Dataset Information

Basal Body Protein TbSAF1 Is Required for Microtubule Quartet Anchorage to the Basal Bodies in Trypanosoma brucei.

ABSTRACT: Sperm flagellar protein 1 (Spef1, also known as CLAMP) is a microtubule-associated protein involved in various microtubule-related functions from ciliary motility to polarized cell movement and planar cell polarity. In Trypanosoma brucei, the causative agent of trypanosomiasis, a single Spef1 ortholog (TbSpef1) is associated with a microtubule quartet (MtQ), which is in close association with several single-copied organelles and is required for their coordinated biogenesis during the cell cycle. Here, we investigated the interaction network of TbSpef1 using BioID, a proximity-dependent protein-protein interaction screening method. Characterization of selected candidates provided a molecular description of TbSpef1-MtQ interactions with nearby cytoskeletal structures. Of particular interest, we identified a new basal body protein TbSAF1, which is required for TbSpef1-MtQ anchorage to the basal bodies. The results demonstrate that MtQ-basal body anchorage is critical for the spatial organization of cytoskeletal organelles, as well as the morphology of the membrane-bound flagellar pocket where endocytosis takes place in this parasite.IMPORTANCE Trypanosoma brucei contains a large array of single-copied organelles and structures. Through extensive interorganelle connections, these structures replicate and divide following a strict temporal and spatial order. A microtubule quartet (MtQ) originates from the basal bodies and extends toward the anterior end of the cell, stringing several cytoskeletal structures together along its path. In this study, we examined the interaction network of TbSpef1, the only protein specifically located to the MtQ. We identified an interaction between TbSpef1 and a basal body protein TbSAF1, which is required for MtQ anchorage to the basal bodies. This study thus provides the first molecular description of MtQ association with the basal bodies, since the discovery of this association ?30?years ago. The results also reveal a general mechanism of the evolutionarily conserved Spef1/CLAMP, which achieves specific cellular functions via their conserved microtubule functions and their diverse molecular interaction networks.

SUBMITTER: Dong X

PROVIDER: S-EPMC7291619 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Basal Body Protein TbSAF1 Is Required for Microtubule Quartet Anchorage to the Basal Bodies in <i>Trypanosoma brucei</i>.

Dong Xiaoduo X Lim Teck Kwang TK Lin Qingsong Q He Cynthia Y CY

mBio 20200609 3

Sperm flagellar protein 1 (Spef1, also known as CLAMP) is a microtubule-associated protein involved in various microtubule-related functions from ciliary motility to polarized cell movement and planar cell polarity. In <i>Trypanosoma brucei</i>, the causative agent of trypanosomiasis, a single Spef1 ortholog (TbSpef1) is associated with a microtubule quartet (MtQ), which is in close association with several single-copied organelles and is required for their coordinated biogenesis during the cell ...[more]

PMID: 32518185

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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

Basal Body Protein TbSAF1 Is Required for Microtubule Quartet Anchorage to the Basal Bodies in Trypanosoma brucei.

Publications

Basal Body Protein TbSAF1 Is Required for Microtubule Quartet Anchorage to the Basal Bodies in <i>Trypanosoma brucei</i>.

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