An evolutionarily conserved coiled-coil protein implicated in polycystic kidney disease is involved in basal body duplication and flagellar biogenesis in Trypanosoma brucei.

Trypanosoma brucei is a flagellated protozoan with a highly polarized cellular structure. TbLRTP is a trypanosomal protein containing multiple SDS22-class leucine-rich repeats and a coiled-coil domain with high similarity to a mammalian testis-specific protein of unknown function. Homologues are present in a wide range of higher eukaryotes including zebra fish, where the gene product has been implicated in polycystic kidney disease. Western blot analysis and immunofluorescence with antibodies against recombinant TbLRTP indicate that the protein is expressed throughout the trypanosome life cycle and localizes to distal zones of the basal bodies. Overexpression and RNA interference demonstrate that TbLRTP is important for faithful basal body duplication and flagellum biogenesis. Expression of excess TbLRTP suppresses new flagellum assembly, while reduction of TbLRTP protein levels often results in the biogenesis of additional flagellar axonemes and paraflagellar rods that, most remarkably, are intracellular and fully contained within the cytoplasm. The mutant flagella are devoid of membrane and are often associated with four microtubules in an arrangement similar to that observed in the normal flagellar attachment zone. Aberrant basal body and flagellar biogenesis in TbLRTP mutants also influences cell size and cytokinesis. These findings demonstrate that TbLRTP suppresses basal body replication and subsequent flagellar biogenesis and indicate a critical role for the LRTP family of proteins in the control of the cell cycle. These data further underscore the role of aberrant flagellar biogenesis as a disease mechanism.

PtdIns-specific MPR pathway association of a novel WD40 repeat protein, WIPI49.

WIPI49 is a member of a previously undescribed family of WD40-repeat proteins that we demonstrate binds 3-phosphorylated phosphoinositides. Immunofluorescent imaging indicates that WIPI49 is localized to both trans-Golgi and endosomal membranes, organelles between which it traffics in a microtubule-dependent manner. Live cell imaging establishes that WIPI49 traffics through the same set of endosomal membranes as that followed by the mannose-6-phosphate receptor (MPR), and consistent with this, WIPI49 is enriched in clathrin-coated vesicles. Ectopic expression of wild-type WIPI49 disrupts the proper functioning of this MPR pathway, whereas expression of a double point mutant (R221,222AWIPI49) unable to bind phosphoinositides does not disrupt this pathway. Finally, suppression of WIPI49 expression through RNAi, demonstrates that its presence is required for normal endosomal organization and distribution of the CI-MPR. We conclude that WIPI49 is a novel regulatory component of the endosomal and MPR pathway and that this role is dependent upon the PI-binding properties of its WD40 domain.

Rab5 and Rab11 mediate transferrin and anti-variant surface glycoprotein antibody recycling in Trypanosoma brucei.

The mammalian-infective bloodstream form of Trypanosoma brucei possesses a highly active endocytotic system. Evasion of the host immune response by T. brucei is dependent on antigenic variation of VSG (variant surface glycoprotein), but additional mechanisms for removal of surface-bound antibody also operate. Four Rab proteins, Tb (trypanosomal) RAB4, 5A, 5B and 11 are located to the endosomal system; TbRAB5A and TbRAB11 co-localize with internalized anti-VSG antibody and transferrin. A live cell assay was used to record a single cycle of endocytosis of anti-VSG IgG and transferrin, their subsequent degradation within the endosomal system and exocytosis of the products. TbRAB5A and TbRAB11 were involved in the overall process of endocytosis, degradation and exocytosis, whereas TbRAB5B and TbRAB4 were not implicated. The kinetics of anti-VSG IgG and transferrin recycling depend on the nucleotide state of TbRAB5A and TbRAB11. These data, together with previous work, suggest that IgG and transferrin initially enter a TbRAB5A sorting endosome and are most probably recycled subsequently via a TbRAB11-dependent step. Analysis of the recycled IgG and transferrin demonstrated extensive degradation of these recycled proteins. Degradation of transferrin was enhanced in cells expressing increased amounts of TbRAB5A or TbRAB11 with a Ser-->Asn mutation, but was decreased when active TbRAB11 was overexpressed. The extent of degradation of anti-VSG IgG was found to be unaffected by mutant Rab protein expression. The presence of an efficient mechanism for the removal of IgG bound to the external surface of T. brucei and its subsequent proteolysis within the recycling system suggests a role for this pathway in immune evasion.

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.