TbRAB18, a developmentally regulated Golgi GTPase from Trypanosoma brucei

The trypanosomal secretory system is broadly similar to that of higher eukaryotes as proteins enter the system via the endoplasmic reticulum and are transported to the Golgi complex for elaboration of glycan chains. Importantly N-glycan processing is stage specific with only the bloodstream form (BSF) processing beyond the oligomannose form. Increased complexity of the BSF Golgi apparatus, as evidenced by morphological studies, may underpin this higher activity, but few trypanosome-specific Golgi proteins have been described that may play a role in this developmental alteration. Here we describe a novel member of the T. brucei Rab family, TbRAB18, which is stage-regulated and highly expressed in the BSF whilst barely detectable in the insect stage. This stage-specific expression suggests the presence of a TbRAB18-dependent transport pathway required for survival in the mammalian host. Furthermore, data indicate that TbRAB18 localises to membranes in close juxtaposition to structures stained with BODIPY-ceramide, a Golgi marker. Wild type TbRAB18, ectopically expressed in insect stage cells colocalises with TbRAB31, and hence is targeted to the Golgi complex, consistent with the location of the endogenous protein in the bloodstream form, whilst GTP and GDP-locked mutant isoforms demonstrate distinct localisations, suggesting that Golgi-targetting of TbRAB18 is nucleotide-state dependent. We also find that ectopic expression of TbRAB18 wild type and mutant isoforms has no detectable effect on the synthetic anteriograde trafficking probe, TbBiPN. Finally, the location, and hence function, of TbRAB18 are distinct from the closest metazoan homologue, murine Rab18; the latter protein is involved in endocytic transport pathways whilst clearly TbRAB18 is not. Our data indicate further complexity in the evolution of small GTPases, and highlight the need for robust functional data prior to assignment of members of complex gene families.