Characterization of the Late Endosomal ESCRT Machinery in Trypanosoma brucei

The multivesicular body (MB) is a specialized Rab7+ late endosome (LE) containing multiple intralumenal esicles that function in targeting ubiquitinylated cell surface proteins to the lysosome for degradation. African trypanosomes lack a morphologically well-defined MB, but contain orthologs of the ESCRT (Endosomal Sorting Complex Required for Transport) machinery that mediates MB formation. We inestigate the role of Tbps23, an early ESCRT component, and Tbps4, 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. Tbps4 silencing results in approximately threefold accumulation of Tbps23 at the LE, consistent with blocking terminal ESCRT disassembly. Trafficking of endocytic and biosynthetic cargo, but not default lysosomal reporters, is also negatiely affected. Others reported that Tbps23 mediates ubiquitin-dependent lysosomal degradation of inariant surface glycoproteins (ISG65) (Leung et al., Traffic 2008;9:1698-1716). In contrast, we find that Tbps23 ablation does not affect ISG65 turnoer, while Tbps4 silencing markedly enhances lysosomal degradation. We propose seeral models to accommodate these results, including that the ESCRT machinery actually retriees ISG65 from the LE to earlier endocytic compartments, and in its absence ISG65 traffics more efficiently to the lysosome. Oerall, these results confirm that the ESCRT machinery is essential in Trypanosoma brucei and plays important and noel role(s) in LE function in trypanosomes.