Structural Characterization of the TOC Protein Translocon Machinery

项目摘要 传染病每年在世界各地引起广泛的疾病，是世界第二大疾病。 主要死因，特别是在不发达国家。随着多种药物的出现 对于耐药菌株，迫切需要新的、更有效和更可持续的治疗方法。 这些传染病中包括尖复合体，包括弓形虫和 恶性疟原虫，分别是引起弓形虫病和疟疾的寄生虫。这些寄生虫 含有一种独特的类质体细胞器，称为顶生质体，它含有四层膜，因此 已经进化出一种复杂的系统，可以通过这些膜进出口蛋白质。这些 必需的进出口机械是针对这些病原体的新型抗生素的理想目标。许多 这些转位机制在其他高等真核细胞器中也是保守的，如 叶绿体和线粒体，其中绝大多数基因是核编码的，因此必须 翻译后导入。一种这样的机制是外膜的保守易位。 在拟南芥叶绿体(TOC)复合体中，一个研究叶绿体生物学的模式系统。 TOC复合体主要由三个组分组成，Toc33/34和Toc159，都是GTP酶 含有将它们固定在外膜上的N-末端跨膜螺旋，以及Toc75，一种16- 链状β桶跨膜易位子。虽然已经提出了机械论模型来解释 TOC的复杂功能，由于缺乏结构，它们在很大程度上仍未得到证实 定性，这是缝合机械拼图的所有碎片所必需的。在我们的研究中， 我们将使用生物物理方法、X射线结晶学、低温电子显微镜和小角X射线 分散在结构上和功能上是这一专门机器的特征。我们的结果将填补一个 这是该领域长期存在的差距，对于拼凑TOC如何实现的机制至关重要 在顶端复合体和叶绿体中的蛋白质输入中的复杂功能。

Project Summary Infectious diseases cause widespread sickness throughout the world each year and are the second leading cause of death, particularly in underdeveloped countries. And with the emergence of multi-drug resistance strains, the necessity for new, more effective, and more sustainable therapies is immediate. Included in these infectious diseases are the apicomplexa which includes Toxoplasma gondii and Plasmodium falciparum, the parasites causing toxoplasmosis and malaria, respectively. These parasites contain a unique plastid-like organelle called an apicoplast which contains four membranes and therefore have evolved a complex system for importing and exporting proteins across these membranes. These essential import/export machineries are ideal targets for novel antibiotics against these pathogens. Many of these translocon machineries are also conserved in other higher eukaryotic organelles such as chloroplasts and mitochondria, where a large majority of genes are nuclear encoded and therefore must be imported post-translationally. One such machinery is the conserved translocon of the outer membrane in chloroplasts (TOC) complex from Arabidopsis thaliana, a model system for studying chloroplast biology. The TOC complex consists of primarily three components, Toc33/34 and Toc159, both GTPases containing an N-terminal transmembrane helix anchoring them into the outer membrane, and Toc75, a 16- stranded β-barrel membrane-spanning translocon. While mechanistic models have been put forth for how the TOC complex functions, they have remained largely unproven due to the lack of structural characterization, which is needed to stitch together all the pieces of the mechanistic puzzle. In our studies, we will use biophysical methods, X-ray crystallography, cryo-electron microscopy, and small-angle X-ray scattering to structurally and functionally characterize this specialized machinery. Our results will fill a longstanding gap in the field and will be essential for piecing together the mechanism for how the TOC complex functions in protein import in apicomplexa and chloroplasts.