Trypanosoma brucei RAP1 Has Essential Functional Domains That Are Required for Different Protein Interactions

Abstract

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. Tb RAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although Tb RAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for Tb RAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of Tb RAP1 point mutation phenotypes. We have also determined functional domains of Tb RAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of Tb RAP1-mediated VSG silencing. RAP1 is a telomere protein that is well conserved from protozoa to mammals. It plays important roles in chromosome end protection, telomere length control, and gene expression/silencing at both telomeric and nontelomeric loci. Interaction with different partners is an important mechanism by which RAP1 executes its different functions in yeast. The RAP1 ortholog in Trypanosoma brucei is essential for variant surface glycoprotein (VSG) monoallelic expression, an important aspect of antigenic variation, where T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. Like other RAP1 orthologs, T. brucei RAP1 ( Tb RAP1) has conserved functional domains, including BR CA1 C t erminus (BRCT), Myb, MybLike, and R AP1 C t erminus (RCT). To study functions of various Tb RAP1 domains, we established a strain in which one endogenous allele of Tb RAP1 is flanked by loxP repeats, enabling its conditional deletion by Cre-mediated recombination. We replaced the other TbRAP1 allele with various mutant alleles lacking individual functional domains and examined their nuclear localization and protein interaction abilities. The N terminus, BRCT, and RCT of Tb RAP1 are required for normal protein levels, while the Myb and MybLike domains are essential for normal cell growth. Additionally, the Myb domain of Tb RAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor ( Tb TRF), while the BRCT domain is required for its self-interaction. Furthermore, the Tb RAP1 MybLike domain contains a bipartite nuclear localization signal that is required for its interaction with importin α and its nuclear localization. Interestingly, RAP1’s self-interaction and the interaction between RAP1 and TRF are conserved from kinetoplastids to mammals. However, details of the interaction interfaces have changed throughout evolution. IMPORTANCE Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. Tb RAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although Tb RAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for Tb RAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of Tb RAP1 point mutation phenotypes. We have also determined functional domains of Tb RAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of Tb RAP1-mediated VSG silencing.