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将蛋白质输出到自身质膜之外的宿主红细胞内的不同位置，包括红细胞细胞质和表面，以及寄生虫起源的膜结构，称为毛雷尔裂隙。介导这种独特的“细胞外”蛋白质分泌途径的运输信号和因子在很大程度上是未知的。我们实验室之前的工作表明，毛勒氏裂孔的一种跨膜蛋白STEVOR的转运是由三个信号组成的多步骤信号通路介导的：n端信号肽、包含55个氨基酸的隐性靶向信号和跨膜结构域。这些信号必须协同工作，以允许适当地将STEVOR贩运到毛雷尔的裂口。这些数据是通过研究各种steve - gfp融合蛋白的亚细胞定位而产生的。在这里，我们建议对含有靶向信号的55个氨基酸进行详细的突变分析，并进一步确定恶性疟原虫分泌途径中与该信号相互作用的成分，这些成分在将STEVOR运输到毛氏裂口中。该实验设计包括：对靶向信号进行丙氨酸扫描、转染技术以表达GFP融合嵌合体、酵母双杂交筛选以识别与靶向信号相互作用的因子，以及产生抗体以表征恶性疟原虫分泌途径中这些可能的因子。

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.