Discussion

All endo- and exocytosis in the African trypanosome Trypanosoma brucei occurs at a single point on its plasma membrane. This plasma membrane subdomain, the flagellar pocket, is a small vase-shaped invagination containing the root of the cell's single flagellum. A number of cytoskeleton-associated multiprotein complexes are coiled around the  neck of the flagellar pocket on its cytoplasmic face. One of these, the hook complex, has been proposed to affect the entry of cargo into the flagellar pocket. Previous characterisation of the hook complex component TbMORN1 showed that its depletion resulted in an apparent size exclusion effect, with larger cargo now unable to enter the flagellar pocket. In this study, the hook complex component TbSmee1 was characterised in bloodstream form Trypanosoma brucei.TbSmee1 localised to both the hook complex and the flagellum attachment zone tip of replicating cells, with different parts of its primary structure mediating targeting to each. Depletion of TbSmee1 by RNAi was lethal, and resulted in an enlargement of the flagellar pocket. Like TbMORN1, depletion of TbSmee1 was found to affect the entry of cargo, with small fluid-phase cargo still able to enter the flagellar pocket lumen, but larger surface-bound molecules trapped outside. Unexpectedly, the same effect was also produced by depletion of clathrin, suggesting that endocytic activity is a prerequisite for the entry of surface-bound cargo into the flagellar pocket.