Assessing functional divergence in EF-1alpha and its paralogs in eukaryotes and archaebacteria

A number of methods have recently been published that use phylogenetic information extracted from large multiple sequence alignments to detect sites that have changed properties in related protein families. In this study we use such methods to assess functional divergence between eukaryotic EF-1α (eEF-1α), archaebacterial EF-1α (aEF-1α) and two eukaryote-specific EF-1α paralogs—eukaryotic release factor 3 (eRF3) and Hsp70 subfamily B suppressor 1 (HBS1). Overall, the evolutionary modes of aEF-1α, HBS1 and eRF3 appear to significantly differ from that of eEF-1α. However, functionally divergent (FD) sites detected between aEF-1α and eEF-1α only weakly overlap with sites implicated as putative EF-1β or aminoacyl-tRNA (aa-tRNA) binding residues in EF-1α, as expected based on the shared ancestral primary translational functions of these two orthologs. In contrast, FD sites detected between eEF-1α and its paralogs significantly overlap with the putative EF-1β and/or aa-tRNA binding sites in EF-1α. In eRF3 and HBS1, these sites appear to be released from functional constraints, indicating that they bind neither eEF-1β nor aa-tRNA. These results are consistent with experimental observations that eRF3 does not bind to aa-tRNA, but do not support the ‘EF-1α-like’ function recently proposed for HBS1. We re-assess the available genetic data for HBS1 in light of our analyses, and propose that this protein may function in stop codon-independent peptide release.     

War of the Iguanas: conflicting molecular and morphological phylogenies and long-branch attraction in iguanid lizards

Recent studies based on different types of data (i.e., morphology, molecules) have found strongly conflicting phylogenies for the genera of iguanid lizards but have been unable to explain the basis for this incongruence. We reanalyze published data from morphology and from the mitochondrial ND4, cytochrome b, 12S, and 16S genes to explore the sources of incongruence and resolve these conflicts. Much of the incongruence centers on the genus Cyclura, which is the sister taxon of Iguana, according to parsimony analyses of the morphology and the ribosomal genes, but is the sister taxon of all other Iguanini, according to the protein-coding genes. Maximum likelihood analyses show that there has been an increase in the rate of nucleotide substitution in Cyclura in the two protein-coding genes (ND4 and cytochrome b), although this increase is not as clear when parsimony is used to estimate branch lengths. Parametric simulations suggest that Cyclura may be misplaced by the protein-coding genes as a result of long-branch attraction; even when Cyclura and Iguana are sister taxa in a simulated phylogeny, Cyclura is still placed as the basal member of the Iguanini by parsimony analysis in 55% of the replicates. A similar long-branch attraction problem may also exist in the morphological data with regard to the placement of Sauromalus with the Galápagos iguanas (Amblyrhynchus and Conolophus). The results have many implications for the analysis of diverse data sets, the impact of long branches on parsimony and likelihood methods, and the use of certain protein-coding genes in phylogeny reconstruction.     

Heterotachy and long-branch attraction in phylogenetics

Background  

Probabilistic methods have progressively supplanted the Maximum Parsimony (MP) method for inferring phylogenetic trees. One of the major reasons for this shift was that MP is much more sensitive to the Long Branch Attraction (LBA) artefact than is Maximum Likelihood (ML). However, recent work by Kolaczkowski and Thornton suggested, on the basis of simulations, that MP is less sensitive than ML to tree reconstruction artefacts generated by heterotachy, a phenomenon that corresponds to shifts in site-specific evolutionary rates over time. These results led these authors to recommend that the results of ML and MP analyses should be both reported and interpreted with the same caution. This specific conclusion revived the debate on the choice of the most accurate phylogenetic method for analysing real data in which various types of heterogeneities occur. However, variation of evolutionary rates across species was not explicitly incorporated in the original study of Kolaczkowski and Thornton, and in most of the subsequent heterotachous simulations published to date, where all terminal branch lengths were kept equal, an assumption that is biologically unrealistic.     

Suppression of long-branch attraction artefacts in the animal phylogeny using a site-heterogeneous model

Background

Thanks to the large amount of signal contained in genome-wide sequence alignments, phylogenomic analyses are converging towards highly supported trees. However, high statistical support does not imply that the tree is accurate. Systematic errors, such as the Long Branch Attraction (LBA) artefact, can be misleading, in particular when the taxon sampling is poor, or the outgroup is distant. In an otherwise consistent probabilistic framework, systematic errors in genome-wide analyses can be traced back to model mis-specification problems, which suggests that better models of sequence evolution should be devised, that would be more robust to tree reconstruction artefacts, even under the most challenging conditions.

Methods

We focus on a well characterized LBA artefact analyzed in a previous phylogenomic study of the metazoan tree, in which two fast-evolving animal phyla, nematodes and platyhelminths, emerge either at the base of all other Bilateria, or within protostomes, depending on the outgroup. We use this artefactual result as a case study for comparing the robustness of two alternative models: a standard, site-homogeneous model, based on an empirical matrix of amino-acid replacement (WAG), and a site-heterogeneous mixture model (CAT). In parallel, we propose a posterior predictive test, allowing one to measure how well a model acknowledges sequence saturation.

Results

Adopting a Bayesian framework, we show that the LBA artefact observed under WAG disappears when the site-heterogeneous model CAT is used. Using cross-validation, we further demonstrate that CAT has a better statistical fit than WAG on this data set. Finally, using our statistical goodness-of-fit test, we show that CAT, but not WAG, correctly accounts for the overall level of saturation, and that this is due to a better estimation of site-specific amino-acid preferences.

Conclusion

The CAT model appears to be more robust than WAG against LBA artefacts, essentially because it correctly anticipates the high probability of convergences and reversions implied by the small effective size of the amino-acid alphabet at each site of the alignment. More generally, our results provide strong evidence that site-specificities in the substitution process need be accounted for in order to obtain more reliable phylogenetic trees.

     

Convergence among cave catfishes: long-branch attraction and a Bayesian relative rates test

Convergence has long been of interest to evolutionary biologists. Cave organisms appear to be ideal candidates for studying convergence in morphological, physiological, and developmental traits. Here we report apparent convergence in two cave-catfishes that were described on morphological grounds as congeners: Prietella phreatophila and Prietella lundbergi. We collected mitochondrial DNA sequence data from 10 species of catfishes, representing five of the seven genera in Ictaluridae, as well as seven species from a broad range of siluriform outgroups. Analysis of the sequence data under parsimony supports a monophyletic Prietella. However, both maximum-likelihood and Bayesian analyses support polyphyly of the genus, with P. lundbergi sister to Ictalurus and P. phreatophila sister to Ameiurus. The topological difference between parsimony and the other methods appears to result from long-branch attraction between the Prietella species. Similarly, the sequence data do not support several other relationships within Ictaluridae supported by morphology. We develop a new Bayesian method for examining variation in molecular rates of evolution across a phylogeny.     

Rate acceleration and long-branch attraction in a conserved gene of cryptic daphniid (Crustacea) species.

The nuclear large subunit (LSU) rRNA gene is a rich source of phylogenetic characters because of its large size, mosaic of slowly and rapidly evolving regions, and complex secondary structure variation. Nevertheless, many studies have indicated that inconsistency, bias, and gene-specific error (e.g., within-individual gene family variation, cryptic sequence simplicity, and sequence coevolution) can complicate animal phylogenies based on LSU rDNA sequences. However, most of these studies sampled small gene fragments from expansion segments--among animals only five nonchordate complete LSU sequences are published. In this study, we sequenced near-complete nuclear LSU genes from 11 representative daphniids (Crustacea). The daphniid expansion segment V6 was larger and showed more length variation (90-351 bp) than is found in all other reported LSU V6 sequences. Daphniid LSU (without the V6 region) phylogenies generally agreed with the existing phylogenies based on morphology and mtDNA sequences. Nevertheless, a major disagreement between the LSU and the expected trees involved a positively misleading association between the two taxa with the longest branches, Daphnia laevis and D. occidentalis. Both maximum parsimony (MP) and maximum likelihood (ML) optimality criteria recovered this association, but parametric simulations indicated that MP was markedly more sensitive to this bias than ML. Examination of data partitions indicated that the inconsistency was caused by increased nucleotide substitution rates in the branches leading to D. laevis and D. occidentalis rather than among-taxon differences in base composition or distribution of sites that are free to vary. These results suggest that lineage-specific rate acceleration can lead to long-branch attraction even in the conserved genes of animal species that are almost morphologically indistinguishable.     

Interaction of subunits of polypeptide chain elongation factor I from pig liver. Formation of EF-1alpha.EF-1betagamma and EF-1beta complexes

In the preceding papers, we showed that one of the two complementar factors of polypeptide chain elongation factor 1 (EF-1) from pig liver, EF-1alpha, functionally corresponds to bacterial EF-Tu (Nagata, S., Iwasaki, K., and Kaziro, Y. (1976) Arch. Biochem. Biophys. 172, 168), while the other, EF-1betagamma, as well as one of its subunits, EF-1beta, corresponds to bacterial EF-Ts (Motoyoshi, K. and Iwasaki, K. (1977) J. Biochem. 82, 703). Therefore, the interaction between EF-1alpha and EF-1 betagamma or EF-1beta was was examined and the following results were obtained. i) EF-1betagamma catalytically promoted the exchange of [14C]GDP bound to EF-1alpha with exogenous [3H]GDP. ii). In the absence of the exogenous guanine nucleotide, EF-1betagamma as well as EF-1beta could displace GDP bound to EF-1alpha to form an EF-1alpha.EF-1betagamma as well as an EF-1alpha.EF-1beta complex. iii) The occurrence of EF-1alpha.EF-1betagamma and EF-1alpha.EF-1beta complexes was demonstrated by gel filtration on Sephadex G-150. These results strongly indicate that the mechanism of the action of EF-1betagamma or EF-1beta in converting EF-1alpha.GDP into EF-1alpha.GTP is analogous to bacterial EF-Ts, and the reaction is accomplished by the following reactions; EF-1alpha.GDP + EF-1betagamma (or EF-1beta) in equilibrium EF-1alpha.EF-1betagamma (or EF-1beta) + GDP; EF-1alpha.EF-1beta (or EF-1beta) + GTP IN EQUILIBRIUM EF-1alpha.GTP + EF-1betagamma (or EF-1beta).     

Molecular architecture of leishmania EF-1alpha reveals a novel site that may modulate protein translation: a possible target for drug development

Elongation factor-1alpha plays an essential role in eukaryotic protein biosynthesis. Recently, we have shown by protein structure modeling the presence of a hairpin-loop of 12 amino acids in mammalian EF-1alpha that is absent in the leishmania homologue [D. Nandan, A. Cherkasov, R. Sabouti, T. Yi, N.E. Reiner, Molecular cloning, biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: comparison with the mammalian homologue, Biochem. Biophys. Res. Commun. 302 (2003) 646-652]. As a consequence of this deletion, an exposed region is available on the main body of leishmania EF-1alpha. Here we report the generation of an anti-EF-1alpha antibody (DN-3) which bound selectively to the exposed region of leishmania EF-1alpha, with no reactivity with human EF-1alpha. In a leishmania cell-free protein translation system, DN-3 substantially inhibited protein translation. A similar inhibitory effect was observed when a specific peptide based on the exposed region was used in the cell-free protein translation assay. The application of structure-based in silico methods to identify potential ligands to target the exposed region identified a small molecule that selectively attenuated in vitro translation using leishmania extracts. Moreover, this small molecule showed selective suppressive effect on multiplication of leishmania in culture. Taken together, these findings identify a novel, exposed region in leishmania EF-1alpha that may be involved in protein synthesis and a potential site for drug targeting.     

Purification and characterization of a germ cell-specific form of elongation factor 1 alpha (EF-1 alpha) from Xenopus laevis.

Elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from full-grown oocytes of Xenopus laevis. This protein is encoded by a gene previously shown to be expressed in male and female germ cells, and repressed in somatic cells. The purified protein was identified with EF-1 alpha on criteria of molecular mass, cross-reaction with antibodies raised against Artemia salina EF-1 alpha, affinity for guanine nucleotides, and ability to promote the mRNA-dependent binding of aminoacyl tRNA to 80S ribosomes.     

EFL GTPase in cryptomonads and the distribution of EFL and EF-1alpha in chromalveolates

EFL (EF-like protein) is a member of the GTPase superfamily that includes several translation factors. Because it has only been found in a few eukaryotic lineages and its presence correlates with the absence of the related core translation factor EF-1alpha, its distribution is hypothesized to be the result of lateral gene transfer and replacement of EF-1alpha. In one supergroup of eukaryotes, the chromalveolates, two major lineages were found to contain EFL (dinoflagellates and haptophytes), while the others encode EF-1alpha (apicomplexans, ciliates, heterokonts and cryptomonads). For each of these groups, this distribution was deduced from whole genome sequence or expressed sequence tag (EST) data from several species, with the exception of cryptomonads from which only a single EF-1alpha PCR product from one species was known. By sequencing ESTs from two cryptomonads, Guillardia theta and Rhodomonas salina, and searching for all GTPase translation factors, we revealed that EFL is present in both species, but, contrary to expectations, we found EF-1alpha in neither. On balance, we suggest the previously reported EF-1alpha from Rhodomonas salina is likely an artefact of contamination. We also identified EFL in EST data from two members of the dinoflagellate lineage, Karlodinium micrum and Oxyrrhis marina, and from an ongoing genomic sequence project from a third, Perkinsus marinus. Karlodinium micrum is a symbiotic pairing of two lineages that would have both had EFL (a dinoflagellate and a haptophyte), but only the dinoflagellate gene remains. Oxyrrhis marina and Perkinsus marinus are early diverging sister-groups to dinoflagellates, and together show that EFL originated early in this lineage. Phylogenetic analysis confirmed that these genes are all EFL homologues, and showed that cryptomonad genes are not detectably related to EFL from other chromalveolates, which collectively form several distinct groups. The known distribution of EFL now includes a third group of chromalveolates, cryptomonads. Of the six major subgroups of chromalveolates, EFL is found in half and EF-1alpha in the other half, and none as yet unambiguously possess both genes. Phylogenetic analysis indicates EFL likely arose early within each subgroup where it is found, but suggests it may have originated multiple times within chromalveolates as a whole.     

Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects

We report the complete sequence of a paralogous copy of elongation factor-1 alpha (EF-1 alpha) in the honeybee, Apis mellifera (Hymenoptera: Apidae). This copy differs from a previously described copy in the positions of five introns and in 25% of the nucleotide sites in the coding regions. The existence of two paralogous copies of EF-1 alpha in Drosophila and Apis suggests that two copies of EF-1 alpha may be widespread in the holometabolous insect orders. To distinguish between a single, ancient gene duplication and parallel, independent fly and bee gene duplications, we performed a phylogenetic analysis of hexapod EF-1 alpha sequences. Unweighted parsimony analysis of nucleotide sequences suggests an ancient gene duplication event, whereas weighted parsimony analysis of nucleotides and unweighted parsimony analysis of amino acids suggests the contrary: that EF-1 alpha underwent parallel gene duplications in the Diptera and the Hymenoptera. The hypothesis of parallel gene duplication is supported both by congruence among nucleotide and amino acid data sets and by topology-dependent permutation tail probability (T-PTP) tests. The resulting tree topologies are also congruent with current views on the relationships among the holometabolous orders included in this study (Diptera, Hymenoptera, and Lepidoptera). More sequences, from diverse orders of holometabolous insects, will be needed to more accurately assess the historical patterns of gene duplication in EF-1 alpha.      

EF-1[alpha] Is Associated with a Cytoskeletal Network Surrounding Protein Bodies in Maize Endosperm Cells

By using indirect immunofluorescence and confocal microscopy, we documented changes in the distribution of elongation factor-1[alpha] (EF-1[alpha]), actin, and microtubules during the development of maize endosperm cells. In older interphase cells actively forming starch grains and protein bodies, the protein bodies are enmeshed in EF-1[alpha] and actin and are found juxtaposed with a multidirectional array of microtubules. Actin and EF-1[alpha] appear to exist in a complex, because we observed that the two are colocalized, and treatment with cytochalasin D resulted in the redistribution of EF-1[alpa]. These data suggest that EF-1[alpha] and actin are associated in maize endosperm cells and may help to explain the basis of the correlation we found between the concentration of EF-1[alpha] and lysine content. The data also support the hypothesis that the cytoskeleton plays a role in storage protein deposition. The distributions of EF-1[alpha] actin, and microtubules change during development. We observed that in young cells before the accumulation of starch and storage protein, EF-1[alpha], actin, and microtubules are found mainly in the cell cortex or in association with nuclei.     

42Sp48 in previtellogenic Xenopus oocytes is structurally homologous to EF-1 alpha and may be a stage-specific elongation factor

We have isolated the cDNA for 42Sp48 and EF-1 alpha from mixed stage oocytes and tailbud (stage 22) Xenopus laevis cDNA libraries by use of the cDNA for human elongation factor-1 alpha (EF-1 alpha) as probe. The nucleotide and deduced amino acid sequences of the entire coding region of 42Sp48 and EF-1 alpha cDNA were established. The proposed functional homology of the proteins is reflected in highly conserved amino acid sequences (91% identity), while the large number of silent mutations at the gene level may serve to prevent recombination at their loci. 42Sp48 is apparently encoded by two genes in Xenopus, while no sequences corresponding to 42Sp48 could be found in murine or human genomic DNA. 42Sp48 has been proposed to act as a stage-specific elongation factor in Xenopus. Comparison of the deduced amino acid sequences of 42Sp48 and EF-1 alpha with that of elongation factor Tu from E. coli, for which the three-dimensional structure including that of the GTP binding sites have been determined, supports this hypothesis.     

Elongation factor 1 alpha (EF-1 alpha) is concentrated in the Balbiani body and accumulates coordinately with the ribosomes during oogenesis of Xenopus laevis

In Xenopus laevis oocytes two distinct systems catalyze the mRNA-dependent binding of aminoacyl tRNA to the A site of ribosomes. These systems are elongation factor 1 alpha (EF-1 alpha) and the 42S nucleoprotein particle. This particle is also implicated in the long-term storage of 5S RNA and aminoacyl tRNA during early oogenesis. We report here that the ribosomes and the storage particles are distributed uniformly in the cytoplasm of previtellogenic (stage I) oocytes. In contrast, EF-1 alpha is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body. When the Balbiani body disperses in early vitellogenic oocytes (stage II), EF-1 alpha becomes evenly distributed in the cytoplasm. The main phase of EF-1 alpha accumulation follows the disappearance of the 42S particles (stage II), but coincides with the main phase of ribosome accumulation (stages III and IV).     

Actin crosslinking protein EF-1a of Dictyostelium discoideum has a unique bonding rule that allows square-packed bundles.

The elongation factor 1a (EF-1a) of Dictyostelium discoideum is an actin crosslinking protein that gives rise to a unique kind of actin bundle. Purified actin and EF-1a were allowed to form bundles and then were characterized by electron microscopy, computed diffraction analysis, and modeling. In these bundles crosslinked actin filaments are rotated by 90 degrees relative to each other, whereas other known crosslinking proteins require filaments to be unrotated. Bundles of actin EF-1a would tend to exclude other actin bundling proteins. EF-1a can thus regulate the state of the actin cytoskeleton as well as regulate protein synthesis.     

Detection and characterization of glutathione S-transferase activity in rice EF-1betabeta'gamma and EF-1gamma expressed in Escherichia coli.

Plant elongation factor EF-1 consists of four subunits (EF-1alphabetabeta'gamma). EF-1alpha. GTP catalyses the binding of aminoacyl-tRNA to the ribosome. EF-1beta and EF-1beta' catalyze the GDP/GTP exchange on EF-1alpha. GDP. However, the function of EF-1gamma, a subunit detected in eukaryotes, but not in prokaryotes remained unknown. This report demonstrates that rice EF-1betabeta'gamma and recombinant EF-1gamma possess glutathione S-transferase (GST) activity. The EF-1betabeta'gamma- or EF-1gamma-dependent GST activity is about one-fiftieth of the rice GST activity. The Km values of EF-1betabeta'gamma, EF-1gamma, and rice GST for glutathione and 1-chloro-2,4-dinitrobenzene are of about the same order. Although recombinant EF-1gamma is heat labile, active EF-1gamma was obtained by purifying it in the presence of 20% glycerol.