Comprehensive analysis reveals dynamic and evolutionary plasticity of Rab GTPases and membrane traffic in Tetrahymena thermophila.

Cellular sophistication is not exclusive to multicellular organisms, and unicellular eukaryotes can resemble differentiated animal cells in their complex network of membrane-bound structures. These comparisons can be illuminated by genome-wide surveys of key gene families. We report a systematic analysis of Rabs in a complex unicellular Ciliate, including gene prediction and phylogenetic clustering, expression profiling based on public data, and Green Fluorescent Protein (GFP) tagging. Rabs are monomeric GTPases that regulate membrane traffic. Because Rabs act as compartment-specific determinants, the number of Rabs in an organism reflects intracellular complexity. The Tetrahymena Rab family is similar in size to that in humans and includes both expansions in conserved Rab clades as well as many divergent Rabs. Importantly, more than 90% of Rabs are expressed concurrently in growing cells, while only a small subset appears specialized for other conditions. By localizing most Rabs in living cells, we could assign the majority to specific compartments. These results validated most phylogenetic assignments, but also indicated that some sequence-conserved Rabs were co-opted for novel functions. Our survey uncovered a rare example of a nuclear Rab and substantiated the existence of a previously unrecognized core Rab clade in eukaryotes. Strikingly, several functionally conserved pathways or structures were found to be associated entirely with divergent Rabs. These pathways may have permitted rapid evolution of the associated Rabs or may have arisen independently in diverse lineages and then converged. Thus, characterizing entire gene families can provide insight into the evolutionary flexibility of fundamental cellular pathways.

The Rab7 subfamily across Paramecium aurelia species; evidence of high conservation in sequence and function.

We examined sequence conservation and signatures of selection in Rab7 proteins across 11 Paramecium aurelia species, and determined the localization patterns of two P. tetraurelia Rab7 paralogs when expressed as GFP fusions in live cells. We found that, while there is a variable number of Rab7 paralogs per genome, Rab7 genes are highly conserved in sequence and appear to be under strong purifying selection across aurelias. Additionally, and surprisingly based on earlier studies, we found that two P. tetraurelia Rab7 proteins have virtually identical localization patterns. Consistent with this, when we examined the gene family of a highly conserved Rab binding partner across aurelias (Rab-Interacting Lysosomal Protein, or RILP), we found that residues in key binding sites in RILPs were absolutely conserved in 13 of 21 proteins, representing genes from 9 of the 11 species examined. Of note, RILP gene number appears to be even more constrained than Rab7 gene number per genome. Abbreviation: WGD: Whole genome duplication.

Early stages of functional diversification in the Rab GTPase gene family revealed by genomic and localization studies in Paramecium species.

New gene functions arise within existing gene families as a result of gene duplication and subsequent diversification. To gain insight into the steps that led to the functional diversification of paralogues, we tracked duplicate retention patterns, expression-level divergence, and subcellular markers of functional diversification in the Rab GTPase gene family in three Paramecium aurelia species. After whole-genome duplication, Rab GTPase duplicates are more highly retained than other genes in the genome but appear to be diverging more rapidly in expression levels, consistent with early steps in functional diversification. However, by localizing specific Rab proteins in Paramecium cells, we found that paralogues from the two most recent whole-genome duplications had virtually identical localization patterns, and that less closely related paralogues showed evidence of both conservation and diversification. The functionally conserved paralogues appear to target to compartments associated with both endocytic and phagocytic recycling functions, confirming evolutionary and functional links between the two pathways in a divergent eukaryotic lineage. Because the functionally diversifying paralogues are still closely related to and derived from a clade of functionally conserved Rab11 genes, we were able to pinpoint three specific amino acid residues that may be driving the change in the localization and thus the function in these proteins.

One genome's junk is another's garbage.

Experiments on a single-celled ciliate reveal how mobile genetic elements can shape a genome, even one which is not transcriptionally active.

The LATD gene of Medicago truncatula is required for both nodule and root development.

The evolutionary origins of legume root nodules are largely unknown. We have identified a gene, LATD, of the model legume Medicago truncatula, that is required for both nodule and root development, suggesting that these two developmental processes may share a common evolutionary origin. The latd mutant plants initiate nodule formation but do not complete it, resulting in immature, non-nitrogen-fixing nodules. Similarly, lateral roots initiate, but remain short stumps. The primary root, which initially appears to be wild type, gradually ceases growth and forms an abnormal tip that resembles that of the mutant lateral roots. Infection by the rhizobial partner, Sinorhizobium meliloti, can occur, although infection is rarely completed. Once inside latd mutant nodules, S. meliloti fails to express rhizobial genes associated with the developmental transition from free-living bacterium to endosymbiont, such as bacA and nex38. The infecting rhizobia also fail to express nifH and fix nitrogen. Thus, both plant and bacterial development are blocked in latd mutant roots. Based on the latd mutant phenotype, we propose that the wild-type function of the LATD gene is to maintain root meristems. The strong requirement of both nodules and lateral roots for wild-type LATD gene function supports lateral roots as a possible evolutionary origin for legume nodules.

Conservation and innovation in Tetrahymena membrane traffic: proteins, lipids, and compartments.

The past decade has seen a significant expansion in our understanding of membrane traffic in Tetrahymena thermophila, facilitated by the development of new experimental tools and by the availability of the macronuclear genome sequence. Here we review studies on multiple pathways of uptake and secretion, as well as work on metabolism of membrane lipids. We discuss evidence for conservation versus innovation in the mechanisms used in ciliates compared with those in other eukaryotic lineages, and raise the possibility that existing gene expression databases can be exploited to analyze specific pathways of membrane traffic in these cells.

A Rab-based view of membrane traffic in the ciliate Tetrahymena thermophila.

Biologists have long recognized that some single-celled organisms show striking morphological and behavioral complexity, and details of the genetic underpinnings can be mined from the trove of newly-sequenced genomes. Ciliates, among which Tetrahymena thermophila and Paramecium tetraurelia have received most attention, provide clear examples of a lineage in which, as in animal cells, the core pathways of membrane traffic have undergone dramatic expansion and elaboration to facilitate multiple modes of exocytosis and endocytosis. Recent surveys of the Rab GTPases in T. thermophila, including analysis of a large set of GFP-tagged copies, provide a new set of compartmental markers for this lineage, as well as striking views of membrane dynamics in these cells. In addition, phylogenetic analysis of the Tetrahymena Rabs suggests that different eukaryotic lineages may have independently evolved some functionally similar pathways.