The malaria parasite Plasmodium invades a host erythrocyte, multiplies within a parasitophorous vacuole (PV) and then ruptures the PV and erythrocyte membranes in a process known as egress. Both egress and invasion are controlled by effector proteins discharged from specialized secretory organelles. The aspartic protease plasmepsin X (PM X) regulates activity for many of these effectors, but it is unclear how PM X accesses its diverse substrates that reside in different organelles. PM X also autoprocesses to generate different isoforms. The function of this processing is not understood. We have mapped the self-cleavage sites and have constructed parasites with cleavage site mutations. Surprisingly, a quadruple mutant that remains full-length retains in vitro activity, is trafficked normally, and supports normal egress, invasion and parasite growth. The N-terminal half of the prodomain stays bound to the catalytic domain even after processing and is required for proper intracellular trafficking of PM X. We find that this enzyme cleaves microneme and exoneme substrates before discharge, while the rhoptry substrates that are dependent on PM X activity are cleaved after exoneme discharge into the PV. The data give insight into the temporal, spatial and biochemical control of this unusual but important aspartic protease.

疟原虫疟原虫侵入宿主红细胞，在寄生液泡 (PV) 内繁殖，然后在一个称为出口的过程中破裂 PV 和红细胞膜。外出和入侵均由专门的分泌细胞器释放的效应蛋白控制。天冬氨酸蛋白酶纤溶酶 X (PM X) 调节许多效应子的活性，但尚不清楚 PM X 如何访问不同细胞器中的不同底物。 PM X 还自动处理以生成不同的亚型。此处理的功能尚不清楚。我们已经绘制了自切割位点图并构建了具有切割位点突变的寄生虫。令人惊讶的是，保持全长的四重突变体保留了体外活性，正常运输，并支持正常的外出、入侵和寄生虫生长。即使在处理后，前结构域的 N 端一半仍与催化结构域结合，并且是 PM X 适当的细胞内运输所必需的。我们发现，这种酶在放电前裂解微线体和外线体底物，而依赖于 PM 的棒状体底物X 活性在外显子释放到 PV 后被裂解。这些数据让我们深入了解这种不寻常但重要的天冬氨酸蛋白酶的时间、空间和生化控制。