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Protein trafficking through endo/lysosomal compartments is critically important to the biology of the protozoan parasite Trypanosoma brucei , but the routes material may take to the lysosome, as well as the molecular factors regulating those routes, remain incompletely understood. Phosphoinositides are signaling phospholipids that regulate many trafficking events by recruiting specific effector proteins to discrete membrane subdomains. In this study, we investigate the role of one phosphoinositide, PI(3,5)P 2 in T. brucei . We find a low steady state level of PI(3,5)P 2 in bloodstream form parasites comparable to that of other organisms. RNAi knockdown of the putative PI(3)P-5 kinase TbFab1 decreases the PI(3,5)P 2 pool leading to rapid cell death. TbFab1 and PI(3,5)P 2 both localize strongly to late endo/lysosomes. While most trafficking functions were intact in TbFab1 deficient cells, including both endocytic and biosynthetic trafficking to the lysosome, lysosomal turnover of an endogenous ubiquitinylated membrane protein, ISG65, was completely blocked suggesting that TbFab1 plays a role in the ESCRT-mediated late endosomal/multivesicular body degradative pathways. Knockdown of a second component of PI(3,5)P 2 metabolism, the PI(3,5)P 2 phosphatase TbFig4, also resulted in delayed turnover of ISG65. Together, these results demonstrate an essential role for PI(3,5)P 2 in the turnover of ubiquitinylated membrane proteins and in trypanosome endomembrane biology. [ABSTRACT FROM AUTHOR]

• GPI-PLC is a non-essential membrane bound enzyme in African trypanosomes. • Biosynthetic and endocytic trafficking to the lysosome are normal in GPI-PLC kncokout. • GPI-PLC knockout does affect the rate of receptor mediated endocytosis of transferrin. Glycosylphosphatidylinositol-phospholipase C (GPI-PLC) is an enzyme that has been implicated in GPI-dependent protein trafficking and phosphoinositide metabolism in the bloodstream stage of African trypanosomes. However, despite the fact that it is associated with the cytoplasmic face of internal organellar compartments, its role in general membrane trafficking has not been investigated. Using a GPI-PLC null cell line, we determine the effect of GPI-PLC deficiency on these processes. Biosynthetic trafficking of lysosomal cargo, soluble cathepsin L and membrane bound p67, are unaffected. Likewise, secretory transport, recycling and ultimate lysosomal turnover of the GPI-anchored and transmembrane glycoproteins, transferrin receptor and invariant surface glycoprotein 65, respectively, were unaffected. A significant decrease in the endocytic uptake of transferrin was observed, confirming a prior report, but ultimate delivery to the lysosome was unimpacted. These results contribute to our understanding of the roles of this enigmatic enzyme in trypanosome cell biology. [ABSTRACT FROM AUTHOR]