoding orthologous proteins between Arabidopsis, yeast, and humans indicated that yeast and humans had a higher percentage of transcripts with shorter half-lives and that the relative stability of transcripts from genes encoding proteins involved in cell cycle, transcription, translation, and energy metabolism is conserved. Comparison of decay rates with changes in transcript abundance under a variety of abiotic stresses reveal that a set of transcription factors are downregulated with similar kinetics to decay rates, suggesting that inhibition of their transcription is an important early response to abiotic stress.

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, β-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.

Divalent Metal Ions in Plant Mitochondria and Their Role in Interactions with Proteins and Oxidative Stress-Induced Damage to Respiratory Function

Understanding the metal ion content of plant mitochondria and metal ion interactions with the proteome are vital for insights into both normal respiratory function and the process of protein damage during oxidative stress. We have analyzed the metal content of isolated Arabidopsis (Arabidopsis thaliana) mitochondria, revealing a 26:8:6:1 molar ratio for iron:zinc:copper:manganese and trace amounts of cobalt and molybdenum. We show that selective changes occur in mitochondrial copper and iron content following in vivo and in vitro oxidative stresses. Immobilized metal affinity chromatography charged with Cu2+, Zn2+, and Co2+ was used to identify over 100 mitochondrial proteins with metal-binding properties. There were strong correlations between the sets of immobilized metal affinity chromatography-interacting proteins, proteins predicted to contain metal-binding motifs, and protein sets known to be oxidized or degraded during abiotic stress. Mitochondrial respiratory chain pathways and matrix enzymes varied widely in their susceptibility to metal-induced loss of function, showing the selectivity of the process. A detailed study of oxidized residues and predicted metal interaction sites in the tricarboxylic acid cycle enzyme aconitase identified selective oxidation of residues in the active site and showed an approach for broader screening of functionally significant oxidation events in the mitochondrial proteome.

Approaches to defining dual-targeted proteins in Arabidopsis

A variety of approaches were used to predict dual-targeted proteins in Arabidopsis thaliana. These predictions were experimentally tested using GFP fusions. Twelve new dual-targeted proteins were identified: five that were dual-targeted to mitochondria and plastids, six that were dual-targeted to mitochondria and peroxisomes, and one that was dual-targeted to mitochondria and the nucleus. Two methods to predict dual-targeted proteins had a high success rate: (1) combining the AraPerox database with a variety of subcellular prediction programs to identify mitochondrial- and peroxisomal-targeted proteins, and (2) using a variety of prediction programs on a biochemical pathway or process known to contain at least one dual-targeted protein. Several technical parameters need to be taken into account before assigning subcellular localization using GFP fusion proteins. The position of GFP with respect to the tagged polypeptide, the tissue or cells used to detect subcellular localization, and the portion of a candidate protein fused to GFP are all relevant to the expression and targeting of a fusion protein. Testing all gene models for a chromosomal locus is required if more than one model exists.

Phage-Type RNA Polymerase RPOTmp Performs Gene-Specific Transcription in Mitochondria of Arabidopsis thaliana

Transcription of mitochondrial genes in animals, fungi, and plants relies on the activity of T3/T7 phage-type RNA polymerases. Two such enzymes, RPOTm and RPOTmp, are present in the mitochondria of eudicotyledonous plants; RPOTmp is additionally found in plastids. We have characterized the transcriptional role of the dual-targeted RNA polymerase in mitochondria of Arabidopsis thaliana. Examination of mitochondrial transcripts in rpoTmp mutants revealed major differences in transcript abundances between wild-type and rpoTmp plants. Decreased levels of specific transcripts were correlated with reduced abundances of the respiratory chain complexes I and IV. Altered transcript levels in rpoTmp were found to result from gene-specific transcriptional changes, establishing that RPOTmp functions in distinct transcriptional processes within mitochondria. Decreased transcription of specific genes in rpoTmp was not associated with changes in promoter utilization; therefore, RPOTmp function is not promoter specific but gene specific. This implies that additional gene-specific elements direct the transcription of a subset of mitochondrial genes by RPOTmp.

Heterogeneity of the Mitochondrial Proteome for Photosynthetic and Non-photosynthetic Arabidopsis Metabolism

Heterogeneity of the mitochondrial proteome in plants underlies fundamental differences in the roles of these organelles in different tissues. We quantitatively compared the mitochondrial proteome isolated from a non-photosynthetic cell culture model with more specialized mitochondria isolated from photosynthetic shoots. Differences in intact mitochondrial respiratory rates with various substrates and activities of specific enzymes provided a backdrop of the functional variation between these mitochondrial populations. Proteomics comparisons provided a deep insight into the different steady-state abundances of specific mitochondrial proteins. Combined these data showed the elevated level of the photorespiratory apparatus and its complex interplay with glycolate, cysteine, formate, and one-carbon metabolism as well as the decrease of selected parts of the tricarboxylic acid cycle, alterations in amino acid metabolism focused on 2-oxoglutarate generation, and degradation of branched chain amino acids. Comparisons with microarray analysis of these tissue types showed a positive, mild correlation between mRNA and mitochondrial protein abundance, a tighter correlation for specific biochemical pathways, but over 78% concordance in direction between changes in protein and transcript abundance in the two tissues. Overall these results indicated that the majority of the variation in the plant mitochondrial proteome occurred in the matrix, highlighted the constitutive nature of the respiratory apparatus, and showed the differences in substrate choice and/or availability during photosynthetic and non-photosynthetic metabolism.

Insights into female sperm storage from the spermathecal fluid proteome of the honeybee Apis mellifera

BackgroundFemale animals are often able to store sperm inside their body - in some species even for several decades. The molecular basis of how females keep non-own cells alive is largely unknown, but since sperm cells are reported to be transcriptionally silenced and, therefore, limited in their ability to maintain their own function, it is likely that females actively participate in sperm maintenance. Because female contributions are likely to be of central importance for sperm survival, molecular insights into the process offer opportunities to observe mechanisms through which females manipulate sperm.ResultsWe used the honeybee, Apis mellifera, in which queens are highly polyandrous and able to maintain sperm viable for several years. We identified over a hundred proteins representing the major constituents of the spermathecal fluid, which females contribute to sperm in storage. We found that the gel profile of proteins from spermathecal fluid is very similar to the secretions of the spermathecal gland and concluded that the spermathecal glands are the main contributors to the spermathecal fluid proteome. A detailed analysis of the spermathecal fluid proteins indicate that they fall into a range of different functional groups, most notably enzymes of energy metabolism and antioxidant defense. A metabolic network analysis comparing the proteins detected in seminal fluid and spermathecal fluid showed a more integrated network is present in the spermathecal fluid that could facilitate long-term storage of sperm.ConclusionsWe present a large-scale identification of proteins in the spermathecal fluid of honeybee queens and provide insights into the molecular regulation of female sperm storage.

Large-scale characteristics of the energy landscape in protein-protein interactions

Characterization of intermolecular energy landscapes in protein–protein interactions is important for understanding the mechanisms of these interactions as well as for designing better protein docking methods. A simplified representation of the landscape was used for a systematic study of its large‐scale characteristics in a large nonredundant dataset of protein complexes. The focus of the study is on the basic features of the low‐resolution energy basins and their distribution on the landscape. The results clearly show that, in general, the number of such basins is small, these basins are well formed, correlated with actual binding modes, and the pattern of basins distribution depends on the type of the complex. For docking studies, the results suggest that adequate starting points for the structural refinement are detectable by low‐resolution procedures and the number of such starting points is relatively small. Proteins 2008.

Synchrotron X-ray analysis of the electron density in CoF2 and ZnF2

Accurate structure factors for small crystals of the rutile-type structures CoF(2), cobalt difluoride, and ZnF(2), zinc difluoride, have been measured with focused lambda = 0.8400 (2) A synchrotron X-radiation at room temperature. Phenomenological structural trends across the full series of rutile-type transition metal difluorides are analysed, showing the importance of the metal atom in the degree of distortion of the metal-F(6) octahedra in these structures. Multipole models reveal strong asphericities in the electron density surrounding the transition metals, which are consistent with expectations from crystal field theory and the structural trends in these compounds. Transition metal 3d-orbital populations were computed from the multipole refinement parameters, showing significant repopulation of orbitals compared with the free atom, particularly for CoF(2).