恶性疟原虫和伯氏疟原虫基因组发表20年以来,其蛋白质编码基因的三分之一仍然缺乏功能注释。在缺乏序列和结构同源性的情况下,蛋白质-蛋白质相互作用可以通过在其天然细胞环境中绘制蛋白质复合物来促进这些孤儿基因的功能预测。疟原虫核孔复合物(NPC)就是一个很好的例子:它的定义仍然不明确;它的成分缺乏与NPC中描述的30种蛋白质的保守性。包括真菌和动物在内的真核生物进化枝,但不是疟原虫.这里,我们开发了一种基于TurboID融合蛋白的标记方法,这允许P.bergheiNPC的可视化,并有助于其成分的识别。亲和纯化和质谱分析后,我们确定了4种已知的核孔蛋白(Nups)(138、205、221和诱饵313),并验证与推定的苯丙氨酸-甘氨酸(FG)Nup637的相互作用;我们将5种缺乏注释的蛋白质(因此与属外的蛋白质有意义的同源性)分配给NPC,这通过绿色荧光蛋白(GFP)标记得到证实。基于基因缺失的尝试,所有新的Nups-Nup176、269、335、390和434-对寄生虫的生存至关重要。它们与疟原虫属以外的蛋白质缺乏一级序列同源性;尽管2包含与人Nup155和酵母Nup157具有结构同源性的短结构域,以及缩合蛋白SMC(染色体4的结构维持)。此处开发的方案展示了邻近标记的功能,可阐明疟原虫中蛋白质复合物的组成和分类学限制基因的注释。它为探索疟原虫NPC的功能和了解其进化位置打开了大门。重要性核孔复合物(NPC)是不断进化的平台,已用于研究早期分支真核生物的进化模式。疟原虫NPC由于其进化差异的性质而定义不清,因此无法通过同源性搜索对其进行表征。尽管疟原虫基因组的发表已经过去了20年,30%的基因仍缺少功效注释。我们的研究证明了使用TurboID进行邻近标记以将功能分配给疟疾寄生虫中的孤儿蛋白的能力。我们已经确定了总共10个Nups,可以进一步研究NPC动力学,结构元素,参与核质运输,和核孔蛋白的独特非运输功能,为这种疟疾寄生虫提供适应性。
Twenty years since the publication of the Plasmodium falciparum and P. berghei genomes one-third of their protein-coding genes still lack functional annotation. In the absence of sequence and structural homology, protein-protein interactions can facilitate functional prediction of such orphan genes by mapping protein complexes in their natural cellular environment. The Plasmodium nuclear pore complex (NPC) is a case in point: it remains poorly defined; its constituents lack conservation with the 30+ proteins described in the NPC of many opisthokonts, a clade of eukaryotes that includes fungi and animals, but not Plasmodium. Here, we developed a labeling methodology based on TurboID fusion proteins, which allows visualization of the P. berghei NPC and facilitates the identification of its components. Following affinity purification and mass spectrometry, we identified 4 known nucleoporins (Nups) (138, 205, 221, and the bait 313), and verify interaction with the putative phenylalanine-glycine (FG) Nup637; we assigned 5 proteins lacking annotation (and therefore meaningful homology with proteins outside the genus) to the NPC, which is confirmed by green fluorescent protein (GFP) tagging. Based on gene deletion attempts, all new Nups - Nup176, 269, 335, 390, and 434 - are essential to parasite survival. They lack primary sequence homology with proteins outside the Plasmodium genus; albeit 2 incorporate short domains with structural homology to human Nup155 and yeast Nup157, and the condensin SMC (Structural Maintenance Of Chromosomes 4). The protocols developed here showcase the power of proximity labeling for elucidating protein complex composition and annotation of taxonomically restricted genes in Plasmodium. It opens the door to exploring the function of the Plasmodium NPC and understanding its evolutionary position. IMPORTANCE The nuclear pore complex (NPC) is a platform for constant evolution and has been used to study the evolutionary patterns of early-branching eukaryotes. The Plasmodium NPC is poorly defined due to its evolutionary divergent nature making it impossible to characterize it via homology searches. Although 2 decades have passed since the publication of the Plasmodium genome, 30% of the genes still lack functional annotation. Our study demonstrates the ability of proximity labeling using TurboID to assign function to orphan proteins in the malaria parasite. We have identified a total of 10 Nups that will allow further study of NPC dynamics, structural elements, involvement in nucleocytoplasmic transport, and unique non-transport functions of nucleoporins that provide adaptability to this malaria parasite.