ABSTRACTTheEndosomalSortingComplexRequired forTransport machinery consists of four protein complexes (ESCRT 0-IV) and the post ESCRT ATPase Vps4. ESCRT mediates cargo delivery for lysosomal degradation via formation of multivesicular bodies.Trypanosoma bruceicontains orthologues of ESCRT I-III and Vps4. Trypanosomes also have a ubiquitinylated invariant surface glycoprotein (ISG65) that is delivered to the lysosome by ESCRT, however, we previously implicated TbVps4 in rescue and recycling of ISG65. Here we use conditional silencing to investigate the role of TbVps24, a phosphoinositide-binding ESCRT III component, on protein trafficking. TbVps24 localizes to the TbRab7+late endosome, and binds PI(3,5)P2, the product of the TbFab1 kinase, both of which also localize to late endosomes. TbVps24 silencing is lethal, and negatively affects biosynthetic trafficking of the lysosomal markers p67 and TbCathepsin L. However, the major phenotype of silencing is accelerated degradation and depletion of the surface pool of ISG65. Thus, TbVps24 silencing phenocopies that of TbVps4 in regard to ISG65 trafficking. This presents a paradox since we have previously found that depletion of TbFab1 completely blocks ISG65 turnover. We propose a model in which late ESCRT components operate at two sites, one PI(3,5)P2-dependent (degradation) and one PI(3,5)P2-independent (recycling), to regulate ISG65 homeostasis.