Extensive molecular tinkering in the evolution of the membrane attachment mode of the Rheb GTPase

Rheb is a conserved and widespread Ras-like GTPase involved in cell growth regulation mediated by the (m)TORC1 kinase complex and implicated in tumourigenesis in humans. Rheb function depends on its association with membranes via prenylated C-terminus, a mechanism shared with many other eukaryotic GTPases. Strikingly, our analysis of a phylogenetically rich sample of Rheb sequences revealed that in multiple lineages this canonical and ancestral membrane attachment mode has been variously altered. The modifications include: (1) accretion to the N-terminus of two different phosphatidylinositol 3-phosphate-binding domains, PX in Cryptista (the fusion being the first proposed synapomorphy of this clade), and FYVE in Euglenozoa and the related undescribed flagellate SRT308; (2) acquisition of lipidic modifications of the N-terminal region, namely myristoylation and/or S-palmitoylation in seven different protist lineages; (3) acquisition of S-palmitoylation in the hypervariable C-terminal region of Rheb in apusomonads, convergently to some other Ras family proteins; (4) replacement of the C-terminal prenylation motif with four transmembrane segments in a novel Rheb paralog in the SAR clade; (5) loss of an evident C-terminal membrane attachment mechanism in Tremellomycetes and some Rheb paralogs of Euglenozoa. Rheb evolution is thus surprisingly dynamic and presents a spectacular example of molecular tinkering

The Plastid Genome of Eutreptiella Provides a Window into the Process of Secondary Endosymbiosis of Plastid in Euglenids

Euglenids are a group of protists that comprises species with diverse feeding modes. One distinct and diversified clade of euglenids is photoautotrophic, and its members bear green secondary plastids. In this paper we present the plastid genome of the euglenid Eutreptiella, which we assembled from 454 sequencing of Eutreptiella gDNA. Comparison of this genome and the only other available plastid genomes of photosynthetic euglenid, Euglena gracilis, revealed that they contain a virtually identical set of 57 protein coding genes, 24 genes fewer than the genome of Pyramimonas parkeae, the closest extant algal relative of the euglenid plastid. Searching within the transcriptomes of Euglena and Eutreptiella showed that 6 of the missing genes were transferred to the nucleus of the euglenid host while 18 have been probably lost completely. Euglena and Eutreptiella represent the deepest bifurcation in the photosynthetic clade, and therefore all these gene transfers and losses must have happened before the last common ancestor of all known photosynthetic euglenids. After the split of Euglena and Eutreptiella only one additional gene loss took place. The conservation of gene content in the two lineages of euglenids is in contrast to the variability of gene order and intron counts, which diversified dramatically. Our results show that the early secondary plastid of euglenids was much more susceptible to gene losses and endosymbiotic gene transfers than the established plastid, which is surprisingly resistant to changes in gene content

Evolution of nuclear and plastid genomes in euglenids

Algae form a diverse group of simple photosynthetic eukaryotes of polyphyletic origin. Algae with a primary plastid (Archaeplastida) acquired it by ingesting cyanobacterium, a prokaryote; algae with a complex plastid acquired their plastid by ingesting another eukaryote with a primary or already complex plastid. Algae with a complex plastid are chimeras containing genes derived from the host genome, as well as genes derived from the genome of the endosymbiont, and also genetic material derived from genomes of their previous stable or transient endosymbionts. One of the groups with plastid derived from green algae are euglenophytes. This thesis deals with the genomes of three organisms that represent individual actors in the endosymbiotic process in euglenophytes. These are a heterotrophic host from the class Euglenida, a phototrophic endosymbiont from the class of green algae Prasinophyceae and the resulting phototrophic euglenid from the group Euglenophyceae. Knowledge of their genomes should illuminate the course of endosymbiotic gene transfer (EGT) in the formation of algae with a complex plastid. We annotated the plastid genome of a phototrophic euglenid Eutreptiella gymnastica and published it as the third plastome of Euglenophytes after the iconic and economically important Euglena gracilis..

Simplified maps of the plastid genomes of <i>Eutreptiella gymnastica</i>, <i>Euglena gracilis</i>, <i>Euglena longa</i> and <i>Pyramimonas parkeae</i>.

<p>The maps are in scale to their sizes. The colors indicate the coding strands (plus-green and minus-violet), the ribosomal RNAs (blue) and introns (yellow). The inverted repeats IRA and IRB in <i>Pyramimonas</i> and <i>Eutreptiella</i> are marked in red. The ori site in <i>Euglena gracilis</i> is marked by an arrow.</p

Phylogenies of plastid genomes of green algae, euglenids and <i>Bigelowiella</i> based on 70 genes.

<p><b>A.</b> This phylogenetic tree was constructed using the maximum likelihood method implemented in RAxML, using the LG+I+G model selected by ProtTest. The bootstraps were estimated in 500 replicates. <b>B.</b> This tree was constructed in Beast v 1.6.1 using the WAG+I+Γ model of substitution and an uncorrelated exponential model of relaxed molecular clock. MCMCs were run for 10*10⁶ generations; trees from the first 2*10⁶ generations were discarded as the burn-in. Node labels represent posterior probabilities, node bars represent the 95% confidence interval of relative node ages.</p

General features of euglenid and <i>Pyramimonas</i> cpDNA.

<p>General features of euglenid and <i>Pyramimonas</i> cpDNA.</p

Venn diagrams showing overlaps in protein coding capacities between known euglenid plastid genomes and the plastid genome of <i>Pyramimonas parkeae</i>.

<p>The schematic representation of genome relationships is indicated in the left. Arrows indicate the probable fate of the genes absent from euglenid genomes. The genes are colour coded in respect to the functional group of their products: housekeeping proteins (black), proteins involved in photosynthesis (green), maturases of introns (red) and genes with unknown function (gray). Maturases of introns included in the phylogenetic tree of maturases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033746#pone.0033746.s001" target="_blank">Figure S1</a>) are marked by asterisks.</p