Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion.
Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion.
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DOI：
10.1371/journal.ppat.1000309
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发表时间：
2009-02
期刊：
影响因子：
6.7
通讯作者：
Lebrun M
中科院分区：
文献类型：
--
作者：
Besteiro S;Michelin A;Poncet J;Dubremetz JF;Lebrun M
顶复门寄生虫入侵过程中最保守的特征之一是在寄生虫顶端和宿主细胞膜之间形成移动连接（MJ），该连接沿着寄生虫移动并作为将其推进宿主细胞内的支撑。直到最近一段时期，MJ 在分子水平上还是完全未知的。最近，源自两种不同的后高尔基体专门分泌细胞器的蛋白质，即微线体（AMA1）和棒状体颈（RON2/RON4/RON5 蛋白质），已被证明可以形成复合物。特别是 AMA1 和 RON4，在入侵过程中已定位于 MJ。使用生化方法，我们已确定 RON8 是该复合物的另一个成员。我们还证明了所有 RON 蛋白在侵袭过程中都存在于 MJ 中。使用代谢标记和免疫沉淀，我们证明 RON2 和 AMA1 能够在其他成员不存在的情况下相互作用。我们还发现，所有 MJ 蛋白在运输到各自细胞器的过程中都会经历蛋白水解成熟，并且它们可以在体外以非成熟形式结合。最后，虽然 AMA1 先前已被证明在分泌时插入寄生虫膜，但我们通过使用差异透化和将 RON 特异性抗体加载到宿主细胞中证明，RON 复合物靶向宿主细胞膜，其中 RON4/5/8 仍然与细胞质面相关。从全球范围来看，这些结果指向了一种 MJ 组织模型，其中寄生虫会在 MJ 的两侧（在宿主和其自身的质膜上）分泌和插入相互作用的成分。顶端复门寄生虫的一个独特特征是寄生虫的顶端与宿主细胞膜之间形成紧密接触，称为移动连接，在入侵期间沿着寄生虫移动。源自两种不同分泌细胞器（AMA1 的微线体和 RON2/4/5 蛋白的棒状体颈）的蛋白质相关联，形成连接。在这里，我们通过描述另一种蛋白质成分 RON8，进一步表征了 MJ 复合物。先前已证明 AMA1 在分泌时会插入寄生虫膜中。我们的研究表明，所有 RON 蛋白都易位到宿主细胞中，其中 RON4/5/8 仍然与宿主细胞质膜的细胞质面相关。此外，我们在体外发现了跨膜 MJ 蛋白 AMA1 和 RON2 之间的特殊相互作用。总的来说，这促使我们提出第一个模型来描述宿主细胞和弓形虫之间界面上假定的 MJ 组织。在这个最初的概念中，寄生虫会在 MJ 的两侧输出自己的受体 (RON2) 和配体 (AMA1)。
One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes. A unique feature of apicomplexan parasites is the formation of an intimate contact between the apex of the parasite and the host cell membrane called the moving junction that moves along the parasite during invasion. Proteins originated from two distinct secretory organelles, the microneme for AMA1 and the rhoptry neck for RON2/4/5 proteins, are associated to form the junction. Here, we have furthered the characterization of the MJ complex by describing RON8, an additional protein component. AMA1 has previously been shown to be inserted into the parasite membrane upon secretion. Our study demonstrates that all the RON proteins are translocated into the host cell, where RON4/5/8 remain associated with the cytoplasmic face of the host cell plasma membrane. Furthermore, we identified a privileged interaction between transmembrane MJ proteins AMA1 and RON2 in vitro. Overall, this led us to propose the first model describing the putative MJ organisation at the interface between the host cell and Toxoplasma. In this original concept, the parasite would export its own receptor (RON2) and ligand (AMA1) on either side of the MJ.
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