Summary We present the first functional analysis of the small GTPase, TbRab7, in Trypanosoma brucei. TbRab7 defines discrete late endosomes closely juxtaposed to the terminal p67+ lysosome. RNAi indicates that TbRab7 is essential in bloodstream trypanosomes. Initial rates of endocytosis were unaffected, but lysosomal delivery of cargo, including tomato lectin (TL) and trypanolytic factor (TLF) were blocked. These accumulate in a dispersed internal compartment of elevated pH, likely derived from the late endosome. Surface binding of TL but not TLF was reduced, suggesting that cellular distribution of flagellar pocket receptors is differentially regulated by TbRab7. TLF activity was reduced approximately threefold confirming that lysosomal delivery is critical for trypanotoxicity. Unexpectedly, delivery of endogenous proteins, p67 and TbCatL, were unaffected indicating that TbRab7 does not regulate biosynthetic lysosomal trafficking. Thus, unlike mammalian cells and yeast, lysosomal trafficking of endocytosed and endogenous proteins occur via different routes and/or are regulated differentially. TbRab7 silencing had no effect on a cryptic default pathway to the lysosome, suggesting that the default lysosomal reporters p67ΔTM, p67ΔCD and VSGΔGPI do not utilize the endocytic pathway as previously proposed. Surprisingly, conditional knockout indicates that TbRab7 may be non-essential in procyclic insect form trypanosomes.

The multivesicular body (MVB) is a specialized Rab7+ late endosome (LE) containing multiple intralumenal vesicles that function in targeting ubiquitinylated cell surface proteins to the lysosome for degradation. African trypanosomes lack a morphologically well-defined MVB, but contain orthologues of the ESCRT machinery that mediates MVB formation. We investigate the role of TbVps23, an early ESCRT component, and TbVps4, the terminal ESCRT ATPase, in lysosomal trafficking in bloodstream form trypanosomes. Both localize to the TbRab7+ LE and RNAi silencing of each rapidly blocks growth. TbVps4 silencing results in ∼3-fold accumulation of TbVps23 at the LE, consistent with blocking terminal ESCRT disassembly. Trafficking of endocytic and biosynthetic cargo, but not default lysosomal reporters, is also negatively affected. Others reported that TbVps23 mediates ubiquitin-dependent lysosomal degradation of invariant surface glycoproteins (ISG65) (Traffic 2008, 9:1698). In contrast, we find that TbVps23 ablation does not affect ISG65 turnover, while TbVps4 silencing markedly enhances lysosomal degradation. We propose several models to accommodate these results, including that the ESCRT machinery actually retrieves ISG65 from the LE to earlier endocytic compartments, and in its absence ISG65 traffics more efficiently to the lysosome. Overall, these results confirm that the ESCRT machinery is essential in T. brucei and plays important and novel role(s) in LE function in trypanosomes.

The twin-arginine translocation (Tat) pathway is a system used by some bacteria to export proteins out from the cytosol to the cell surface or extracellular environment. A functional Tat

African trypanosomes are the causative agents of human trypanosomiasis (sleeping sickness). The pathogenic stage of the parasite has unique adaptations to life in the bloodstream of the mammalian host, including upregulation of endocytic and lysosomal activities. We investigated stage-specific requirements for cytoplasmic adaptor/clathrin machinery in post-Golgi apparatus biosynthetic sorting to the lysosome using RNA interference silencing of the Tb1 subunit of adaptor complex 1 (AP-1), in conjunction with immunolocalization, kinetic analyses of reporter transport, and quantitative endocytosis assays. Tb1 silencing was lethal in both stages, indicating a critical function(s) for the AP-1 machinery. Transport of soluble and membrane-bound secretory cargoes was Tb1 independent in both stages. In procyclic parasites, trafficking of the lysosomal membrane protein, p67, was disrupted, leading to cell surface mislocalization. The lysosomal protease trypanopain was also secreted, suggesting a transmembrane-sorting receptor for this soluble hydrolase. In bloodstream trypanosomes, both p67 and trypanopain trafficking were unaffected by Tb1 silencing, suggesting that AP-1 is not necessary for biosynthetic lysosomal trafficking. Endocytosis in bloodstream cells was also unaffected, indicating that AP-1 does not function at the flagellar pocket. These results indicate that post-Golgi apparatus sorting to the lysosome is critically dependent on the AP-1/clathrin machinery in procyclic trypanosomes but that this machinery is not necessary in bloodstream parasites. We propose a simple model for stage-specific default secretory trafficking in trypanosomes that is consistent with the behavior of other soluble and glycosylphosphatidylinositol-anchored cargos and which is influenced by upregulation of endocytosis in bloodstream parasites as an adaptation to life in the mammalian bloodstream.