The mature N-termini of Plasmodium effector proteins confer specificity of export.
The mature N-termini of Plasmodium effector proteins confer specificity of export.
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红细胞内疟原虫恶性疟原虫将数百种蛋白质输出到宿主红细胞（RBC）中。大多数通过一段疏水性氨基酸靶向内质网（ER），并在称为疟原虫输出元件（PEXEL）的保守基序处通过ER蛋白酶plasmepsin V（PM V）进一步下游切割。成熟的效应物然后通过分泌途径到达寄生虫周围的寄生虫空泡（PV）。在那里，PEXEL蛋白以某种方式被识别为出口目的蛋白，而不是PV驻留蛋白，并选择性地转移到RBC中。成熟的N-末端似乎对于输出是重要的。关于是否需要PM V切割以进行适当的输出或是否有任何产生成熟N-末端的方法就足够，存在相互矛盾的数据。我们用信号肽序列替换了含有PEXEL的输出GFP报告基因的N-末端序列，并表明信号肽酶的精确切割，产生适当的成熟N-末端，产生输出能力。表达仅具有天然ER靶向信号而没有PMV切割位点的构建体显著降低了成熟PEXEL报告基因的量，表明疏水段缺乏有效的切割信号。因此，当适当地位于ER靶向序列之后时，PEXEL基序用作专门的信号切割位点。我们的数据表明，PMV切割和RBC输出是PEXEL蛋白的两个独立事件。我们还测试并拒绝了α-螺旋成熟N-末端是输出所必需的假设。疟疾寄生虫将数百种蛋白质输出到宿主红细胞的细胞质中以供其生存。被称为PEXEL基序的五个氨基酸序列在许多输出蛋白质中是保守的，并且被认为是输出信号。然而，该基序在寄生虫的内质网内被切割，并且从第四个PEXEL残基开始的成熟蛋白质行进到寄生虫外周用于输出。我们表明，PEXEL基序是不允许输出的，只要相同的成熟蛋白质可以通过替代方法在ER中有效地产生。我们还表明，输出和非输出蛋白质在寄生虫外周基于其成熟的N-末端进行区分;然而，该区域内的任何可辨别的输出信号仍然是隐蔽的。我们的研究解决了PEXEL蛋白运输中长期存在的矛盾。
The intraerythrocytic malaria parasite Plasmodium falciparum exports hundreds of proteins into the host red blood cell (RBC). Most are targeted to the endoplasmic reticulum (ER) by a stretch of hydrophobic amino acids and cleaved further downstream at a conserved motif called the Plasmodium Export Element (PEXEL) by the ER protease plasmepsin V (PM V). The mature effectors then travel through the secretory pathway to the parasitophorous vacuole (PV) that surrounds the parasite. There, PEXEL proteins are somehow recognized as export-destined proteins, as opposed to PV-resident proteins, and are selectively translocated out into the RBC. The mature N-terminus appears to be important for export. There is conflicting data on whether PM V cleavage is needed for proper export or whether any means of generating the mature N-terminus would suffice. We replaced the PEXEL-containing N-terminal sequence of an exported GFP reporter with a signal peptide sequence and showed that precise cleavage by signal peptidase, generating the proper mature N-terminus, yields export competence. Expressing a construct with only the native ER targeting signal without the PM V cleavage site dramatically decreased the amount of a mature PEXEL reporter, indicating that the hydrophobic stretch lacks an efficient cleavage signal. Therefore, the PEXEL motif functions as a specialized signal cleavage site when appropriately located after an ER targeting sequence. Our data suggest that PM V cleavage and RBC export are two independent events for PEXEL proteins. We also tested and rejected the hypothesis that an alpha-helical mature N-terminus is necessary for export. Malaria parasites export hundreds of proteins to the cytoplasm of the host red blood cells for their survival. A five amino acid sequence, called the PEXEL motif, is conserved among many exported proteins and is thought to be a signal for export. However, the motif is cleaved inside the endoplasmic reticulum of the parasite, and mature proteins starting from the fourth PEXEL residue travel to the parasite periphery for export. We showed that the PEXEL motif is dispensable for export as long as identical mature proteins can be efficiently produced via alternative means in the ER. We also showed that the exported and non-exported proteins are differentiated at the parasite periphery based on their mature N-termini; however, any discernible export signal within that region remained cryptic. Our study resolves a longstanding paradox in PEXEL protein trafficking.