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.
The mechanisms by which pathogens interact with their environment are of major importance, both for fulfilling the basic needs of the parasite and understanding immune evasion. For African trypanosomes, the surface is dominated by the variant surface glycoprotein (VSG), but recent data has demonstrated an important role for ubiquitylation in mediating turnover of invariant surface glycoproteins (ISGs) and maintaining ISG copy number independent of VSG. Further, ISG expression is required for suramin-sensitivity. Here we describe mechanisms mediating ISG turnover, uncovered using a screen for genes involved in sensitivity to suramin. These involve multiple aspects of the ubiquitylation machinery, and connect ISG turnover with additional surface proteins. Our data provide a first insight into the complexity of regulation of the ISG family, identifying further aspects to the control of a drug-sensitivity pathway in trypanosomes, and offering insights into metabolism of the parasite surface proteome.