Structural requirements for protein transport across the vacuolar membrane of Plasmodium falciparum

恶性疟原虫是一种强制性的细胞内寄生虫，可诱导宿主红细胞的形态和生理改变。这些修饰是由寄生虫合成和分泌的蛋白质介导的。为了到达它们的最终目的地，蛋白质必须穿过包裹着寄生虫的液泡膜，液泡膜形成了红细胞细胞质的屏障。蛋白质信号和允许特定寄生虫蛋白通过空泡膜运输的机制在很大程度上是未知的。是否需要特定的蛋白质信号来介导这一过程存在矛盾的实验数据。我们希望通过构建编码嵌合蛋白的基因来解决这种差异。这些基因将在被转染的寄生虫中表达，基因产物的定位将通过分离感染红细胞和共聚焦激光扫描显微镜进行分析。在一种补充的方法中，在红细胞膜选择性渗透后，将分析蛋白质在空泡膜上的易位。这个实验系统将允许操纵液泡和红细胞胞浆的生化组成。因此，可以分析蛋白质结构和生化参数的协同效应。

Plasmodium falciparum is an obligatory intracellular parasite that induces morphological and physiological alterations of its host erythrocyte. These modifications are mediated by proteins which are synthesised and secreted by the parasite. In order to reach their final destinations the proteins have to be transported across the membrane of a vacuole which surrounds the parasite and which forms a barrier to the erythrocyte cytosol. The protein signals and the mechanisms that allow transport of specific parasite proteins across the vacuolar membrane are largely unknown. Conflicting experimental data exist whether specific protein signals are required to mediate this process. We want to resolve this discrepancy by constructing genes encoding chimeric proteins. The genes will be expressed in transfected parasites and the localisation of the gene products will be analysed by fractionation of infected erythrocytes and by confocal laserscan microscopy. In a complementary approach, translocation of proteins across the vacuolar membrane will be analysed after selective permeabilization of the erythrocyte plasma membrane. This experimental system will allow to manipulate the biochemical composition of the vacuole and of the erythrocyte cytosol. Thus, synergistic effects of protein structure and biochemical parameters can be analysed.