BIOGENESIS OF RHOPTRIES IN TOXOPLASMA GONDII

描述：在免疫抑制患者中， 弓形虫可通过快速侵入，繁殖， 以及宿主细胞，特别是中枢神经系统的细胞的裂解。为了 为了侵入并操纵宿主细胞以满足其需要，寄生虫分泌 三个专门的细胞器的内容-微线，棒状体和致密 颗粒。尽管这些细胞器在寄生虫的生存中起着核心作用， 它们形成的机制和途径知之甚少。我们 最近发现，在哺乳动物细胞中，T.弓形虫具有高度的 靶向跨膜蛋白的通用和区分机制， 不同的细胞内位置的基础上识别酪氨酸含有 在它们的细胞质尾部有氨基酸基序。在一个案例中， 表明棒状蛋白ROP 2需要这样的基序来靶向 从一个成熟的棒状体的假定的前棒状体室， 识别这些基序的蛋白质与棒状体密切相关 生物起源我们建议进一步阐明蛋白质的要求， 通过制备和分析ROP 2的一系列突变体， 其他棒状体蛋白，并通过表征其寡聚化。在 此外，我们建议使用ROP 2突变体作为标记，以彻底表征 通过电子显微镜，亚细胞分级， 和体外贩运分析，这将大大提高我们的 了解棒状体的生物发生途径和机制 这些目标大大增加了父母补助金的三个具体目标， 其为：I.）表征棒状体之间的分子相互作用 PVM和宿主细胞蛋白质/细胞器中的蛋白质， 靶向T.弓形虫致密颗粒，和III.）阐明 其特点是控制致密颗粒分泌。通过阐明 棒状体生物发生和靶向的机制，这是一个类似于 具体目标二在父赠款致密颗粒，拟议的研究 提供了一个关键的窗口到棒状蛋白质靶向之间的联系， 棒状体蛋白功能

DESCRIPTION: In immunosuppressed patients the protozoan parasite Toxoplasma gondii can cause severe tissue destruction by rapid invasion, multiplication, and lysis of host cells, notably those of the central nervous system. In order to invade and manipulate the host cell to its needs the parasite secretes the contents of three specialized organelles - micronemes, rhoptries and dense granules. Despite the central role of these organelles in parasite survival, the mechanisms and pathways by which they are formed are poorly understood. We have recently found that, as in mammalian cells, T. gondii has a highly versatile and discriminatory mechanism for targeting transmembrane proteins to diverse intracellular locations based on the recognition of tyrosine-containing amino acid motifs in their cytoplasmic tails. In one case, the evidence suggested that a rhoptry protein, ROP2, requires such a motif for targeting from a putative pre-rhoptry compartment of mature rhoptries, and that adaptor proteins that recognize these motifs are closely involved in rhoptry biogenesis. We propose to further elucidate the protein requirements for rhoptry targeting by preparing and analyzing a series of mutants of ROP2 and other rhoptry proteins and by characterizing their oligomerization. In addition, we propose to use ROP2 mutants as markers to thoroughly characterize the pre-rhoptry compartment by electron microscopy, subcellular fractionation, and in vitro trafficking assays, which would considerably advance our understanding of the rhoptry biogenesis pathway and mechanisms These goals add substantially to the three specific aims of the parent grant, which are to: I.) Characterize the molecular interactions between rhoptry proteins in the PVM and host cell proteins/organelles, Identify the unique features of protein targeting to T. gondii dense granules, and III.) Elucidate the novel features controlling dense granule secretion. By elucidating the mechanisms of rhoptry biogenesis and targeting, a goal which is analogous to specific aim II in the parent grant for dense granules, the proposed studies provide a critical window into the link between rhoptry protein targeting and rhoptry protein function.