Identification of Gene Encoding Plasmodium knowlesi Phosphatidylserine Decarboxylase by Genetic Complementation in Yeast and Characterization of in Vitro Maturation of Encoded Enzyme
Identification of Gene Encoding Plasmodium knowlesi Phosphatidylserine Decarboxylase by Genetic Complementation in Yeast and Characterization of in Vitro Maturation of Encoded Enzyme
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DOI：
10.1074/jbc.m111.313676
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发表时间：
2012-01-02
影响因子：
4.8
通讯作者：
Voelker, Dennis R.
中科院分区：
文献类型：
--
作者：
Choi, Jae-Yeon;Augagneur, Yoann;Voelker, Dennis R.
恶性疟原虫（Plasmodium falciparum）是严重人类疟疾的病原体，其23兆碱基的基因组包含5300个相似的基因，其中大部分基因在其他生物中功能未知或缺乏同源物。这些基因功能的鉴定将有助于发现新的抗疟药物和疫苗的开发目标。恶性疟原虫基因组异常富含A + T，这阻碍了在异源系统中克隆和表达这些基因以进行功能分析。酿酒酵母的遗传工具的大剧目，使这种酵母的寄生虫基因的大规模功能互补分析的理想系统。在这里，我们报告的cDNA文库的构建诺氏疟原虫，具有较低的A + T含量相比，恶性疟原虫。该文库应用于酵母互补试验，以鉴定参与磷脂酰丝氨酸脱羧的疟疾基因。用诺氏毕赤酵母文库转化磷脂酰乙醇胺合成缺陷的psd 1 Delta psd 2 Delta dpl 1 Delta酵母菌株，鉴定出一种新的寄生虫磷脂酰丝氨酸脱羧酶（PkPSD）。与其他真核生物中与膜紧密相关的磷脂酰丝氨酸脱羧酶不同，酵母中表达的PkPSD酶在膜和可溶性组分之间均匀分布。体外研究表明，截短形式的PkPSD是可溶性的，并在磷脂酰丝氨酸依赖性反应中进行自动蛋白水解成熟，该反应被其他阴离子磷脂抑制。这项研究定义了一种通过基于库的遗传互补来探测疟原虫基因功能的新系统，及其在揭示编码蛋白质新生化特性方面的有用性。
The 23-megabase genome of Plasmodium falciparum, the causative agent of severe human malaria, contains similar to 5300 genes, most of unknown function or lacking homologs in other organisms. Identification of these gene functions will help in the discovery of novel targets for the development of antimalarial drugs and vaccines. The P. falciparum genome is unusually A + T-rich, which hampers cloning and expressing these genes in heterologous systems for functional analysis. The large repertoire of genetic tools available for Saccharomyces cerevisiae makes this yeast an ideal system for large scale functional complementation analyses of parasite genes. Here, we report the construction of a cDNA library from P. knowlesi, which has a lower A + T content compared with P. falciparum. This library was applied in a yeast complementation assay to identify malaria genes involved in the decarboxylation of phosphatidylserine. Transformation of a psd1 Delta psd2 Delta dpl1 Delta yeast strain, defective in phosphatidylethanolamine synthesis, with the P. knowlesi library led to identification of a new parasite phosphatidylserine decarboxylase (PkPSD). Unlike phosphatidylserine decarboxylase enzymes from other eukaryotes that are tightly associated with membranes, the PkPSD enzyme expressed in yeast was equally distributed between membrane and soluble fractions. In vitro studies reveal that truncated forms of PkPSD are soluble and undergo auto-endoproteolytic maturation in a phosphatidylserine-dependent reaction that is inhibited by other anionic phospholipids. This study defines a new system for probing the function of Plasmodium genes by library-based genetic complementation and its usefulness in revealing new biochemical properties of encoded proteins.