Trypanosoma brucei belongs to a group of unicellular, flagellated parasites that are responsible for human African trypanosomiasis. An essential aspect of parasite pathogenicity is cytoskeleton remodelling, which occurs during the life cycle of the parasite and is accompanied by major changes in morphology and organelle positioning. The flagellum originates from the basal bodies and exits the cell body through the flagellar pocket (FP) but remains attached to the cell body via the flagellum attachment zone (FAZ). The FP is an invagination of the pellicular membrane and is the sole site for endo- and exocytosis. The FAZ is a large complex of cytoskeletal proteins, plus an intracellular set of four specialised microtubules (MtQ) that elongate from the basal bodies to the anterior end of the cell. At the distal end of the FP, an essential, intracellular, cytoskeletal structure called the flagellar pocket collar (FPC) circumvents the flagellum. Overlapping the FPC is the hook complex (HC) (a sub-structure of the previously named bilobe) that is also essential and is thought to be involved in protein FP entry. BILBO1 is the only functionally characterised FPC protein and is necessary for FPC and FP biogenesis. Here, we used a combination of in vitro and in vivo approaches to identify and characterize a new BILBO1 partner protein—FPC4. We demonstrate that FPC4 localises to the FPC, the HC, and possibly to a proximal portion of the MtQ. We found that the C-terminal domain of FPC4 interacts with the BILBO1 N-terminal domain, and we identified the key amino acids required for this interaction. Interestingly, the FPC4 N-terminal domain was found to bind microtubules. Over-expression studies highlight the role of FPC4 in its association with the FPC, HC and FPC segregation. Our data suggest a tripartite association between the FPC, the HC and the MtQ.

布氏锥虫属于一组单细胞、有鞭毛的寄生虫，是人类非洲锥虫病的罪魁祸首。寄生虫致病性的一个重要方面是细胞骨架重塑，它发生在寄生虫的生命周期中，并伴随着形态和细胞器定位的重大变化。鞭毛起源于基体并通过鞭毛袋（FP）离开细胞体，但通过鞭毛附着区（FAZ）保持附着在细胞体上。 FP 是表膜的内陷，是胞吞和胞吐的唯一部位。 FAZ 是一个大型的细胞骨架蛋白复合体，加上一组细胞内的四个专门的微管 (MtQ)，这些微管从基体延伸到细胞的前端。在 FP 的远端，一种称为鞭毛口袋领 (FPC) 的重要细胞内细胞骨架结构包围鞭毛。与 FPC 重叠的是钩复合体 (HC)（以前称为双叶的子结构），它也是必不可少的，被认为参与蛋白质 FP 的进入。 BILBO1 是唯一具有功能特征的 FPC 蛋白，并且是 FPC 和 FP 生物发生所必需的。在这里，我们结合使用体外和体内方法来鉴定和表征新的 BILBO1 伴侣蛋白 - FPC4。我们证明 FPC4 定位于 FPC、HC，并且可能定位于 MtQ 的近端部分。我们发现 FPC4 的 C 端结构域与 BILBO1 N 端结构域相互作用，并确定了这种相互作用所需的关键氨基酸。有趣的是，FPC4 N 端结构域被发现可以结合微管。 过表达研究强调了 FPC4 在与 FPC、HC 和 FPC 分离相关中的作用。我们的数据表明 FPC、HC 和 MtQ 之间存在三方关联。