Modulation of the Surface Proteome through Multiple Ubiquitylation Pathways in African Trypanosomes

Abstract

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes.

Exploiting the Achilles’ heel of membrane trafficking in trypanosomes

“…Additional clathrin adaptor proteins are present and include both ENTH and ANTH-domain phosphoinositide-binding proteins [17] and a cohort of trypanosomatid-specific proteins [18]. Sorting surface trans -membrane proteins requires ubiquitylation, likely performed by divergent ubiquitin ligases, although these remain unidentified [19 •• ]. Whilst these details indicate a distinct endocytic system, the level of conservation with, for example, Saccharomyces cerevisiae , is considerable and the divergence certainly appears less extreme than in Apicomplexan parasites, where entire compartments and pathways have been repurposed [20, 21•].…”

“…Genome-wide loss-of-function screens identified multiple genes that sensitize trypanosomes to suramin [26 •• ] (Figure 2), many of which have roles and/or locations at the endocytic pathway, for example, invariant surface glycoprotein 75 (ISG75), two deubiquitylating enzymes (DUBs) Usp7 and Vdu1, the AP-1 adaptin complex and the lysosomal protein p67 [27]. An ISG75-dependent pathway is required for lysosomal delivery of suramin while an AP-1-dependent path is likely connected to lysosomal composition and a requirement for transport of p67 and other factors (Figure 1) [19 •• ]. ISG75 stability is regulated by ubiquitylation [28] and evidence that trypanosome Usp7 and Vdu1 modulate ISG75 turnover is consistent with this model [19 •• ].…”

“…An ISG75-dependent pathway is required for lysosomal delivery of suramin while an AP-1-dependent path is likely connected to lysosomal composition and a requirement for transport of p67 and other factors (Figure 1) [19 •• ]. ISG75 stability is regulated by ubiquitylation [28] and evidence that trypanosome Usp7 and Vdu1 modulate ISG75 turnover is consistent with this model [19 •• ]. Significantly, the suramin-uptake pathway is highly specific and does not involve the closely related invariant surface glycoprotein 65 (ISG65) family.…”

“…Indeed, for both pentamidine and melarsoprol, the screens identified proteins likely involved in phosphorylation (Phosphatase 2C) and ubiquitylation (Cullin1) (Figure 2), post-translational modifications implicated in regulating endocytosis of mammalian AQP [49]. This is reminiscent of the DUBs that sensitize trypanosomes to suramin and regulate ISG75 [19 •• ]. However, if these proteins are involved in the promotion of AQP2 internalization and turnover, knockdown could change pentamidine sensitivity regardless of whether the drug binds the channel or translocates through it.…”

Resolving the homology—function relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology

“…AP-1 is involved in lysosomal delivery of p67, the major lysosomal glycoprotein, in T. brucei and there is evidence that this is developmentally regulated [52,53]. More recently AP-1 was implicated in sensitivity of T. brucei to suramin, an important frontline drug, and this appears to synergize with endocytosis of surface components, presumably to “condition” the lysosome in some manner to maintain sensitivity to the drug [52]. …”