Late ESCRT Machinery Mediates The Recycling And Rescue of Invariant Surface Glycoprotein 65 inTrypanosoma brucei

Abstract

Running title: Vps24 and ISG65 trafficking in trypanosomes Key Words: Trypanosome, ESCRT, Vps24, invariant surface glycoprotein 65, recycling, endocytosis Vps24 and ISG65 trafficking in trypanosomes 2 ABSTRACT (200) The Endosomal Sorting Complex Required for Transport 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 brucei contains 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)P 2 , 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 p67and 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)P 2 -dependent (degradation) and one PI(3,5)P 2 -independent (recycling), to regulate ISG65 homeostasis.

To the Surface and Back: Exo- and Endocytic Pathways in Trypanosoma brucei

“…TbRab7 depletion does not affect the endocytosis rate, but leads to complete cessation of delivery of the endocytosed cargo to the lysosome, a similar effect to that observed upon silencing of TbVps23 and TbVps4 (Silverman et al, 2011(Silverman et al, , 2013. Interestingly, while TbRab7 has no influence on biosynthetic trafficking of the lysosomal markers p67 and TbCathepsin L (TbCatL), the ESCRT components TbVps4, TbVps23 and TbVps24 do (Silverman et al, 2011(Silverman et al, , 2013Umaer and Bangs, 2020). Another Rab, TbRab28, seems to be involved in transport to the lysosome.…”

“…Orthologs of the ESCRT machinery, such as TbVps4, TbVps23, TbVps24, and TbVps28 were found in T. brucei (Leung et al, 2008;Silverman et al, 2013;Gilden et al, 2017;Umaer and Bangs, 2020). These ESCRT components as well as TbFab1 kinase and its product PI(3,5)P 2 , one of the ligands of TbVps24, localize in LE membranes of T. brucei (Leung et al, 2008;Silverman et al, 2013;Gilden et al, 2017;Umaer and Bangs, 2020). Despite the role of ESCRT in ILV formation and MVB development in mammalian cells, T. brucei lacks a well-defined MVB (Silverman et al, 2013).…”

A multifaceted strategy to improve recombinant expression and structure determination of complex proteins: A Trypanosoma surface protein as an example

“…As an example we use ISG65 (Invariant surface glycoproteins 65) from the eukaryotic parasite Trypanosoma brucei. Invariant surface glycoproteins have been discovered about 30 years ago [7] and since then have been the subject of numerous studies [8][9][10][11][12][13]. Nevertheless, despite many efforts, no structure of an ISG has been be determined yet.…”