Ubiquitylation and Developmental Regulation of Invariant Surface Protein Expression in Trypanosomes

Abstract

The cell surface of Trypanosoma brucei is dominated by the glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG), which is essential for immune evasion. VSG biosynthesis, trafficking, and turnover are well documented, but trans-membrane domain (TMD) proteins, including the invariant surface glycoproteins (ISGs), are less well characterized. Internalization and degradation of ISG65 depend on ubiquitylation of conserved cytoplasmic lysines. Using epitope-tagged ISG75 and reporter chimeric proteins bearing the cytoplasmic and trans-membrane regions of ISG75, together with multiple mutants with lysine-to-arginine mutations, we demonstrate that the cytoplasmic tail of ISG75 is both sufficient and necessary for endosomal targeting and degradation. The ISG75 chimeric reporter protein localized to endocytic organelles, while lysine-null versions were significantly stabilized at the cell surface. Importantly, ISG75 cytoplasmic lysines are modified by extensive oligoubiquitin chains and ubiquitylation is abolished in the lysine-null version. Furthermore, we find evidence for differential modes of turnover of ISG65 and ISG75. Full-length lysine-null ISG65 localization and protein turnover are significantly perturbed, but ISG75 localization and protein turnover are not, while ubiquitin conjugates can be detected for full-length lysine-null ISG75 but not ISG65. We find that the ISG75 ectodomain has a predicted coiled-coil, suggesting that ISG75 could be part of a complex, while ISG65 behaves independently. We also demonstrate a developmental stage-specific mechanism for exclusion of surface ISG expression in insect-stage cells by a ubiquitin-independent mechanism. We suggest that ubiquitylation may be a general mechanism for regulating trans-membrane domain surface proteins in trypanosomes.In higher eukaryotes, internalization and sorting of surface trans-membrane domain (TMD) proteins require specific signals within the cytoplasmic domain that are recognized by multiple factors within the endocytic machinery (5). One pathway involves covalent attachment of ubiquitin to substrate lysine residues within the cytoplasmic domain of TMD proteins. Ubiquitylation is mediated by an enzymatic cascade terminating in an E3 ubiquitin ligase, which transfers thioesterlinked ubiquitin to the substrate lysine-NH 2 group, forming an isopeptide bond (23).

Rab28 function in trypanosomes: interactions with retromer and ESCRT pathways

“…An in trans decrease in expression levels of ESCRT I (Vps23 and Vps28) and retromer complex (Vps26) proteins on Rab28 knockdown suggests a functional connection between Rab28, ESCRT and retromer. The effects on ESCRT are fully consistent with the observed partial protection of ISG65 and ISG75, as both require ubiquitylation for turnover and, hence, are probably sorted by the ESCRT system (Chung et al, 2008;Leung et al, 2011). The effects on the Rab11 recycling compartment suggest increased cycling of cargo between endosomes, as reported upon silencing of Vps26 and Vps23 in mammalian cells (Seaman, 2004;Razi and Futter, 2006;Raiborg et al, 2008).…”

“…Specifically, both retromer and ESCRT participate in transport at late endosomal compartments, but their very distinct functions necessitate coordination. T. brucei Rab28 could be required for maintaining structural boundaries at endosomes or for correct assembly of ESCRT and/or retromer complexes, and potentially in controlling turnover of ubiquitylated cargo, which is probably a major route for degradation of surface proteins in trypanosomes (Leung et al, 2011). Depletion of T. brucei Rab28 resulted in extensive morphological defects, suggesting that Rab28 is required to maintain endosome structure.…”

“…A possible role for T. brucei Rab28 in turnover of internalized surface proteins was examined. Invariant surface glycoproteins 65 and 75 (ISG65 and ISG75) are abundant type I cell surface trans-membrane proteins possessing multiple cytoplasmic lysine residues and undergo ubiquitin-dependent degradation (Chung et al, 2004;Leung et al, 2011).…”

Evidence for Recycling of Invariant Surface Transmembrane Domain Proteins in African Trypanosomes

“…Radioimmunoprecipitation assays (RIPAs) were performed as described previously (12). Briefly, 1 ϫ 10 7 cells were washed in PBS, and resuspended in 500 l of methionine-cysteine (Met/Cys)-free RPMI 1640 (Sigma) medium supplemented with 10% dialyzed fetal bovine serum (FBS), followed by incubation at 37°C for 1 h. Cells were pulse-labeled for 1 h with EasyTAg EXPRESS 35 S protein labeling mix (PerkinElmer) at a specific activity of 200 Ci/ml and chased by addition of 4.5 ml HMI-9 medium for 4 h. Cells were washed in ice-cold PBS, lysed in 100 l RIPA buffer (25 mM Tris-Cl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) for 15 min on ice, and incubated for 5 min at 95°C in RIPA buffer containing 1% SDS.…”

“…Recently, the trafficking and functions of a family of invariant surface glycoproteins (ISGs) have been described. These proteins have type I transmembrane topology and intercalate with VSG on the surface, but are rapidly turned over by a ubiquitylation-dependent mechanism (10)(11)(12). Most recently, the ISG75 subfamily have been implicated in the uptake of the first-line defense compound suramin, indicating an important role in drug interactions and also that ISGs may have physiological roles as receptors, although the ligand(s) remains to be identified (13).…”