Evolution of elongation factor-like (EFL) protein in Rhizaria is revised by radiolarian EFL gene sequences

Elongation factor 1α (EF-1α) and elongation factor-like (EFL) proteins are considered to carry out equivalent functions in translation in eukaryotic cells. Elongation factor 1α and EFL genes are patchily distributed in the global eukaryotic tree, suggesting that the evolution of these elongation factors cannot be reconciled without multiple lateral gene transfer and/or ancestral co-occurrence followed by differential loss of either of the two factors. Our current understanding of the EF-1α/EFL evolution in the eukaryotic group Rhizaria, composed of Foraminifera, Radiolaria, Filosa, and Endomyxa, remains insufficient, as no information on EF-1α/EFL gene is available for any members of Radiolaria. In this study, EFL genes were experimentally isolated from four polycystine radiolarians (i.e. Dictyocoryne, Eucyrtidium, Collozoum, and Sphaerozoum), as well as retrieved from publicly accessible expressed sequence tag data of two acantharean radiolarians (i.e. Astrolonche and Phyllostaurus) and the endomyxan Gromia. The EFL homologs from radiolarians, foraminiferans, and Gromia formed a robust clade in both maximum-likelihood and Bayesian phylogenetic analyses, suggesting that EFL genes were vertically inherited from their common ancestor. We propose an updated model for EF-1α/EFL evolution in Rhizaria by incorporating new EFL data obtained in this study.     

A New Divergent Type of Eukaryotic Methionine Adenosyltransferase is Present in Multiple Distantly Related Secondary Algal Lineages

ABSTRACT. S‐adenosylmethionine is one of the most important metabolites in living cells and is synthesized in a single reaction catalyzed by methionine adenosyltransferase (MAT). At the sequence and structural level, this enzyme is one of the most conserved proteins known. Here we show that some representatives of three distantly related eukaryotic lineages—dinoflagellates, haptophytes, and euglenids—possess a highly divergent type of MAT, which we call MATX. Even though MATX contains all the sites known to be involved in catalysis and the association of monomers, it also has four insertions throughout the protein that are not observed in other MAT homologs. The phylogenetic distribution and affinities of MATX suggest that it originated in a single eukaryotic lineage and was spread via multiple events of eukaryote‐to‐eukaryote lateral gene transfer. We suggest a tentative model in which the origin of MATX is connected with the progression of secondary endosymbiosis.     

Cercozoa comprises both EF-1α-containing and EFL-containing members

Elongation factor 1α (EF-1α) and elongation factor-like protein (EFL) are considered to be functionally equivalent proteins involved in peptide synthesis. Eukaryotes can be fundamentally divided into ‘EF-1α-containing’ and ‘EFL-containing’ types. Recently, EF-1α and EFL genes have been surveyed across the diversity of eukaryotes to explore the origin and evolution of EFL genes. Although the phylum Cercozoa is a diverse group, gene data for either EFL or EF-1α are absent from all cercozoans except chlorarachniophytes which were previously defined as EFL-containing members. Our survey revealed that two members of the cercozoan subphylum Filosa (Thaumatomastix sp. and strain YPF610) are EFL-containing members. Importantly, we identified EF-1α genes from two members of Filosa (Paracercomonas marina and Paulinella chromatophora) and a member of the other subphylum Endomyxa (Filoreta japonica). All cercozoan EFL homologues could not be recovered as a monophyletic group in maximum-likelihood and Bayesian analyses, suggesting that lateral gene transfer was involved in the EFL evolution in this protist assemblage. In contrast, EF-1α analysis successfully recovered a monophyly of three homologues sampled from the two cercozoan subphyla. Based on the results, we postulate that cercozoan EF-1α genes have been vertically inherited, and the current EFL-containing species may have secondarily lost their EF-1α genes.     

The gut fungus Basidiobolus ranarum has a large genome and different copy numbers of putatively functionally redundant elongation factor genes

Fungal genomes range in size from 2.3 Mb for the microsporidian Encephalitozoon intestinalis up to 8000 Mb for Entomophaga aulicae, with a mean genome size of 37 Mb. Basidiobolus, a common inhabitant of vertebrate guts, is distantly related to all other fungi, and is unique in possessing both EF-1α and EFL genes. Using DNA sequencing and a quantitative PCR approach, we estimated a haploid genome size for Basidiobolus at 350 Mb. However, based on allelic variation, the nuclear genome is at least diploid, leading us to believe that the final genome size is at least 700 Mb. We also found that EFL was in three times the copy number of its putatively functionally overlapping paralog EF-1α. This suggests that gene or genome duplication may be an important feature of B. ranarum evolution, and also suggests that B. ranarum may have mechanisms in place that favor the preservation of functionally overlapping genes.     

Expanded phylogenies of canonical and non-canonical types of methionine adenosyltransferase reveal a complex history of these gene families in eukaryotes

Most eukaryotes possess the highly-conserved enzyme methionine adenosyltransferase (MAT) that produces S-adenosyl-l-methionine, a molecule essential to a variety of cellular processes. However, a recent study revealed that genomes of a very few eukaryote lineages encode a highly divergent type of MAT (called MATX), instead of the canonical MAT enzyme. Since MATX-containing eukaryotes are phylogenetically interspersed with MAT-containing organisms, it is likely that the MATX gene was spread into the MAT-containing groups via multiple eukaryote-to-eukaryote lateral gene transfer events. Here, we further investigate the evolutionary history of these gene families by vastly increasing the sampling of species containing MAT (22 new taxa) and MATX (8 new taxa). Our expanded analyses reveal the first example of lateral transfer of a MAT gene between the pelagophycean alga Aureococcus anophagefferens and a cryptomonad. The increased MATX sampling also provided new insights into the evolution of MATX. Specifically, our MATX phylogeny robustly grouped the haptophyte homologues with the Aureococcus homologue to the exclusion of the diatom homologues, suggesting a transfer of the MATX gene between haptophytes and pelagophytes. Various scenarios of MAT and MATX gene family evolution in diatoms are re-evaluated in light of the new data.     

Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases

Eukaryotes and archaea both possess multiple genes coding for family B DNA polymerases. In animals and fungi, three family B DNA polymerases, alpha, delta, and epsilon, are responsible for replication of nuclear DNA. We used a PCR-based approach to amplify and sequence phylogenetically conserved regions of these three DNA polymerases from Giardia intestinalis and Trichomonas vaginalis, representatives of early-diverging eukaryotic lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs suggests that the gene duplications that gave rise to the three replicative paralogs occurred before the divergence of the earliest eukaryotic lineages, and that all eukaryotes are likely to possess these paralogs. One eukaryotic paralog, epsilon, consistently branches within archaeal sequences to the exclusion of other eukaryotic paralogs, suggesting that an epsilon-like family B DNA polymerase was ancestral to both archaea and eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form monophyletic groups in phylogenetic analysis, it is possible that archaeal family B paralogs themselves evolved by a series of gene duplications independent of the gene duplications that gave rise to eukaryotic paralogs.      

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.     

Gain and loss of elongation factor genes in green algae

Background  

Two key genes of the translational apparatus, elongation factor-1 alpha (EF-1α) and elongation factor-like (EFL) have an almost mutually exclusive distribution in eukaryotes. In the green plant lineage, the Chlorophyta encode EFL except Acetabularia where EF-1α is found, and the Streptophyta possess EF-1α except Mesostigma, which has EFL. These results raise questions about evolutionary patterns of gain and loss of EF-1α and EFL. A previous study launched the hypothesis that EF-1α was the primitive state and that EFL was gained once in the ancestor of the green plants, followed by differential loss of EF-1α or EFL in the principal clades of the Viridiplantae. In order to gain more insight in the distribution of EF-1α and EFL in green plants and test this hypothesis we screened the presence of the genes in a large sample of green algae and analyzed their gain-loss dynamics in a maximum likelihood framework using continuous-time Markov models.     

Interaction of the low molecular weight form of elongation factor 1 with guanine nucleotides and aminoacyl-tRNA.

A low molecular weight form of the eukaryotic polypeptide chain elongation factor 1 (EF-1α) has been extensively purified from pig liver to give an apparently homogeneous preparation, which seemed to be analogous to the bacterial elongation factor, EF-Tu (Iwasaki, K., Nagata, S., Mizumoto, K., and Kaziro, Y. (1974) J. Biol. Chem. 249, 5008). Thus, the interaction of the purified EF-1α with guanine nucleotides as well as aminoacyl-tRNA has been investigated and the following results have been obtained. (1) EF-1α when kept in the absence of glycerol lost its activity to promote the binding of aminoacylt-RNA to ribosomes though it retained the ability to bind guanine nucleotides. However, the former activity could be stabilized by the addition of 25% ($\text{v}\text{v}$) glycerol to the solution. (2) EF-1α formed a binary complex with guanine nucleotides such as GTP, GDP, 5′-guanylyl methylenediphosphonate or 5′-guanylyl imidodiphosphate. The molar ratio of EF-1α to GTP or GDP in the binary complex was shown to be 1. (3) The presence of a ternary complex containing EF-1α, GTP and aminoacyl-tRNA was demonstrated by several methods, i.e., (i) an increased heat stability of EF-1α in the presence of GTP and Phe-tRNA, (ii) a decrease in the amount of the EF-1α·GTP complex in the presence of aminoacyl-tRNA, (iii) a protection of the ester linkage of Phe-tRNA from hydrolysis at alkaline pH by the presence of both EF-1α and GTP, and (iv) the isolation of the complex by gel filtration.     

Tracing back EFL gene evolution in the cryptomonads-haptophytes assemblage: separate origins of EFL genes in haptophytes, photosynthetic cryptomonads, and goniomonads

A recently identified GTPase, elongation factor-like (EFL) protein is proposed to bear the principal functions of translation elongation factor 1alpha (EF-1alpha). Pioneering studies of EF-1alpha/EFL evolution have revealed the phylogenetically scattered distribution of EFL amongst eukaryotes, suggesting frequent eukaryote-to-eukaryote EFL gene transfer events and subsequent replacements of EF-1alpha functions by EFL. We here determined/identified seven new EFL sequences of the photosynthetic cryptomonad Cryptomonas ovata, the non-photosynthetic cryptomonad (goniomonad) Goniomonas amphinema, the foraminifer Planoglabratella opecularis, the haptophyte Chrysochromulina sp., the centroheliozoan Raphidiophrys contractilis, and two red algae Chondrus crispus and Gracilaria changii. The analyses of these EFL sequences successfully brought new insights into lateral EFL gene transfer amongst eukaryotes. Of most interest is a complex EFL evolution in a monophyletic assemblage comprised of cryptomonads and haptophytes. Since our analyses rejected any phylogenetic affinity amongst the EFL sequences from Goniomonas, photosynthetic cryptomonads, and haptophytes, the EFL genes of the three lineages most likely originated from different phylogenetic sources.     

Retention of protein complex membership by ancient duplicated gene products in budding yeast

To investigate functional divergence of gene duplicates, we examined the protein-protein interactions and coexistence in complexes of paralogs resulting from an ancient whole-genome duplication in yeast. Strikingly, half the surveyed paralog pairs were found to be co-clustered in protein complexes, and were more conserved and highly expressed than non-co-clustered paralogs; however, their discordant expression patterns and conservation rates indicate differential regulation of subfunctionalized paralogs. These results highlight the value of protein complex membership in studying functional divergence among gene duplicates.     

5′‐N‐ethylcarboxamidoadenosine is not a paralog‐specific Hsp90 inhibitor

The molecular chaperone Hsp90 facilitates the folding and modulates activation of diverse substrate proteins. Unlike other heat shock proteins such as Hsp60 and Hsp70, Hsp90 plays critical regulatory roles by maintaining active states of kinases, many of which are overactive in cancer cells. Four Hsp90 paralogs are expressed in eukaryotic cells: Hsp90α/β (in the cytosol), Grp94 (in the endoplasmic reticulum), Trap1 (in mitochondria). Although numerous Hsp90 inhibitors are being tested in cancer clinical trials, little is known about why different Hsp90 inhibitors show specificity among Hsp90 paralogs. The paralog specificity of Hsp90 inhibitors is likely fundamental to inhibitor efficacy and side effects. In hopes of gaining insight into this issue we examined NECA (5′‐N‐ethylcarboxamidoadenosine), which has been claimed to be an example of a highly specific ligand that binds to one paralog, Grp94, but not cytosolic Hsp90. To our surprise we find that NECA inhibits many different Hsp90 proteins (Grp94, Hsp90α, Trap1, yeast Hsp82, bacterial HtpG). NMR experiments demonstrate that NECA can bind to the N‐terminal domains of Grp94 and Hsp82. We use ATPase competition experiments to quantify the inhibitory power of NECA for different Hsp90 proteins. This scale: Hsp82 > Hsp90α > HtpG ≈ Grp94 > Trap1, ranks Grp94 as less sensitive to NECA inhibition. Because NECA is primarily used as an adenosine receptor agonist, our results also suggest that cell biological experiments utilizing NECA may have confounding effects from cytosolic Hsp90 inhibition.     

Biological characterization of various forms of elongation factor 1 from rabbit reticulocytes

Two forms of elongation factor 1 (EF-1) have been tested for a variety of biological functions. One form, EF-1H, is a high-molecular-weight aggregate (M_r > 500,000) containing four distinct polypeptides (α, β, γ, δ). The other form, EF-1α, consists of a single polypeptide which is the same as the α subunit of EF-1H. Both EF-1α and EF-1H function catalytically in binding Phe-tRNA to ribosomes, and in poly(U)-directed polyphenylalanine synthesis. The activity of EF-1α is enhanced in polyphenylalanine synthesis by a complementary component, EF-1βδ. It is also shown that EF-1βδ can facilitate an exchange of EF-1α-bound GDP for GTP. The EF-1α dissociation constants for GDP and GTP were 0.47 and 0.55 μm respectively, while the EF-1H dissociation constants for GDP and GTP were 2.0 and 1.6 μm, respectively. Thus, while EF-1α and EF-1H had approximately the same affinities for GDP and GTP, the EF-1α dissociation constants were about fourfold lower than the EF-1H dissociation constants. Attempts to isolate complexes of EF-1α or EF-1H with GTP and Phe-tRNA or with GTP, Phe-tRNA, and ribosomes were unsuccessful using either Millipore filters, gel filtration, or sucrose density gradients. The results presented in this report, along with studies from other laboratories, strengthen the hypothesis that the general mechanism of the elongation cycle is similar in eucaryotes and procaryotes.     

Rooting phylogenies using gene duplications: an empirical example from the bees (Apoidea)

The placement of the root node in a phylogeny is fundamental to characterizing evolutionary relationships. The root node of bee phylogeny remains unclear despite considerable previous attention. In order to test alternative hypotheses for the location of the root node in bees, we used the F1 and F2 paralogs of elongation factor 1-alpha (EF-1α) to compare the tree topologies that result when using outgroup versus paralogous rooting. Fifty-two taxa representing each of the seven bee families were sequenced for both copies of EF-1α. Two datasets were analyzed. In the first (the "concatenated" dataset), the F1 and F2 copies for each species were concatenated and the tree was rooted using appropriate outgroups (sphecid and crabronid wasps). In the second dataset (the "duplicated" dataset), the F1 and F2 copies were aligned to each another and each copy for all taxa were treated as separate terminals. In this dataset, the root was placed between the F1 and F2 copies (e.g., paralog rooting). Bayesian analyses demonstrate that the outgroup rooting approach outperforms paralog rooting, recovering deeper clades and showing stronger support for groups well established by both morphological and other molecular data. Sequence characteristics of the two copies were compared at the amino acid level, but little evidence was found to suggest that one copy is more functionally conserved. Although neither approach yields an unambiguous root to the tree, both approaches strongly indicate that the root of bee phylogeny does not fall near Colletidae, as has been previously proposed. We discuss paralog rooting as a general strategy and why this approach performs relatively poorly with our particular dataset.     

The RAB Family GTPase Rab1A from Plasmodium falciparum Defines a Unique Paralog Shared by Chromalveolates and Rhizaria

ABSTRACT. The RAB GTPases, which are involved in regulation of endomembrane trafficking, exhibit a complex but incompletely understood evolutionary history. We elucidated the evolution of the RAB1 subfamily ancestrally implicated in the endoplasmic reticulum-to-Golgi traffic. We found that RAB1 paralogs have been generated over the course of eukaryotic evolution, with some duplications coinciding with the advent of major eukaryotic lineages (e.g. Metazoa, haptophytes). We also identified a unique, derived RAB1 paralog, orthologous to the Plasmodium Rab1A, that occurs in stramenopiles, alveolates, and Rhizaria, represented by the chlorarachniophyte Gymnochlora stellata . This finding is consistent with the recently documented existence of a major eukaryotic clade ("SAR") comprising these three lineages. We further found a Rab1A-like protein in the cryptophyte Guillardia theta , but it exhibits unusual features among RAB proteins: absence of a C-terminal prenylation motif and an N-terminal extension with two MSP domains; and its phylogenetic relationships could not be established convincingly due to its divergent nature. Our results nevertheless point to a unique membrane trafficking pathway shared by at least some lineages of chromalveolates and Rhizaria, an insight that has implications towards interpreting the early evolution of eukaryotes and the endomembrane system.     

Evolutionary relationships between Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici isolates inferred from mating type, elongation factor-1α and exopolygalacturonase sequences

Fusarium oxysporum is a ubiquitous species complex of soilborne plant pathogens that comprises many different formae speciales, each characterized by a high degree of host specificity. In this study, the evolutionary relationships between different isolates of the F. oxysporum species complex have been examined, with a special emphasis on the formae speciales lycopersici and radicis-lycopersici, sharing tomato as host while causing different symptoms. Phylogenetic analyses of partial sequences of a housekeeping gene, the elongation factor-1α (EF-1α) gene, and a gene encoding a pathogenicity trait, the exopolygalacturonase (pgx4) gene, were conducted on a worldwide collection of F. oxysporum strains representing the most frequently observed vegetative compatibility groups of these formae speciales. Based on the reconstructed phylogenies, multiple evolutionary lineages were found for both formae speciales. However, different tree topologies and statistical parameters were obtained for the cladograms as several strains switched from one cluster to another depending on the locus that was used to infer the phylogeny. In addition, mating type analysis showed a mixed distribution of the MAT1-1 and MAT1-2 alleles in the F. oxysporum species complex, irrespective of the geographic origin of the tested isolates. This observation, as well as the topological conflicts that were detected between EF-1α and pgx4, are discussed in relation to the evolutionary history of the F. oxysporum species complex.     

An evaluation of elongation factor 1 alpha as a phylogenetic marker for eukaryotes

Elongation factor 1 alpha (EF-1 alpha) is a highly conserved ubiquitous protein involved in translation that has been suggested to have desirable properties for phylogenetic inference. To examine the utility of EF-1 alpha as a phylogenetic marker for eukaryotes, we studied three properties of EF-1 alpha trees: congruency with other phyogenetic markers, the impact of species sampling, and the degree of substitutional saturation occurring between taxa. Our analyses indicate that the EF-1 alpha tree is congruent with some other molecular phylogenies in identifying both the deepest branches and some recent relationships in the eukaryotic line of descent. However, the topology of the intermediate portion of the EF-1 alpha tree, occupied by most of the protist lineages, differs for different phylogenetic methods, and bootstrap values for branches are low. Most problematic in this region is the failure of all phylogenetic methods to resolve the monophyly of two higher-order protistan taxa, the Ciliophora and the Alveolata. JACKMONO analyses indicated that the impact of species sampling on bootstrap support for most internal nodes of the eukaryotic EF-1 alpha tree is extreme. Furthermore, a comparison of observed versus inferred numbers of substitutions indicates that multiple overlapping substitutions have occurred, especially on the branch separating the Eukaryota from the Archaebacteria, suggesting that the rooting of the eukaryotic tree on the diplomonad lineage should be treated with caution. Overall, these results suggest that the phylogenies obtained from EF-1 alpha are congruent with other molecular phylogenies in recovering the monophyly of groups such as the Metazoa, Fungi, Magnoliophyta, and Euglenozoa. However, the interrelationships between these and other protist lineages are not well resolved. This lack of resolution may result from the combined effects of poor taxonomic sampling, relatively few informative positions, large numbers of overlapping substitutions that obscure phylogenetic signal, and lineage-specific rate increases in the EF-1 alpha data set. It is also consistent with the nearly simultaneous diversification of major eukaryotic lineages implied by the "big-bang" hypothesis of eukaryote evolution.     

Exchange of free GTP with EF-1α·GDP complex promoted by a factor EF-1β from pig liver

Eukaryotic polypeptide chain elongation factor 1 (EF-1) has been resolved into two complementary factors, EF-1α and EF-1β, both of which were purified. Recently, we find that [³H] GDP bound to purified EF-1α is replaced by exogenous GTP rather slowly when the reaction is carried out at ionic strength optimal for polyphenylalanine synthesis. EF-1β stimulates the exchange of free GTP with EF-1α·GDP, indicating that the function of EF-1β is, at least in part, similar to that of bacterial EF-Ts.     

EVOLUTION IN A PUTATTVELY ANCIENT ASEXUAL APHID LINEAGE: RECOMBINATION AND RAPID KARYOTYPE CHANGE

Ancient asexual lineages are of great potential significance for understanding the evolutionary biology of sex, but their existence is controversial. In part, this is because claims of ancient asexuality have rested on negative evidence—a mere absence of evidence for sexuality in a taxon. M. Meselson has suggested a method, discussed by Judson and Normark (1996) and by Birky (1996), that has the potential to uncover positive evidence of ancient asexuality. Phylogenetic relationships between alleles and interallelic divergences are predicted to be very different in diploid lineages that lack recombination from those in diploid lineages that undergo recombination. I have applied Meselson's method to the putatively ancient asexual aphid tribe Tramini (Homoptera: Aphidoidea: Lachnidae), using the intron-bearing nuclear protein-coding gene elongation factor 1α (EF-1α). I found heterozygosities much lower than intraspecific divergences, indicating that some recombination has occurred, but not discriminating between recombination within an asexual lineage (automixis or mitotic recombination) and outcrossing sex. Species of Tramini (especially in the genus Trama) typically have highly structurally heterozygous karyotypes that appear to be incompatible with regular successful meiosis, and have very high levels of karyotype variability within species. I found very high levels of karyotype variability within lineages with identical EF-1α and mitochondrial (cytochrome oxidase 1 and 2) genotypes, indicating a high rate of karyotype evolution compared to the rate of nucleotide substitution.