Department of Microbiology and Immunology, University at Buffalo School of Medicine, Buffalo, New York, United States of America.
Department of Biochemistry & Molecular Biology, University of Georgia, Athens, Georgia, United States of America.
PLoS Pathog. 2019 Jul 26;15(7):e1007946. doi: 10.1371/journal.ppat.1007946. eCollection 2019 Jul.
By binding to the adaptor protein SKP1 and serving as substrate receptors for the SKP1 Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasma genome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function.
通过与衔接蛋白 SKP1 结合，并作为 SKP1 Cullin 泛素连接酶复合物的底物受体，F -box 蛋白调节包括细胞周期进程和膜运输在内的关键细胞过程。虽然 F -box 蛋白在真核生物中是保守的，并且在酵母、植物和动物中得到了很好的研究，但在寄生原生动物中的研究却滞后了。我们在弓形虫基因组中鉴定了 18 种推定的 F-box 蛋白，其中 4 种在疟原虫中有预测的同源物。两个保守的 F-box 蛋白被证明对弓形虫的适应性很重要，在这里我们关注一个 F-box 蛋白，命名为 TgFBXO1，因为它在复制性速殖子中表达水平最高，并且在互作组筛选中也被鉴定为弓形虫 SKP1 结合蛋白。TgFBXO1 与弓形虫 SKP1 相互作用，证实它是一种真正的 F-box 蛋白。在间期中，TgFBXO1 是内膜复合物 (IMC) 的一个组成部分，IMC 是位于质膜下的一个细胞器。在复制早期，TgFBXO1 定位于正在发育的子细胞支架，这是子细胞 IMC 和微管形成和延伸的部位。TgFBXO1 定位于子细胞支架需要中心体复制，但在动粒分离完成之前。子细胞支架定位需要 TgFBXO1 的 N-豆蔻酰化，并且依赖于小分子 GTP 酶 TgRab11b。最后，我们证明 TgFBXO1 由于其作为子细胞支架效应物的功能而对寄生虫的生长是必需的。TgFBXO1 是第一个在顶复门寄生虫中研究的 F-box 蛋白，也是第一个被证明对子细胞支架功能重要的蛋白。
