Identification of signals and factors facilitating transport of STEVOR to the Maurer`s clefts of Plasmodium falciparum

人类疟疾寄生虫恶性疟原虫（Plasmodium falciparum）将其自身质膜以外的蛋白质输出到其宿主红细胞内的不同位置，包括红细胞细胞质和表面，以及称为Maurer's clefts的寄生虫起源的膜结构。运输信号和介导这种独特的“细胞外”蛋白质分泌途径的因素在很大程度上是未知的。我们实验室以前的工作表明，STEVOR，毛雷尔裂的居民跨膜蛋白的运输，是由三个信号组成的多步信号通路介导的：N-末端信号肽，包含在55个氨基酸内的凹陷的靶向信号，和跨膜结构域。这些信号必须协同工作，才能将STEVOR正确地贩运到毛雷尔的裂缝中。通过研究各种STEVOR-GFP融合蛋白的亚细胞定位产生数据。在这里，我们建议进行一个详细的突变分析的55个氨基酸的靶向信号，并进一步确定组件的恶性疟原虫分泌途径，与此信号的运输STEVOR毛雷尔的裂缝。所提出的实验设计包括，除了丙氨酸扫描整个靶向信号，转染技术表达GFP融合嵌合体，酵母双杂交筛选，以确定与靶向信号相互作用的因素，并产生抗体，以表征这些推定的因素。恶性疟原虫分泌途径。

The human malarial parasite Plasmodium falciparum exports proteins beyond its own plasma membrane to distinct locations within its host erythrocyte, including the erythrocyte cytoplasm and surface, as well as to membranous structures of parasite origin termed Maurer's clefts. The trafficking signals and factors mediating this unique "extracellular" protein secretory pathway are largely uncharacterized. Previous work from our laboratory has suggested that transport of STEVOR, a resident trans-membrane protein of Maurer's clefts, is mediated by a multi-step signaling pathway composed of three signals: an N-terminal signal peptide, a recessed targeting signal contained within 55 amino acids, and a trans-membrane domain. These signals have to work in concert to allow proper trafficking of STEVOR to Maurer's clefts. The data were generated by investigating the sub-cellular localization of various STEVOR-GFP fusion proteins. Here, we propose to carry out a detailed mutational analysis of the 55 amino acids containing the targeting signal, and furthermore, identify components of the P. falciparum secretory pathway that interacts with this signal in trafficking of STEVOR to the Maurer's clefts. The proposed experimental design includes, apart from an alanine scan across the targeting signal, transfection technology to express GFP fusion chimeras, a yeast two-hybrid screen to identify factors interacting with the targeting signal, and the generation of antibodies to characterize these putative factors of the P. falciparum secretory pathway.