Evolution of Tre-2/Bub2/Cdc16 (TBC) Rab GTPase-activating proteins.

Rab GTPases serve as major control elements in the coordination and definition of specific trafficking steps and intracellular compartments. Rab activity is modulated in part by GTPase-activating proteins (GAPs), and many RabGAPs share a Tre-2/Bub2/Cdc16 (TBC)-domain architecture, although the majority of TBC proteins are poorly characterized. We reconstruct the evolutionary history of the TBC family using ScrollSaw, a method for the phylogenetic analysis of pan-eukaryotic data sets, and find a sophisticated, ancient TBC complement of at least 10 members. Significantly, the TBC complement is nearly always smaller than the Rab cohort in any individual genome but also suggests Rab/TBC coevolution. Further, TBC-domain architecture has been well conserved in modern eukaryotes. The reconstruction also shows conservation of ancestral TBC subfamilies, continuing evolution of new TBCs, and frequent secondary losses. These patterns give additional insights into the sculpting of the endomembrane system.

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

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.

Rab11 function in Trypanosoma brucei: identification of conserved and novel interaction partners.

The Ras-like GTPase Rab11 is implicated in multiple aspects of intracellular transport, including maintenance of plasma membrane composition and cytokinesis. In metazoans, these functions are mediated in part via coiled-coil Rab11-interacting proteins (FIPs) acting as Rab11 effectors. Additional interaction between Rab11 and the exocyst subunit Sec15 connects Rab11 with exocytosis. We find that FIPs are metazoan specific, suggesting that other factors mediate Rab11 functions in nonmetazoans. We examined Rab11 interactions in Trypanosoma brucei, where endocytosis is well studied and the role of Rab11 in recycling well documented. TbSec15 and TbRab11 interact, demonstrating evolutionary conservation. By yeast two-hybrid screening, we identified additional Rab11 interaction partners. Tb927.5.1640 (designated RBP74) interacted with both Rab11 and Rab5. RBP74 shares a coiled-coil architecture with metazoan FIPs but is unrelated by sequence and appears to play a role in coordinating endocytosis and recycling. A second coiled-coil protein, Tb09.211.4830 (TbAZI1), orthologous to AZI1 in Homo sapiens, interacts exclusively with Rab11. AZI1 is restricted to taxa with motile cilia/flagella. These data suggest that Rab11 functions are mediated by evolutionarily conserved (i.e., AZI1 and Sec15) and potentially lineage-specific (RBP74) interactions essential for the integration of the endomembrane system.

The single ENTH-domain protein of trypanosomes; endocytic functions and evolutionary relationship with epsin.

Epsin N-terminal homology (ENTH) domains occur in proteins of either the epsin or epsin-related (epsinR) form. They principally function in clathrin-mediated trafficking and membrane deformation. Both epsin and epsinR possess clathrin-binding motifs, but only epsin incorporates a ubiquitin-interaction motif (UIM). To better understand the origins of ENTH-domain proteins and their functions, we performed detailed comparative genomics and phylogenetics on the epsin family. The epsin ENTH-UIM configuration is an architecture restricted to yeast and animals. Further, we undertook functional analysis in Trypanosoma brucei (T. brucei), a divergent organism possessing a single ENTH-domain protein (TbEpsinR). TbEpsinR has a cellular location similar to both epsin and epsinR at plasma membrane clathrin budding sites and endosomal compartments, and associates with clathrin, as demonstrated by coimmunoprecipitation. Knockdown of TbEpsinR leads to a significant decrease in the intracellular pools of multiple surface antigens, without affecting bulk membrane internalization. Therefore, despite lacking the UIM, TbEpsinR maintains a similar role to metazoan epsin in endocytosis and participates as a clathrin-associated adaptor. We suggest that recruitment of a UIM to the ENTH-domain proteins was not essential for participation in endocytosis of ubiquitylated molecules, and is presumably a specific innovation restricted to higher eukaryotes.

Intracellular trafficking in the trypanosomatids.

Trypanosomes are members of the kinetoplastida, a group of divergent protozoan parasites responsible for considerable morbidity and mortality worldwide. These organisms have highly complex life cycles requiring modification of their cell surface together with engagement of immune evasion systems to effect survival; both processes intimately involve the membrane trafficking system. The completion of three trypanosomatid and several additional protist genomes in the last few years is providing an exciting opportunity to evaluate, at the molecular level, the evolution and diversity of membrane trafficking across deep evolutionary time as well as to analyse in unprecedented detail the membrane trafficking systems of trypanosomes.

TbVps34, the trypanosome orthologue of Vps34, is required for Golgi complex segregation.

Phosphoinositides are important regulators of numerous cellular functions. The yeast class III phosphatidylinositol 3-kinase Vps34p, and its human orthologue hVPS34, are implicated in control of several key pathways, including endosome to lysosome transport, retrograde endosome to Golgi traffic, multivesicular body formation, and autophagy. We have identified the Vps34p orthologue in the African trypanosome, TbVps34. Knockdown of TbVps34 expression by RNA interference induces a severe growth defect, with a post-mitotic block to cytokinesis accompanied by a variety of morphological abnormalities. GFP2xFYVE, a chimeric protein that specifically binds phosphatidylinositol 3-phosphate, localizes to the trypanosome endosomal system and is delocalized under TbVps34 RNA interference (RNAi), confirming that TbVps34 is an authentic phosphatidylinositol 3-kinase. Expression of GFP2xFYVE enhances the TbVps34 RNAi-associated growth defect, suggesting a synthetic interaction via competition for phosphatidylinositol 3-phosphate-binding sites with endogenous FYVE domain proteins. Endocytosis of a fluid phase marker is unaffected by TbVps34 RNAi, but receptor-mediated endocytosis of transferrin and transport of concanavalin A to the lysosome are both impaired, confirming a role in membranous endocytic trafficking for TbVps34. TbVps34 knockdown inhibits export of variant surface glycoprotein, indicating a function in exocytic transport. Ultrastructural analysis revealed a highly extended Golgi apparatus following TbVps34 RNAi, whereas expression of the Golgi marker red fluorescent protein-GRASP (Grp1 (general receptor for phosphoinositides-1)-associated scaffold protein) demonstrated that trypanosomes are able to duplicate the Golgi complex but failed to complete segregation during mitosis, despite faithful replication and segregation of basal bodies and the kinetoplast. These observations implicate TbVps34 as having a role in coordinating segregation of the Golgi complex at cell division.