Sculpting the endomembrane system in deep time: high resolution phylogenetics of Rab GTPases

Abstract

The presence of a nucleus and other membrane-bounded intracellular compartments is the defining feature of eukaryotic cells. Endosymbiosis accounts for the origins of mitochondria and plastids, but the evolutionary ancestry of the remaining cellular compartments is incompletely documented. Resolving the evolutionary history of organelle-identity encoding proteins within the endomembrane system is a necessity for unravelling the origins and diversification of the endogenously derived organelles. Comparative genomics reveals events after the last eukaryotic common ancestor (LECA), but resolution of events prior to LECA, and a full account of the intracellular compartments present in LECA, has proved elusive. We have devised and exploited a new phylogenetic strategy to reconstruct the history of the Rab GTPases, a key family of endomembrane-specificity proteins. Strikingly, we infer a remarkably sophisticated organellar composition for LECA, which we predict possessed as many as 23 Rab GTPases. This repertoire is significantly greater than that present in many modern organisms and unexpectedly indicates a major role for secondary loss in the evolutionary diversification of the endomembrane system. We have identified two Rab paralogues of unknown function but wide distribution, and thus presumably ancient nature; RabTitan and RTW. Furthermore, we show that many Rab paralogues emerged relatively suddenly during early metazoan evolution, which is in stark contrast to the lack of significant Rab family expansions at the onset of most other major eukaryotic groups. Finally, we reconstruct higher-order ancestral clades of Rabs primarily linked with endocytic and exocytic process, suggesting the presence of primordial Rabs associated with the establishment of those pathways and giving the deepest glimpse to date into pre-LECA history of the endomembrane system.

Fig. 1.

Increased phylogenetic resolution using ScrollSaw versus a traditional (Trad) approach. Two test datasets (Trad.M1 and Trad.M2 or NN.R1 and NN.R3) were analysed in each instance, and the numbers of clades reconstructed by each analysis are given. Blue bars are moderate stringency, and red bars high stringency cutoff for assigning confidence to a clade. Statistical support is given for MrBayes (posterior probability) or ML (bootstrap) as indicated.

Fig. 2.

The ScrollSaw workflow. A dataset, here curated predicted Rab protein sequences, is subdivided on taxonomic grounds (1). All pairwise comparisons are made between the relationships for each data subset to identify the minimal distance pairs (2), and the resultant data used to reconstruct a ScrollSaw dataset, which now includes representatives across the whole sampled taxonomic range (3). This analysis allows reconstruction of supergroup-specific expansions and reconstruction of the LECA. Additional taxa are introduced into the analysis to address specific issues (4) that could not be resolved adequately using the main data subset (3).

Fig. 3.

Evolutionary relationships of core Rab clades predicted to be present in the LECA. Phylogenetic tree for the Rab clades predicted to have been present in the last eukaryotic common ancestor following ScrollSaw. Clades are indicated by vertical bars, and putative functional groupings for primordial endocytic and exocytic Rabs are shaded. Ran is used as an outgroup and the best MrBayes tree topology is shown. Individual leaves are colour-coded according to supergroup, and statistical support is indicated by the raw values for important nodes defining Rab clades, or are iconized as indicated (upper right). Supergroup divisions are sensu Adl et al. (Adl et al., 2005), except the SAR + CCTH supergroup, which contains the stramenopiles, alveolates and Rhizaria together with the cryptophyte-centrohelid-telonemid-haptophyte grouping (Burki et al., 2009).

Fig. 4.

Rab representation for select eukaryotes. Individual Rab clades, inferred as present in the LECA, are shown as columns. Taxa are shown as rows, with the hypothetical LECA as the lowest row (grey box). A schematic phylogeny for the taxa is drawn on the left and derived from Walker et al. and references therein (Walker et al., 2011). The total number of Rabs found in each genome is also indicated on the right of the taxon labels, and by a hash. Here, and in Fig. 5, black circles indicate at least one member of the clade has been identified with phylogenetic support (>0.80/50/50 MrBayes/PhyML/RaxML) and grey circles indicate naming based on BLAST results. Taxa are colour-coded by supergroup as in Fig. 3.

Fig. 5.

Evolutionarily novel Rab clades present in Holozoa and Metazoa. For the data shown here and in Fig. 4, either positive identification by phylogenetics or lack of identification of at least one homologue of each Rab clade in each organism by either BLAST or ScrollSaw, was attained in 93% of the cases, with positive assignment by BLAST alone being necessary less than 7% of the time. Data are based on the phylogenetic reconstructions in Fig. 3 and supplementary material Fig. S6 and Fig. S18.

Fig. 6.

The birth and death of Rab subfamilies in eukaryote supergroups. Points of presumed origin (blue) and loss (magenta) are shown as circles overlaid onto a schematic taxonomy of the eukaryotes. Evidence for losses and origins is based on the data in supplementary material Figs S7–S16. Closed circles indicate that two full genomes or more support the predicted event, and open circles with italic text are events where support is derived from only one completed genome plus EST data. Rabs are indicated by numbers except for RTW, IFT27, Titan and Ran; primes indicate that the ancestral clade has divided to generate a new clade but one that is clearly derived from the ancestral Rab clade.