Iowa State Universitygrid.34421.30, Biomedical Sciences, Ames, Iowa, USA.
mBio. 2022 Oct 26;13(5):e0181522. doi: 10.1128/mbio.01815-22. Epub 2022 Aug 30.
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. 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.
疟原虫和伯氏疟原虫基因组发表 20 年来，其三分之一的蛋白质编码基因仍然缺乏功能注释。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自然细胞环境中映射蛋白质复合物来促进对这些孤儿基因的功能预测。疟原虫核孔复合体（NPC）就是一个很好的例子：它仍然定义不明确；其组成部分与许多后生动物 NPC 中描述的 30 多种蛋白质没有保守性，后生动物是一个真核生物的分支，包括真菌和动物，但不包括疟原虫。在这里，我们开发了一种基于 TurboID 融合蛋白的标记方法，该方法可以可视化伯氏疟原虫 NPC，并有助于鉴定其成分。通过亲和纯化和质谱分析，我们鉴定出 4 种已知的核孔蛋白（Nups）（138、205、221 和诱饵 313），并验证了与假定的苯丙氨酸-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具有有意义的同源性）分配给 NPC，这通过绿色荧光蛋白（GFP）标记得到证实。基于基因缺失尝试，所有新的 Nups-Nup176、269、335、390 和 434-对寄生虫的存活都是必不可少的。它们与疟原虫属外的蛋白质没有一级序列同源性；尽管其中 2 种包含与人类 Nup155 和酵母 Nup157 以及凝聚素 SMC（染色体结构维持 4）具有结构同源性的短结构域。这里开发的方案展示了邻近标记在阐明蛋白质复合物组成和注释疟原虫分类受限基因方面的强大功能。它为探索疟原虫 NPC 的功能和理解其进化地位打开了大门。核孔复合体（NPC）是一个不断进化的平台，已被用于研究早期分支真核生物的进化模式。由于疟原虫 NPC 的进化差异，使得通过同源搜索对其进行特征描述变得不可能。尽管疟原虫基因组发表已经过去了 20 年，但仍有 30%的基因缺乏功能注释。我们的研究表明，使用 TurboID 的邻近标记能够为疟原虫中的孤儿蛋白赋予功能。我们总共鉴定了 10 种 Nups，这将允许进一步研究 NPC 动力学、结构元素、参与核质转运以及核孔蛋白的独特非转运功能，这些功能为这种疟原虫提供了适应性。
