A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor.
A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor.
复制标题
DOI：
10.1371/journal.ppat.1002392
复制
发表时间：
2011-12
期刊：
影响因子：
6.7
通讯作者：
Striepen B
中科院分区：
文献类型：
--
作者：
Sheiner L;Demerly JL;Poulsen N;Beatty WL;Lucas O;Behnke MS;White MW;Striepen B
顶复门的寄生虫引起影响全球健康和经济的疾病。这些单细胞真核生物具有残质体，顶质体，这是一个必不可少的细胞器和验证的药物靶点。然而，其生物学的大部分仍然知之甚少，特别是其精心划分：四个膜定义了四个不同的空间。只有少数的细胞器蛋白质已被确定，特别是少数蛋白质是已知的非腔顶质体隔间。我们假设，扩大顶质体蛋白质的目录将有助于确定新的细胞器功能，并扩大范围的目标超出了有限的一组特征的途径。我们开发了一种基于细胞周期和系统分布的mRNA丰度的生物信息学筛选。我们实验评估了57个基因，并成功的30个表位标记的候选人11个新的顶质体蛋白被确定。其中，7个似乎靶向细胞器的内腔，4个定位于外周隔室。为了解决它们的功能，我们开发了一个强大的系统，通过启动子置换策略构建条件突变体。我们证实了该系统的可行性，通过建立两个选定的基因-管腔和外周apicoplast蛋白的条件突变体。后者特别有趣，因为它编码一种假设的蛋白质，该蛋白质在顶复门寄生虫和其他维持红藻内共生体的相关生物中是保守的和独特的。我们的研究表明，这种外周质体蛋白PPP 1可能定位于周质隔室。PPP1的条件性破坏表明，它是寄生虫生存所必需的。该突变体的表型分析与PPP1蛋白在顶质体生物发生中的作用一致，特别是在将核编码蛋白导入细胞器中。顶复体是一组寄生虫，引起重要的疾病，包括疟疾和几种艾滋病相关的机会性感染。寄生虫依赖于藻类内共生体，顶质体，这为药物开发提供了阿喀琉斯之踵。我们使用弓形虫作为一个模型来描述顶质体的生物学和功能。在这项研究中，我们采用一种策略，以确定新的顶质体蛋白，并优先考虑他们作为潜在的目标，通过分析遗传突变体。为了帮助实现这一目标，我们开发了一种新的寄生虫系和一种能够简化条件突变体构建的方案。利用这种新的方法，我们发现了许多新的顶质体蛋白，其中许多还没有指定的功能。我们证明，功能可以推导出使用我们的遗传方法，通过建立一个新的因素有趣的本地化和进化历史的顶质体蛋白进口的重要作用。
Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle. Apicomplexa are a group of parasites that cause important diseases, including malaria and several AIDS associated opportunistic infections. The parasites depend on an algal endosymbiont, the apicoplast, and this provides an Achilles' heel for drug development. We use Toxoplasma gondii as a model to characterize the biology and function of the apicoplast. In this study we apply a strategy to identify new apicoplast proteins and to prioritize them as potential targets through the analysis of genetic mutants. To aid this goal we develop a new parasite line and a protocol enabling the streamlined construction of conditional mutants. Using this new approach we discover numerous new apicoplast proteins, many of them have no assigned function yet. We demonstrate that function can be deduced using our genetic approach by establishing the essential role in apicoplast protein import for a new factor with intriguing localization and evolutionary history.
登录
查看更多内容
影响因子：
3.7
作者：
Behnke MS;Wootton JC;Lehmann MM;Radke JB;Lucas O;Nawas J;Sibley LD;White MW
通讯作者：
White MW
影响因子：
7.4
作者：
Dangoor, Inbal;Peled-Zehavi, Hadas;Danon, Avihai
通讯作者：
Danon, Avihai
影响因子：
9.8
作者：
Bozdech, Zbynek;Llinas, Manuel;Pulliam, Brian Lee;Wong, Edith D;Zhu, Jingchun;DeRisi, Joseph L
通讯作者：
DeRisi, Joseph L
影响因子：
--
作者：
Brossier, Fabien;Starnes, G. Lucas;Sibley, L. David
通讯作者：
Sibley, L. David
影响因子：
1.5
作者：
Gubbels, MJ;Wieffer, M;Striepen, B
通讯作者：
Striepen, B