Novel components of the Toxoplasma inner membrane complex revealed by BioID.
Novel components of the Toxoplasma inner membrane complex revealed by BioID.
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DOI：
10.1128/mbio.02357-14
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发表时间：
2015-02-17
期刊：
影响因子：
6.4
通讯作者：
Bradley PJ
中科院分区：
文献类型：
--
作者：
Chen AL;Kim EW;Toh JY;Vashisht AA;Rashoff AQ;Van C;Huang AS;Moon AS;Bell HN;Bentolila LA;Wohlschlegel JA;Bradley PJ
刚地弓形虫内膜复合物（IMC）是一种外周膜系统，由位于质膜下的扁平肺泡囊组成，与支持细胞骨架网络偶联。IMC在寄生虫复制、运动和宿主细胞侵袭中起重要作用。尽管在寄生虫的生物学中具有这些中心作用，但构成IMC的蛋白质在很大程度上是未知的。在这项研究中，我们已经采用了一种技术，称为邻近依赖性生物素识别（BioID）用于弓形虫，以确定新的组成部分的IMC。使用肺泡和细胞骨架网络中的IMC蛋白作为诱饵，我们在这两个亚细胞器区室中总共发现了19种新的IMC蛋白，其中两种我们通过基因敲除进行了功能评估。重要的是，使用这种方法标记IMC蛋白揭示了一组定位于肺泡囊缝合处的蛋白质，这些蛋白质仅通过冷冻断裂电子显微镜在弓形虫物种中整体可见。总的来说，我们的研究极大地扩展了IMC中已知蛋白质的库，并通过实验验证了BioID作为发现弓形虫特定细胞区室的新成分的策略。结合伴侣的鉴定对于确定细胞区室内的蛋白质功能是至关重要的。然而，在膜或细胞骨架区室中发现蛋白质-蛋白质相互作用是具有挑战性的，特别是对于经常被这些区室的实验操作破坏的瞬时或不稳定的相互作用。为了解决这些问题，我们采用了体内生物素化技术，称为BioID弓形虫物种，以确定结合伙伴和近端蛋白质在天然细胞环境。我们使用BioID来识别寄生虫IMC中的19种新蛋白质，IMC是由融合的膜囊和底层细胞骨架组成的细胞器，其蛋白质组成在很大程度上是未知的。我们还展示了BioID在特定隔室（如IMC细胞骨架）内靶向发现蛋白质的能力。此外，我们发现了一组新的蛋白定位于肺泡缝的IMC。BioID有望揭示弓形虫细胞内蛋白质成分和相互作用的新见解。
The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii. The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma.