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A functional analysis of TOEFAZ1 uncovers protein domains essential for cytokinesis in Trypanosoma brucei.

ABSTRACT: The parasite Trypanosoma brucei is highly polarized, including a flagellum that is attached along the cell surface by the flagellum attachment zone (FAZ). During cell division, the new FAZ positions the cleavage furrow, which ingresses from the anterior tip of the cell towards the posterior. We recently identified TOEFAZ1 (for 'Tip of the Extending FAZ protein 1') as an essential protein in trypanosome cytokinesis. Here, we analyzed the localization and function of TOEFAZ1 domains by performing overexpression and RNAi complementation experiments. TOEFAZ1 comprises three domains with separable functions: an N-terminal ?-helical domain that may be involved in FAZ recruitment, a central intrinsically disordered domain that keeps the morphogenic kinase TbPLK at the new FAZ tip, and a C-terminal zinc finger domain necessary for TOEFAZ1 oligomerization. Both the N-terminal and C-terminal domains are essential for TOEFAZ1 function, but TbPLK retention at the FAZ is not necessary for cytokinesis. The feasibility of alternative cytokinetic pathways that do not employ TOEFAZ1 are also assessed. Our results show that TOEFAZ1 is a multimeric scaffold for recruiting proteins that control the timing and location of cleavage furrow ingression.

SUBMITTER: Sinclair-Davis AN

PROVIDER: S-EPMC5702046 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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A functional analysis of TOEFAZ1 uncovers protein domains essential for cytokinesis in <i>Trypanosoma brucei</i>.

Sinclair-Davis Amy N AN McAllaster Michael R MR de Graffenried Christopher L CL

Journal of cell science 20171009 22

The parasite <i>Trypanosoma brucei</i> is highly polarized, including a flagellum that is attached along the cell surface by the flagellum attachment zone (FAZ). During cell division, the new FAZ positions the cleavage furrow, which ingresses from the anterior tip of the cell towards the posterior. We recently identified TOEFAZ1 (for 'Tip of the Extending FAZ protein 1') as an essential protein in trypanosome cytokinesis. Here, we analyzed the localization and function of TOEFAZ1 domains by perf ...[more]

PMID: 28993462

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Project description: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 (TbRAP1) has conserved functional domains, including BRCA1 C terminus (BRCT), Myb, MybLike, and RAP1 C terminus (RCT). To study functions of various TbRAP1 domains, we established a strain in which one endogenous allele of TbRAP1 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 TbRAP1 are required for normal protein levels, while the Myb and MybLike domains are essential for normal cell growth. Additionally, the Myb domain of TbRAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor (TbTRF), while the BRCT domain is required for its self-interaction. Furthermore, the TbRAP1 MybLike domain contains a bipartite nuclear localization signal that is required for its interaction with importin ? 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. TbRAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although TbRAP1 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 TbRAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of TbRAP1 point mutation phenotypes. We have also determined functional domains of TbRAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of TbRAP1-mediated VSG silencing.

Dataset Information

A functional analysis of TOEFAZ1 uncovers protein domains essential for cytokinesis in Trypanosoma brucei.

Publications

A functional analysis of TOEFAZ1 uncovers protein domains essential for cytokinesis in <i>Trypanosoma brucei</i>.

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