Both of the Rab5 subfamily small GTPases of Trypanosoma brucei are essential and required for endocytosis

Abstract

Endocytosis is an essential process in Trypanosoma brucei and all evidence suggests it is exclusively clathrin-mediated. The trypanosome genome encodes two Rab5 proteins, small GTPases that play a role in very early stages of endocytosis. In the mammalian bloodstream stage TbRAB5A localises to compartments containing internalised antibody, variant surface glycoprotein (VSG) and transferrin, whilst TbRAB5B localises to compartments containing the transmembrane protein ISG(100). Dominant-active forms of TbRAB5A stimulate endocytosis in procyclic forms and alter the kinetics of anti-VSG antibody and transferrin turnover in bloodstream stages. Similar mutants of TbRAB5B increase fluid phase uptake in procyclic cells but do not significantly affect endocytosis in bloodstream forms. Here, we use RNA interference to evaluate the relative importance of TbRAB5A and TbRAB5B and show that both GTPases are essential in the bloodstream form. Depletion of either TbRAB5A or TbRAB5B results in morphological abnormalities, including enlargement of the flagellar pocket, consistent with a potent block to endocytosis. Also, RNAi compromises transferrin accumulation in both cases but induces distinct patterns of mislocalisation of endosomal markers. Finally, RNAi of either TbRAB5A or TbRAB5B results in a decrease in levels of clathrin. Taken together, these data indicate that both TbRAB5A and TbRAB5B are required for endocytosis in trypanosomes and demonstrate that there are multiple essential endocytic routes in this organism.