The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be required for biogenesis and secretion of egress-related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparum endoplasmic reticulum-resident calcium-binding protein (PfERC), a member of the CREC family. Knockdown of the PfERC gene showed that this gene is essential for asexual growth of P. falciparum Analysis of the intraerythrocytic life cycle revealed that PfERC is essential for parasite egress but is not required for protein trafficking or calcium storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease SUB1. Using double mutant parasites, we showed that PfERC is required for the proteolytic maturation of the essential aspartic protease plasmepsin X, which is required for SUB1 cleavage. Further, we showed that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish that the ER-resident CREC family protein PfERC is a key early regulator of the egress proteolytic cascade of malaria parasites.IMPORTANCE The divergent eukaryotic parasites that cause malaria grow and divide within a vacuole inside a host cell, which they have to break open once they finish cell division. The egress of daughter parasites requires the activation of a proteolytic cascade, and a subtilisin-like protease initiates a proteolytic cascade to break down the membranes blocking egress. It is assumed that the parasite endoplasmic reticulum plays a role in this process, but the proteins in this organelle required for egress remain unknown. We have identified an early ER-resident regulator essential for the maturation of the recently discovered aspartic protease in the egress proteolytic cascade, plasmepsin X, which is required for maturation of the subtilisin-like protease. Conditional loss of PfERC results in the formation of immature and inactive egress proteases that are unable to breakdown the vacuolar membrane barring release of daughter parasites.

中文翻译：

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内质网 CREC 家族蛋白调节疟疾寄生虫的出口蛋白水解级联。

内质网 (ER) 被认为在疟疾寄生虫的排出过程中起重要作用，因为 ER 被认为是与排出相关的细胞器的生物发生和分泌所必需的。然而，没有发现定位于寄生虫 ER 的蛋白质在疟疾寄生虫的排出中发挥作用。在这项研究中，我们生成了恶性疟原虫内质网驻留钙结合蛋白 (PfERC) 的条件突变体，它是 CREC 家族的成员。PfERC 基因的敲除表明该基因对于恶性疟原虫的无性生长是必不可少的。我们发现 PfERC 敲低可防止寄生液泡膜破裂。这是因为 PfERC 敲低抑制了枯草杆菌蛋白酶样丝氨酸蛋白酶 SUB1 的蛋白水解成熟。使用双突变体寄生虫，我们表明 PfERC 是必需天冬氨酸蛋白酶 plasmepsin X 的蛋白水解成熟所必需的，而 SUB1 裂解是必需的。此外，我们表明蛋白水解级联下游的底物加工受到 PfERC 敲低的抑制。因此，这些数据表明内质网驻留 CREC 家族蛋白 PfERC 是疟疾寄生虫出口蛋白水解级联反应的关键早期调节剂。一旦它们完成细胞分裂就会裂开。子寄生虫的排出需要激活蛋白水解级联反应，一种类似枯草杆菌蛋白酶的蛋白酶启动蛋白水解级联反应以分解阻止出口的膜。假设寄生虫内质网在此过程中发挥作用，但该细胞器中用于排出所需的蛋白质仍然未知。我们已经确定了一个早期的内质网驻留调节器，对于最近发现的出口蛋白水解级联中的天冬氨酸蛋白酶的成熟至关重要，血浆蛋白酶 X 是枯草杆菌蛋白酶样蛋白酶成熟所必需的。PfERC 的条件性丢失导致形成不成熟和无活性的出口蛋白酶，这些蛋白酶无法破坏液泡膜，从而阻止子寄生虫的释放。但是这种细胞器中用于排出所需的蛋白质仍然未知。我们已经确定了一个早期的 ER 驻留调节器，对于最近发现的天冬氨酸蛋白酶在出口蛋白水解级联反应中的成熟至关重要，血浆蛋白酶 X 是枯草杆菌蛋白酶样蛋白酶成熟所必需的。PfERC 的条件性丢失导致形成不成熟和无活性的出口蛋白酶，这些蛋白酶无法破坏液泡膜，从而阻止子寄生虫的释放。但是这种细胞器中用于排出所需的蛋白质仍然未知。我们已经确定了一个早期的内质网驻留调节器，对于最近发现的出口蛋白水解级联中的天冬氨酸蛋白酶的成熟至关重要，血浆蛋白酶 X 是枯草杆菌蛋白酶样蛋白酶成熟所必需的。PfERC 的条件性丢失导致形成不成熟和无活性的出口蛋白酶，这些蛋白酶无法破坏液泡膜，从而阻止子寄生虫的释放。