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Tyrosine (Tyr) phosphorylation plays an essential role in signaling in animal systems. However, a few studies have also reported Tyr phosphorylation in plants, but the relative contribution of tyrosine phosphorylation to plant signal transduction has remained an open question. We present an approach to selectively measure and quantify Tyr phosphorylation in plant cells, which can also be applied to whole plants. We combined a (15)N stable isotope metabolic labeling strategy with an immuno-affinity purification using phospho-tyrosine (pY) specific antibodies. This single enrichment strategy was sufficient to reproducibly identify and quantify pY containing peptides from total plant cell extract in a single LC-MS/MS run. We succeeded in identifying 149 unique pY peptides originating from 135 proteins, including a large set of different protein kinases and several receptor-like kinases. We used flagellin perception by Arabidopsis cells, a model system for pathogen triggered immune (PTI) signaling, to test our approach. We reproducibly quantified 23 pY peptides in 2 inversely labeled biological replicates identifying 11 differentially phosphorylated proteins. These include a set of 3 well-characterized flagellin responsive MAP kinases and 4 novel MAP kinases. With this targeted approach, we elucidate a new level of complexity in flagellin-induced MAP kinase activation.

The taxonomic class of oomycetes contains numerous pathogens of plants and animals but is related to nonpathogenic diatoms and brown algae. Oomycetes have flexible genomes comprising large gene families that play roles in pathogenicity. The evolutionary processes that shaped the gene content have not yet been studied by applying systematic tree reconciliation of the phylome of these species. We analyzed evolutionary dynamics of ten Stramenopiles. Gene gains, duplications, and losses were inferred by tree reconciliation of 18,459 gene trees constituting the phylome with a highly supported species phylogeny. We reconstructed a strikingly large last common ancestor of the Stramenopiles that contained ~10,000 genes. Throughout evolution, the genomes of pathogenic oomycetes have constantly gained and lost genes, though gene gains through duplications outnumber the losses. The branch leading to the plant pathogenic Phytophthora genus was identified as a major transition point characterized by increased frequency of duplication events that has likely driven the speciation within this genus. Large gene families encoding different classes of enzymes associated with pathogenicity such as glycoside hydrolases are formed by complex and distinct patterns of duplications and losses leading to their expansion in extant oomycetes. This study unveils the large-scale evolutionary dynamics that shaped the genomes of pathogenic oomycetes. By the application of phylogenetic based analyses methods, it provides additional insights that shed light on the complex history of oomycete genome evolution and the emergence of large gene families characteristic for this important class of pathogens.

Plant infection by oomycete pathogens is a complex process. It requires precise expression of a plethora of genes in the pathogen that contribute to a successful interaction with the host. Whereas much effort has been made to uncover the molecular systems underlying this infection process, mechanisms of transcriptional regulation of the genes involved remain largely unknown. We performed the first systematic de-novo DNA motif discovery analysis in Phytophthora. To this end, we utilized the genome sequence of the late blight pathogen Phytophthora infestans and two related Phytophthora species (P. ramorum and P. sojae), as well as genome-wide in planta gene expression data to systematically predict 19 conserved DNA motifs. This catalog describes common eukaryotic promoter elements whose functionality is supported by the presence of orthologs of known general transcription factors. Together with strong functional enrichment of the common promoter elements towards effector genes involved in pathogenicity, we obtained a new and expanded picture of the promoter structure in P. infestans. More intriguingly, we identified specific DNA motifs that are either highly abundant or whose presence is significantly correlated with gene expression levels during infection. Several of these motifs are observed upstream of genes encoding transporters, RXLR effectors, but also transcriptional regulators. Motifs that are observed upstream of known pathogenicity-related genes are potentially important binding sites for transcription factors. Our analyses add substantial knowledge to the as of yet virtually unexplored question regarding general and specific gene regulation in this important class of pathogens. We propose hypotheses on the effects of cis-regulatory motifs on the gene regulation of pathogenicity-related genes and pinpoint motifs that are prime targets for further experimental validation.

The TOR kinase is a major regulator of growth in eukaryotes. Many components of the TOR pathway are implicated in cancer and metabolic diseases in humans. Analysis of the evolution of TOR and its pathway may provide fundamental insight into the evolution of growth regulation in eukaryotes and provide a practical framework on which experimental evidence can be compared between species. Here we performed phylogenetic analyses on the components of the TOR pathway and determined their point of invention. We find that the two TOR complexes and a large part of the TOR pathway originated before the Last Eukaryotic Common Ancestor and form a core to which new inputs have been added during animal evolution. In addition, we provide insight into how duplications and sub-functionalization of the S6K, RSK, SGK and PKB kinases shaped the complexity of the TOR pathway. In yeast we identify novel AGC kinases that are orthologous to the S6 kinase. These results demonstrate how a vital signaling pathway can be both highly conserved and flexible in eukaryotes.

The pattern of plant organ initiation at the shoot apical meristem (SAM), termed phyllotaxis, displays regularities that have long intrigued botanists and mathematicians alike. In the SAM, the central zone (CZ) contains a population of stem cells that replenish the surrounding peripheral zone (PZ), where organs are generated in regular patterns. These patterns differ between species and may change in response to developmental or environmental cues [1]. Expression analysis of auxin efflux facilitators of the PIN-FORMED (PIN) family combined with modeling of auxin transport has indicated that organ initiation is associated with intracellular polarization of PIN proteins and auxin accumulation [2-10]. However, regulators that modulate PIN activity to determine phyllotactic patterns have hitherto been unknown. Here we reveal that three redundantly acting PLETHORA (PLT)-like AP2 domain transcription factors control shoot organ positioning in the model plant Arabidopsis thaliana. Loss of PLT3, PLT5, and PLT7 function leads to nonrandom, metastable changes in phyllotaxis. Phyllotactic changes in plt3plt5plt7 mutants are largely attributable to misregulation of PIN1 and can be recapitulated by reducing PIN1 dosage, revealing that PLT proteins are key regulators of PIN1 activity in control of phyllotaxis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The role of fibroblast growth factor-2 (FGF-2) in maintaining undifferentiated human embryonic stem cells (hESC) was investigated using a targeted phosphoproteomics approach to specifically profile tyrosine phosphorylation events following FGF-2 stimulation. A cumulative total number of 735 unique tyrosine phosphorylation sites on 430 proteins were identified, by far the largest inventory to date for hESC. Early signaling events in FGF-2 stimulated hESC were quantitatively monitored using stable isotope dimethyl labeling, resulting in temporal tyrosine phosphorylation profiles of 316 unique phosphotyrosine peptides originating from 188 proteins. Apart from the rapid activation of all four FGF receptors, trans-activation of several other receptor tyrosine kinases (RTKs) was observed as well as induced tyrosine phosphorylation of downstream proteins such as PI3-K, MAPK and several Src family members. Both PI3-K and MAPK have been linked to hESC maintenance through FGF-2 mediated signaling. The observed activation of the Src kinase family members by FGF-2 and loss of pluripotent marker expression post Src kinase inhibition may point to the regulation of cytoskeletal and actin depending processes to maintain undifferentiated hESC.

Oomycetes comprise a diverse group of organisms that morphologically resemble fungi but belong to the stramenopile lineage within the supergroup of chromalveolates. Recent studies have shown that plant pathogenic oomycetes have expanded gene families that are possibly linked to their pathogenic lifestyle. We analyzed the protein domain organization of 67 eukaryotic species including four oomycete and five fungal plant pathogens. We detected 246 expanded domains in fungal and oomycete plant pathogens. The analysis of genes differentially expressed during infection revealed a significant enrichment of genes encoding expanded domains as well as signal peptides linking a substantial part of these genes to pathogenicity. Overrepresentation and clustering of domain abundance profiles revealed domains that might have important roles in host-pathogen interactions but, as yet, have not been linked to pathogenicity. The number of distinct domain combinations (bigrams) in oomycetes was significantly higher than in fungi. We identified 773 oomycete-specific bigrams, with the majority composed of domains common to eukaryotes. The analyses enabled us to link domain content to biological processes such as host-pathogen interaction, nutrient uptake, or suppression and elicitation of plant immune responses. Taken together, this study represents a comprehensive overview of the domain repertoire of fungal and oomycete plant pathogens and points to novel features like domain expansion and species-specific bigram types that could, at least partially, explain why oomycetes are such remarkable plant pathogens.

Small GTPases are molecular switches at the hub of many signaling pathways and the expansion of this protein family is interwoven with the origin of unique eukaryotic cell features. We have previously reported on the evolution of CDC25 Homology Domain containing proteins, which act as guanine nucleotide exchange factors (GEFs) for Ras-like proteins. We now report on the evolution of both the Ras-like small GTPases as well as the GTPase activating proteins (GAPs) for Ras-like small GTPases. We performed an in depth phylogenetic analysis in 64 genomes of diverse eukaryotic species. These analyses revealed that multiple ancestral Ras-like GTPases and GAPs were already present in the Last Eukaryotic Common Ancestor (LECA), compatible with the presence of RasGEFs in LECA . Furthermore, we endeavor to reconstruct in which order the different Ras-like GTPases diverged from each other. We identified striking differences between the expansion of the various types of Ras-like GTPases and their respective GAPs and GEFs. Altogether, our analysis forms an extensive evolutionary framework for Ras-like signaling pathways and provides specific predictions for molecular biologists and biochemists.

Unraveling the functional dynamics of phosphorylation networks is a crucial step in understanding the way in which biological networks form a living cell. Recently there has been an enormous increase in the number of measured phosphorylation events. Nevertheless, comparative and integrative analysis of phosphoproteomes is confounded by incomplete coverage and biases introduced by different experimental workflows. As a result, we cannot differentiate whether phosphosites indentified in only one or two samples are the result of condition or species specific phosphorylation, or reflect missing data. Here, we evaluate the impact of incomplete phosphoproteomics datasets on comparative analysis, and we present bioinformatics strategies to quantify the impact of different experimental workflows on measured phosphoproteomes. We show that plotting the saturation in observed phosphosites in replicates provides a reproducible picture of the extent of a particular phosphoproteome. Still, we are still far away from a complete picture of the total human phosphoproteome. The impact of different experimental techniques on the similarity between phosphoproteomes can be estimated by comparing datasets from different experimental pipelines to a common reference. Our results show that comparative analysis is most powerful when datasets have been generated using the same experimental workflow. We show this experimentally by measuring the tyrosine phosphoproteome from Caenorhabditis elegans and comparing it to the tyrosine phosphoproteome of HeLa cells, resulting in an overlap of about 4%. This overlap between very different organisms represents a three-fold increase when compared to dataset of older studies, wherein different workflows were used. The strategies we suggest enable an estimation of the impact of differences in experimental workflows on the overlap between datasets. This will allow us to perform comparative analyses not only on datasets specifically generated for this purpose, but also to extract insights through comparative analysis of the ever-increasing wealth of publically available phosphorylation data.

To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic buffering relationships such as redundancy. Three types of genetic buffering relationships are identified: mixed epistasis, complete redundancy, and quantitative redundancy. In mixed epistasis, the most common buffering relationship, different gene sets respond in different epistatic ways. Mixed epistasis arises from pairs of regulators that have only partial overlap in function and that are coupled by additional regulatory links such as repression of one by the other. Such regulatory modules confer the ability to control different combinations of processes depending on condition or context. These properties likely contribute to the evolutionary maintenance of paralogs and indicate a way in which signaling pathways connect for multiprocess control., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The root cap has a central role in root growth, determining the growth trajectory and facilitating penetration into the soil. Root cap cells have specialized functions and morphologies, and border cells are released into the rhizosphere by specific cell wall modifications. Here, we demonstrate that the cellular maturation of root cap is redundantly regulated by three genes, SOMBRERO (SMB), BEARSKIN1 (BRN1), and BRN2, which are members of the Class IIB NAC transcription factor family, together with the VASCULAR NAC DOMAIN (VND) and NAC SECONDARY WALL THICKENING PROMOTING FACTOR (NST) genes that regulate secondary cell wall synthesis in specialized cell types. Lateral cap cells in smb-3 mutants continue to divide and fail to detach from the root, phenotypes that are independent of FEZ upregulation in smb-3. In brn1-1 brn2-1 double mutants, columella cells fail to detach, while in triple mutants, cells fail to mature in all parts of the cap. This complex genetic redundancy involves differences in expression, protein activity, and target specificity. All three genes have very similar overexpression phenotypes to the VND/NST genes, indicating that members of this family are largely functionally equivalent. Our results suggest that Class IIB NAC proteins regulate cell maturation in cells that undergo terminal differentiation with strong cell wall modifications.

Background: Homology is a crucial concept in comparative genomics. The algorithm probably most widely used for homology detection in comparative genomics, is BLAST. Usually a stringent score cutoff is applied to distinguish putative homologs from possible false positive hits. As a consequence, some BLAST hits are discarded that are in fact homologous., Results: Analogous to the use of the genomics context in genome alignments, we test whether conserved functional context can be used to select candidate homologs from insignificant BLAST hits. We make a co-complex network alignment between complex subunits in yeast and human and find that proteins with an insignificant BLAST hit that are part of homologous complexes, are likely to be homologous themselves. Further analysis of the distant homologs we recovered using the co-complex network alignment, shows that a large majority of these distant homologs are in fact ancient paralogs., Conclusions: Our results show that, even though evolution takes place at the sequence and genome level, co-complex networks can be used as circumstantial evidence to improve confidence in the homology of distantly related sequences.

Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.

The members of the Ras-like superfamily of small GTP-binding proteins are molecular switches that are in general regulated in time and space by guanine nucleotide exchange factors and GTPase activating proteins. The Ras-like G-proteins Ras, Rap and Ral are regulated by a variety of guanine nucleotide exchange factors that are characterized by a CDC25 homology domain. Here we study the evolution of the Ras pathway by determining the evolutionary history of CDC25 homology domain coding sequences. We identified CDC25 homology domain coding sequences in animals, fungi and a wide range of protists, but not in plants. This suggests that the CDC25 homology domain originated in or before the last eukaryotic ancestor but was subsequently lost in plant. We provide evidence that at least seven different ancestral Ras guanine nucleotide exchange factors were present in the ancestor of fungi and animals. Differences between present day fungi and animals are the result of loss of ancestral Ras guanine nucleotide exchange factors early in fungal and animal evolution combined with lineage specific duplications and domain acquisitions. In addition, we identify Ral guanine exchange factors and Ral in early diverged fungi, dating the origin of Ral signaling back to before the divergence of animals and fungi. We conclude that the Ras signaling pathway evolved by gradual change as well as through differential sampling of the ancestral CDC25 homology domain repertoire by both fungi and animals. Finally, a comparison of the domain composition of the Ras guanine nucleotide exchange factors shows that domain addition and diversification occurred both prior to and after the fungal-animal split.

Unlabelled: The aim of the study was to evaluate long-term toxicity of adjuvant treatment in early stage ovarian cancer survivors. Data from all patients treated in one hospital for early stage ovarian cancer diagnosed between 1980 and 1990 were collected using a structured data form. In 93 FIGO stages I and II patients, cytoreductive and staging surgery was performed; 15 received no adjuvant treatment (controls), 39 whole abdominal radiotherapy (WART) and 39 platin-based chemotherapy. Median age at diagnosis was 54 years (range 21-83 years). During follow-up, 49/93 (53%) patients have died with a median overall survival of 18.4 years (95% CI 12.8-23.9). In both the radiotherapy and the chemotherapy group, 50% of patients reported long-term side-effects (all grades) versus 13% of controls. Two patients in the WART group died from bowel complications. Secondary malignancies were observed in 16 patients. Of all patients alive at the last follow-up, 12/17 (71%) patients treated with radiotherapy and 11/18 (61%) treated with chemotherapy experienced long-term morbidity versus 2/9 (22%) controls (P=0.03)., In Conclusion: Long-term follow-up of early stage ovarian cancer patients showed lasting GI morbidity in the survivors treated with adjuvant radiotherapy, which has therefore become obsolete. Cisplatin-based chemotherapy caused peripheral neuropathy versus virtual absence of problems in the survivors of just surgery, emphasising the need for strict criteria before instigating adjuvant treatment.

Although functionally related proteins can be reliably predicted from phylogenetic profiles, many functional modules do not seem to evolve cohesively according to case studies and systematic analyses in prokaryotes. In this study we quantify the extent of evolutionary cohesiveness of functional modules in eukaryotes and probe the biological and methodological factors influencing our estimates. We have collected various datasets of protein complexes and pathways in Saccheromyces cerevisiae. We define orthologous groups on 34 eukaryotic genomes and measure the extent of cohesive evolution of sets of orthologous groups of which members constitute a known complex or pathway. Within this framework it appears that most functional modules evolve flexibly rather than cohesively. Even after correcting for uncertain module definitions and potentially problematic orthologous groups, only 46% of pathways and complexes evolve more cohesively than random modules. This flexibility seems partly coupled to the nature of the functional module because biochemical pathways are generally more cohesively evolving than complexes.

Background: Thanks to recent high coverage mass-spectrometry studies and reconstructed protein complexes, we are now in an unprecedented position to study the evolution of biological systems. Gene duplications, known to be a major source of innovation in evolution, can now be readily examined in the context of protein complexes., Results: We observe that paralogs operating in the same complex fulfill different roles: mRNA dosage increase for more than a hundred cytosolic ribosomal proteins, mutually exclusive participation of at least 54 paralogs resulting in alternative forms of complexes, and 24 proteins contributing to bona fide structural growth. Inspection of paralogous proteins participating in two independent complexes shows that an ancient, pre-duplication protein functioned in both multi-protein assemblies and a gene duplication event allowed the respective copies to specialize and split their roles., Conclusion: Variants with conditionally assembled, paralogous subunits likely have played a role in yeast's adaptation to anaerobic conditions. In a number of cases the gene duplication has given rise to one duplicate that is no longer part of a protein complex and shows an accelerated rate of evolution. Such genes could provide the raw material for the evolution of new functions.

Background: Reversible phosphorylation of proteins is involved in a wide range of processes, ranging from signaling cascades to regulation of protein complex assembly. Little is known about the structure and evolution of phosphorylation networks. Recent high-throughput phosphoproteomics studies have resulted in the rapid accumulation of phosphopeptide datasets for many model organisms. Here, we exploit these novel data for the comparative analysis of phosphorylation events between different species of eukaryotes., Results: Comparison of phosphoproteomics datasets of six eukaryotes yields an overlap ranging from approximately 700 sites for human and mouse (two large datasets of closely related species) to a single site for fish and yeast (distantly related as well as two of the smallest datasets). Some conserved events appear surprisingly old; those shared by plant and animals suggest conservation over the time scale of a billion years. In spite of the hypothesized incomprehensive nature of phosphoproteomics datasets and differences in experimental procedures, we show that the overlap between phosphoproteomes is greater than expected by chance and indicates increased functional relevance. Despite the dynamic nature of the evolution of phosphorylation, the relative overlap between the different datasets is identical to the phylogeny of the species studied., Conclusion: This analysis provides a framework for the generation of biological insights by comparative analysis of high-throughput phosphoproteomics datasets. We expect the rapidly growing body of data from high-throughput mass spectrometry analysis to make comparative phosphoproteomics a powerful tool for elucidating the evolutionary and functional dynamics of reversible phosphorylation.

The formation of proteins into stable protein complexes plays a fundamental role in the operation of the cell. The study of the degree of evolutionary conservation of protein complexes between species and the evolution of protein-protein interactions has been hampered by lack of comprehensive coverage of the high-throughput (HTP) technologies that measure the interactome. We show that new high-throughput datasets on protein co-purification in yeast have a substantially lower false negative rate than previous datasets when compared to known complexes. These datasets are therefore more suitable to estimate the conservation of protein complex membership than hitherto possible. We perform comparative genomics between curated protein complexes from human and the HTP data in Saccharomyces cerevisiae to study the evolution of co-complex memberships. This analysis revealed that out of the 5,960 protein pairs that are part of the same complex in human, 2,216 are absent because both proteins lack an ortholog in S. cerevisiae, while for 1,828 the co-complex membership is disrupted because one of the two proteins lacks an ortholog. For the remaining 1,916 protein pairs, only 10% were never co-purified in the large-scale experiments. This implies a conservation level of co-complex membership of 90% when the genes coding for the protein pairs that participate in the same protein complex are also conserved. We conclude that the evolutionary dynamics of protein complexes are, by and large, not the result of network rewiring (i.e. acquisition or loss of co-complex memberships), but mainly due to genomic acquisition or loss of genes coding for subunits. We thus reveal evidence for the tight interrelation of genomic and network evolution.

Preimplantation genetic diagnosis (PGD) is a method for identifying genetic abnormalities in embryos obtained via in-vitro fertilisation before their implantation. There are many indications for PGD, which offers an alternative to prenatal diagnosis, although each modality has its advantages and drawbacks. In three female patients there were clear indications for PGD: carriage of the gene for myotubular myopathy, a balanced complex chromosomal translocation, and a Robertsonian translocation in the male partner, respectively. The first patient eventually abandoned PGD and chose prenatal diagnosis. She had a total of three abortions. A twin pregnancy with a so-called foetal reduction resulted in the birth of a genetically normal child. The second patient had one spontaneous abortion and subsequently underwent two PGD cycles, resulting in an uncomplicated pregnancy and the delivery of a genetically normal child. The third patient had one genetically normal child and six spontaneous abortions. She then underwent the PGD procedure successfully and is expecting twins; the pregnancy has been uncomplicated. During preconception counselling of couples at high genetic risk, physicians should be aware of PGD as an alternative to prenatal diagnosis.

We study gene family coevolution on a tree of life based on a large-scale ancestral gene content reconstruction, which includes gene duplication and deletion events. The insights obtained from this study are threefold: (1) Global properties, such as the distribution of coevolution partners and the formation of disconnected clusters of coevolving families, can be an inevitable consequence of evolution along a tree. (2) Concerted family expansion (gene duplication) and contraction (gene deletion) reflect functional constraints and therefore lead to better function prediction. (3) "Long-range" coevolutionary relationships, caused mostly by large family expansions or contractions, reveal high-level evolutionary organization of cellular processes in prokaryotes.

Background: Homology is a key concept in both evolutionary biology and genomics. Detection of homology is crucial in fields like the functional annotation of protein sequences and the identification of taxon specific genes. Basic homology searches are still frequently performed by pairwise search methods such as BLAST. Vast improvements have been made in the identification of homologous proteins by using more advanced methods that use sequence profiles. However additional improvement could be made by exploiting sources of genomic information other than the primary sequence or tertiary structure., Results: We test the hypothesis that extrinsic gene properties gene length and gene order can be of help in differentiating spurious sequence similarity from homology in the gray zone. Sharing gene order and similarity in size dramatically increase the chance of a query-hit pair being homologous: gray zone query-hit pairs of similar size and with conserved gene order are homologous in 99% of all cases, while for query-hit pairs without gene order conservation and with different sizes this is only 55%., Conclusion: We have shown that using gene length and gene order drastically improves the detection of homologs within the BLAST gray zone. Our findings suggest that the use of such extrinsic gene properties can also improve the performance of homology detection by more advanced methods, and our study thereby underscores the importance of true data integration for fully exploiting genomic information.

Motivation: Phylogenomics integrates the vast amount of phylogenetic information contained in complete genome sequences, and is rapidly becoming the standard for reliably inferring species phylogenies. There are, however, fundamental differences between the ways in which phylogenomic approaches like gene content, superalignment, superdistance and supertree integrate the phylogenetic information from separate orthologous groups. Furthermore, they all depend on the method by which the orthologous groups are initially determined. Here, we systematically compare these four phylogenomic approaches, in parallel with three approaches for large-scale orthology determination: pairwise orthology, cluster orthology and tree-based orthology., Results: Including various phylogenetic methods, we apply a total of 54 fully automated phylogenomic procedures to the fungi, the eukaryotic clade with the largest number of sequenced genomes, for which we retrieved a golden standard phylogeny from the literature. Phylogenomic trees based on gene content show, relative to the other methods, a bias in the tree topology that parallels convergence in lifestyle among the species compared, indicating convergence in gene content., Conclusions: Complete genomes are no guarantee for good or even consistent phylogenies. However, the large amounts of data in genomes enable us to carefully select the data most suitable for phylogenomic inference. In terms of performance, the superalignment approach, combined with restrictive orthology, is the most successful in recovering a fungal phylogeny that agrees with current taxonomic views, and allows us to obtain a high-resolution phylogeny. We provide solid support for what has grown to be a common practice in phylogenomics during its advance in recent years., Supplementary Information: Supplementary data are available at Bioinformatics online.

Background: Orthology is one of the cornerstones of gene function prediction. Dividing the phylogenetic relations between genes into either orthologs or paralogs is however an oversimplification. Already in two-species gene-phylogenies, the complicated, non-transitive nature of phylogenetic relations results in inparalogs and outparalogs. For situations with more than two species we lack semantics to specifically describe the phylogenetic relations, let alone to exploit them. Published procedures to extract orthologous groups from phylogenetic trees do not allow identification of orthology at various levels of resolution, nor do they document the relations between the orthologous groups., Results: We introduce "levels of orthology" to describe the multi-level nature of gene relations. This is implemented in a program LOFT (Levels of Orthology From Trees) that assigns hierarchical orthology numbers to genes based on a phylogenetic tree. To decide upon speciation and gene duplication events in a tree LOFT can be instructed either to perform classical species-tree reconciliation or to use the species overlap between partitions in the tree. The hierarchical orthology numbers assigned by LOFT effectively summarize the phylogenetic relations between genes. The resulting high-resolution orthologous groups are depicted in colour, facilitating visual inspection of (large) trees. A benchmark for orthology prediction, that takes into account the varying levels of orthology between genes, shows that the phylogeny-based high-resolution orthology assignments made by LOFT are reliable., Conclusion: The "levels of orthology" concept offers high resolution, reliable orthology, while preserving the relations between orthologous groups. A Windows as well as a preliminary Java version of LOFT is available from the LOFT website http://www.cmbi.ru.nl/LOFT.

Background: In the post-genomic era various functional genomics, proteomics and computational techniques have been developed to elucidate the protein interaction network. While some of these techniques are specific for a certain type of interaction, most predict a mixture of interactions. Qualitative labels are essential for the molecular biologist to experimentally verify predicted interactions., Results: Of the individual protein-protein interaction prediction methods, some can predict physical interactions without producing other types of interactions. None of the methods can specifically predict metabolic interactions. We have constructed an 'omics evidence landscape' that combines all sources of evidence for protein interactions from various types of omics data for Saccharomyces cerevisiae. We explore this evidence landscape to identify areas with either only metabolic or only physical interactions, allowing us to specifically predict the nature of new interactions in these areas. We combine the datasets in ways that examine the whole evidence landscape and not only the highest scoring protein pairs in both datasets and find specific predictions., Conclusion: The combination of evidence types in the form of the evidence landscape allows for qualitative labels to be inferred and placed on the predicted protein interaction network of S. cerevisiae. These qualitative labels will help in the biological interpretation of gene networks and will direct experimental verification of the predicted interactions.

Our understanding of the tree of life (TOL) is still fragmentary. Until recently, molecular phylogeneticists have built trees based on ribosomal RNA sequences and selected protein sequences, which, however, usually suffered from lack of support for the deeper branches and inconsistencies probably due to limited subsampling of the entire genome. Now, phylogenetic hypotheses can be based on the analysis of full genomes. We used available complete genome data as well as the eukaryote orthologous group (KOG) proteins to reconstruct with confidence basal branches of the fungal TOL. Phylogenetic analysis of a core of 531 KOGs shared among 21 fungal genomes, three animal genomes and one plant genome showed a single tree with high support resulting from four different methods of phylogenetic reconstruction. The single tree that we inferred from our dataset showed excellent nodal support for each branch, suggesting that it reflects the true phylogenetic relationships of the species involved.

The phylogenetic position of the fission yeast Schizosaccharomyces pombe in the fungal Tree of Life is still controversial. Three alternative phylogenetic positions have been proposed in the literature, namely (1) a position basal to the Hemiascomycetes and Euascomycetes, (2) a position as a sister group to the Euascomycetes with the Hemiascomycetes as a basal branch, or (3) a sister group to the Hemiascomycetes with Euascomycetes as a basal branch. Here we compared 91 clusters of orthologous proteins containing a single orthologue that are shared by 19 eukaryote genomes. The major part of these 91 orthologues supports a phylogenetic position of S. pombe as a basal lineage among the Ascomycota, thus supporting the second proposition. Interestingly, part of the orthologous proteins supported a fourth, not yet described alternative, in which S. pombe is basal to both Basidiomycota and Ascomycota. Both topologies of phylogenetic trees are well supported. We believe that both reflect correctly the phylogenetic history of the species concerned. This apparent paradox may point to a heterogeneous nuclear genome of the fungi. Importantly, this needs to be taken in consideration for a correct understanding of the fungal Tree of Life.

Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment. It is also the nitrogen cycle's major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids and the role of cytoplasm differentiation are unique in biology. Here we use environmental genomics--the reconstruction of genomic data directly from the environment--to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis from a complex bioreactor community. The genome data illuminate the evolutionary history of the Planctomycetes and allow us to expose the genetic blueprint of the organism's special properties. Most significantly, we identified candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism, and discovered unexpected metabolic versatility.

Background: Peroxisomes are ubiquitous eukaryotic organelles involved in various oxidative reactions. Their enzymatic content varies between species, but the presence of common protein import and organelle biogenesis systems support a single evolutionary origin. The precise scenario for this origin remains however to be established. The ability of peroxisomes to divide and import proteins post-translationally, just like mitochondria and chloroplasts, supports an endosymbiotic origin. However, this view has been challenged by recent discoveries that mutant, peroxisome-less cells restore peroxisomes upon introduction of the wild-type gene, and that peroxisomes are formed from the Endoplasmic Reticulum. The lack of a peroxisomal genome precludes the use of classical analyses, as those performed with mitochondria or chloroplasts, to settle the debate. We therefore conducted large-scale phylogenetic analyses of the yeast and rat peroxisomal proteomes., Results: Our results show that most peroxisomal proteins (39-58%) are of eukaryotic origin, comprising all proteins involved in organelle biogenesis or maintenance. A significant fraction (13-18%), consisting mainly of enzymes, has an alpha-proteobacterial origin and appears to be the result of the recruitment of proteins originally targeted to mitochondria. Consistent with the findings that peroxisomes are formed in the Endoplasmic Reticulum, we find that the most universally conserved Peroxisome biogenesis and maintenance proteins are homologous to proteins from the Endoplasmic Reticulum Assisted Decay pathway., Conclusion: Altogether our results indicate that the peroxisome does not have an endosymbiotic origin and that its proteins were recruited from pools existing within the primitive eukaryote. Moreover the reconstruction of primitive peroxisomal proteomes suggests that ontogenetically as well as phylogenetically, peroxisomes stem from the Endoplasmic Reticulum., Reviewers: This article was reviewed by Arcady Mushegian, Gáspár Jékely and John Logsdon., Open Peer Review: Reviewed by Arcady Mushegian, Gáspar Jékely and John Logsdon. For the full reviews, please go to the Reviewers' comments section.

We have developed an automatable procedure for reconstructing the tree of life with branch lengths comparable across all three domains. The tree has its basis in a concatenation of 31 orthologs occurring in 191 species with sequenced genomes. It revealed interdomain discrepancies in taxonomic classification. Systematic detection and subsequent exclusion of products of horizontal gene transfer increased phylogenetic resolution, allowing us to confirm accepted relationships and resolve disputed and preliminary classifications. For example, we place the phylum Acidobacteria as a sister group of delta-Proteobacteria, support a Gram-positive origin of Bacteria, and suggest a thermophilic last universal common ancestor.

Background: The massive scale of microarray derived gene expression data allows for a global view of cellular function. Thus far, comparative studies of gene expression between species have been based on the level of expression of the gene across corresponding tissues, or on the co-expression of the gene with another gene., Results: To compare gene expression between distant species on a global scale, we introduce the "expression context". The expression context of a gene is based on the co-expression with all other genes that have unambiguous counterparts in both genomes. Employing this new measure, we show 1) that the expression context is largely conserved between orthologs, and 2) that sequence identity shows little correlation with expression context conservation after gene duplication and speciation., Conclusion: This means that the degree of sequence identity has a limited predictive quality for differential expression context conservation between orthologs, and thus presumably also for other facets of gene function.

A key complication in comparative genomics for reliable gene function prediction is the existence of duplicated genes. To study the effect of gene duplication on function prediction, we analyze orthologs between pairs of genomes where in one genome the orthologous gene has duplicated after the speciation of the two genomes (i.e. inparalogs). For these duplicated genes we investigate whether the gene that is most similar on the sequence level is also the gene that has retained the ancestral gene-neighborhood. Although the majority of investigated cases show a consistent pattern between sequence similarity and gene-neighborhood conservation, a substantial fraction, 29-38%, is inconsistent. The observation of inconsistency is not the result of a chance outcome owing to a lack of divergence time between inparalogs, but rather it seems to be the result of a chance outcome caused by very similar rates of sequence evolution of both inparalogs relative to their ortholog. If one-to-one orthologous relationships are required, it is advisable to combine contextual information (i.e. gene-neighborhood in prokaryotes and co-expression in eukaryotes) with protein sequence information to predict the most probable functional equivalent ortholog in the presence of inparalogs.

The availability of genome sequences and functional genomics data from multiple species enables us to compare the composition of biomolecular systems like biochemical pathways and protein complexes between species. Here, we review small- and large-scale, "genomics-based" approaches to biomolecular systems variation. In general, caution is required when comparing the results of bioinformatics analyses of genomes or of functional genomics data between species. Limitations to the sensitivity of sequence analysis tools and the noisy nature of genomics data tend to lead to systematic overestimates of the amount of variation. Nevertheless, the results from detailed manual analyses, and of large-scale analyses that filter out systematic biases, point to a large amount of variation in the composition of biomolecular systems. Such observations challenge our understanding of the function of the systems and their individual components and can potentially facilitate the identification and functional characterization of sub-systems within a system. Mapping the inter-species variation of complex biomolecular systems on a phylogenetic species tree allows one to reconstruct their evolution.

Genomes, functional genomics data and 3D structure reflect different aspects of protein function. Here, we combine these data to predict that BolA, a widely distributed protein family with unknown function, is a reductase that interacts with a glutaredoxin. Comparisons at the 3D structure level as well as at the sequence profile level indicate homology between BolA and OsmC, an enzyme that reduces organic peroxides. Complementary to this, comparative analyses of genomes and genomics data provide strong evidence of an interaction between BolA and the mono-thiol glutaredoxin family. The interaction between BolA and a mono-thiol glutaredoxin is of particular interest because BolA does not, in contrast to its homolog OsmC, have evolutionarily conserved cysteines to provide it with reducing equivalents. We propose that BolA uses the mono-thiol glutaredoxin as the source for these.

Genome trees are a means to capture the overwhelming amount of phylogenetic information that is present in genomes. Different formalisms have been introduced to reconstruct genome trees on the basis of various aspects of the genome. On the basis of these aspects, we separate genome trees into five classes: (a) alignment-free trees based on statistic properties of the genome, (b) gene content trees based on the presence and absence of genes, (c) trees based on chromosomal gene order, (d) trees based on average sequence similarity, and (e) phylogenomics-based genome trees. Despite their recent development, genome tree methods have already had some impact on the phylogenetic classification of bacterial species. However, their main impact so far has been on our understanding of the nature of genome evolution and the role of horizontal gene transfer therein. An ideal genome tree method should be capable of using all gene families, including those containing paralogs, in a phylogenomics framework capitalizing on existing methods in conventional phylogenetic reconstruction. We expect such sophisticated methods to help us resolve the branching order between the main bacterial phyla.

A full description of a protein's function requires knowledge of all partner proteins with which it specifically associates. From a functional perspective, 'association' can mean direct physical binding, but can also mean indirect interaction such as participation in the same metabolic pathway or cellular process. Currently, information about protein association is scattered over a wide variety of resources and model organisms. STRING aims to simplify access to this information by providing a comprehensive, yet quality-controlled collection of protein-protein associations for a large number of organisms. The associations are derived from high-throughput experimental data, from the mining of databases and literature, and from predictions based on genomic context analysis. STRING integrates and ranks these associations by benchmarking them against a common reference set, and presents evidence in a consistent and intuitive web interface. Importantly, the associations are extended beyond the organism in which they were originally described, by automatic transfer to orthologous protein pairs in other organisms, where applicable. STRING currently holds 730,000 proteins in 180 fully sequenced organisms, and is available at http://string.embl.de/.

Genomic data provide invaluable, yet unreliable information about protein function. However, if the overlap in information among various genomic datasets is taken into account, one observes an increase in the reliability of the protein-function predictions that can be made. Recently published approaches achieved this either by comparing the same type of data from multiple species (horizontal comparative genomics) or by using subtle, Bayesian methods to compare different types of genomic data from a single species (vertical comparative genomics). In this article, we discuss these methods, illustrating horizontal comparative genomics by comparing yeast two-hybrid (Y2H) data from Saccharomyces cerevisiae with Y2H data from Drosophila melanogaster, and illustrating vertical comparative genomics by comparing RNA expression data with proteomic data from Plasmodium falciparum.

Phylogenetic trees based on gene repertoires are remarkably similar to the current consensus of life history. Yet it has been argued that shared gene content is unreliable for phylogenetic reconstruction because of convergence in gene content due to horizontal gene transfer and parallel gene loss. Here we test this argument, by filtering out as noise those orthologous groups that have an inconsistent phylogenetic distribution, using two independent methods. The resulting phylogenies do indeed contain small but significant improvements. More importantly, we find that the majority of orthologous groups contain some phylogenetic signal and that the resulting phylogeny is the only detectable signal present in the gene distribution across genomes. Horizontal gene transfer or parallel gene loss does not cause systematic biases in the gene content tree.

We investigated the gene coexpression network in Saccharomyces cerevisiae, in which genes are linked when they are coregulated. This network is shown to have a scale-free, small-world architecture. Such architecture is typical of biological networks in which the nodes are connected when they are involved in the same biological process. Current models for the evolution of intracellular networks do not adequately reproduce the features that we observe in the network. We therefore derive a new model for its evolution based on the observation that there is a positive correlation between the sequence similarity of paralogues and their probability of coexpression or sharing of transcription factor binding sites (TFBSs). The simple, neutralist's model consists of (1) coduplication of genes with their TFBSs, (2) deletion and duplication of individual TFBSs and (3) gene loss. A network is constructed by connecting genes that share multiple TFBSs. Our model reproduces the scale-free, small-world architecture of the coregulation network and the homology relations between coregulated genes without the need for selection either at the level of the network structure or at the level of gene regulation.

Functional modules are considered the primary building blocks of biomolecular systems. Here we study to what extent functional modules behave cohesively across genomes:That is, are functional modules also evolutionary modules? We probe this question by analyzing for a large collection of functional modules the phyletic patterns of their genes across 110 genomes. The majority of functional modules display limited evolutionary modularity. This result confirms certain comparative genome analyses, but is in contrast to implicit assumptions in the systems analysis of functional genomics data. We show that this apparent interspecies flexibility in the organization of functional modules depends more on functional differentiation within orthologous groups of genes, than on noise in the functional module definitions. When filtering out these sources of nonmodularity, even though very few functional modules behave perfectly modular in evolution, about half behave at least significantly more modular than a random set of genes. There are substantial differences in the evolutionary modularity between individual functional modules as well as between collections of functional modules, partly corresponding to conceptual differences in the functional module definition, which make comparisons between functional module collections biologically difficult to interpret. Analysis within one collection does not suffer from such differences, and we show that within the EcoCyc metabolic pathway database, biosynthetic pathways are evolutionarily more modular than catabolic pathways.

In all genome-sequencing projects completed to date, a considerable number of 'gaps' have been found in the biochemical pathways of the respective species. In many instances, missing enzymes are displaced by analogs, functionally equivalent proteins that have evolved independently and lack sequence and structural similarity. Here we fill such gaps by analyzing anticorrelating occurrences of genes across species. Our approach, applied to the thiamin biosynthesis pathway comprising approximately 15 catalytic steps, predicts seven instances in which known enzymes have been displaced by analogous proteins. So far we have verified four predictions by genetic complementation, including three proteins for which there was no previous experimental evidence of a role in the thiamin biosynthesis pathway. For one hypothetical protein, biochemical characterization confirmed the predicted thiamin phosphate synthase (ThiE) activity. The results demonstrate the ability of our computational approach to predict specific functions without taking into account sequence similarity.

We show that gene co-expression, which generally provides only a very weak signal for the prediction of functional interactions, can provide a reliable signal by exploiting evolutionary conservation. The encoded proteins of conserved co-expressed gene pairs are highly likely to be part of the same pathway not only after speciation (98%), but also after parallel gene duplication (97%). Conserved co-expression combined with homology data enables us to predict specific gene functions. The use of conservation between parallel duplicated gene pairs to predict function is especially promising given that gene duplication is common in eukaryotes, and that data from only a single organism can be used.

In the genomics era, the interactions between proteins are at the center of attention. Genomic-context methods used to predict these interactions have been put on a quantitative basis, revealing that they are at least on an equal footing with genomics experimental data. A survey of experimentally confirmed predictions proves the applicability of these methods, and new concepts to predict protein interactions in eukaryotes have been described. Finally, the interaction networks that can be obtained by combining the predicted pair-wise interactions have enough internal structure to detect higher levels of organization, such as 'functional modules'.

Functional links between proteins can often be inferred from genomic associations between the genes that encode them: groups of genes that are required for the same function tend to show similar species coverage, are often located in close proximity on the genome (in prokaryotes), and tend to be involved in gene-fusion events. The database STRING is a precomputed global resource for the exploration and analysis of these associations. Since the three types of evidence differ conceptually, and the number of predicted interactions is very large, it is essential to be able to assess and compare the significance of individual predictions. Thus, STRING contains a unique scoring-framework based on benchmarks of the different types of associations against a common reference set, integrated in a single confidence score per prediction. The graphical representation of the network of inferred, weighted protein interactions provides a high-level view of functional linkage, facilitating the analysis of modularity in biological processes. STRING is updated continuously, and currently contains 261 033 orthologs in 89 fully sequenced genomes. The database predicts functional interactions at an expected level of accuracy of at least 80% for more than half of the genes; it is online at http://www.bork.embl-heidelberg.de/STRING/.

Bifidobacteria are Gram-positive prokaryotes that naturally colonize the human gastrointestinal tract (GIT) and vagina. Although not numerically dominant in the complex intestinal microflora, they are considered as key commensals that promote a healthy GIT. We determined the 2.26-Mb genome sequence of an infant-derived strain of Bifidobacterium longum, and identified 1,730 possible coding sequences organized in a 60%-GC circular chromosome. Bioinformatic analysis revealed several physiological traits that could partially explain the successful adaptation of this bacteria to the colon. An unexpectedly large number of the predicted proteins appeared to be specialized for catabolism of a variety of oligosaccharides, some possibly released by rare or novel glycosyl hydrolases acting on "nondigestible" plant polymers or host-derived glycoproteins and glycoconjugates. This ability to scavenge from a large variety of nutrients likely contributes to the competitiveness and persistence of bifidobacteria in the colon. Many genes for oligosaccharide metabolism were found in self-regulated modules that appear to have arisen in part from gene duplication or horizontal acquisition. Complete pathways for all amino acids, nucleotides, and some key vitamins were identified; however, routes for Asp and Cys were atypical. More importantly, genome analysis provided insights into the reciprocal interactions of bifidobacteria with their hosts. We identified polypeptides that showed homology to most major proteins needed for production of glycoprotein-binding fimbriae, structures that could possibly be important for adhesion and persistence in the GIT. We also found a eukaryotic-type serine protease inhibitor (serpin) possibly involved in the reported immunomodulatory activity of bifidobacteria.

Comprehensive protein protein interaction maps promise to reveal many aspects of the complex regulatory network underlying cellular function. Recently, large-scale approaches have predicted many new protein interactions in yeast. To measure their accuracy and potential as well as to identify biases, strengths and weaknesses, we compare the methods with each other and with a reference set of previously reported protein interactions.

By combining the pairwise interactions between proteins, as predicted by the conserved co-occurrence of their genes in operons, we obtain protein interaction networks. Here we study the properties of such networks to identify functional modules: sets of proteins that together are involved in a biological process. The complete network contains 3,033 orthologous groups of proteins in 38 genomes. It consists of one giant component, containing 1,611 orthologous groups, and of 516 small disjointed clusters that, on average, contain only 2.7 orthologous groups. These small clusters have a homogeneous functional composition and thus represent functional modules in themselves. Analysis of the giant component reveals that it is a scale-free, small-world network with a high degree of local clustering (C = 0.6). It consists of locally highly connected subclusters that are connected to each other by linker proteins. The linker proteins tend to have multiple functions, or are involved in multiple processes and have an above average probability of being essential. By splitting up the giant component at these linker proteins, we identify 265 subclusters that tend to have a homogeneous functional composition. The rare functional inhomogeneities in our subclusters reflect the mixing of different types of (molecular) functions in a single cellular process, exemplified by subclusters containing both metabolic enzymes as well as the transcription factors that regulate them. Comparative genome analysis, thus, allows identification of a level of functional interaction between that of pairwise interactions, and of the complete genome.

In the course of evolution, genomes are shaped by processes like gene loss, gene duplication, horizontal gene transfer, and gene genesis (the de novo origin of genes). Here we reconstruct the gene content of ancestral Archaea and Proteobacteria and quantify the processes connecting them to their present day representatives based on the distribution of genes in completely sequenced genomes. We estimate that the ancestor of the Proteobacteria contained around 2500 genes, and the ancestor of the Archaea around 2050 genes. Although it is necessary to invoke horizontal gene transfer to explain the content of present day genomes, gene loss, gene genesis, and simple vertical inheritance are quantitatively the most dominant processes in shaping the genome. Together they result in a turnover of gene content such that even the lineage leading from the ancestor of the Proteobacteria to the relatively large genome of Escherichia coli has lost at least 950 genes. Gene loss, unlike the other processes, correlates fairly well with time. This clock-like behavior suggests that gene loss is under negative selection, while the processes that add genes are under positive selection.

Much has been learned about the cellular pathology of Friedreich's ataxia, a recessive neurodegenerative disease resulting from insufficient expression of the mitochondrial protein frataxin. However, the biochemical function of frataxin has remained obscure, hampering attempts at therapeutic intervention. To predict functional interactions of frataxin with other proteins we investigated whether its gene specifically co-occurs with any other genes in sequenced genomes. In 56 available genomes we identified two genes with identical phylogenetic distributions to the frataxin/cyaY gene: hscA and hscB/JAC1. These genes have not only emerged in the same evolutionary lineage as the frataxin gene, they have also been lost at least twice with it, and they have been horizontally transferred with it in the evolution of the mitochondria. The proteins encoded by hscA and hscB, the chaperone HSP66 and the co-chaperone HSP20, have been shown to be required for the synthesis of 2Fe-2S clusters on ferredoxin in proteobacteria. JAC1, an ortholog of hscB, and SSQ1, a paralog of hscA, have been shown to be required for iron-sulfur cluster assembly in mitochondria of Saccharomyces cerevisiae. Combining data on the co-occurrence of genes in genomes with experimental and predicted cellular localization data of their proteins supports the hypothesis that frataxin is directly involved in iron-sulfur cluster protein assembly. They indicate that frataxin is specifically involved in the same sub-process as HSP20/Jac1p.