The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.

中文翻译：

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恶性疟原虫青蒿素易感性相关的 AP-2 适应蛋白 μ 亚基独立于网格蛋白，对裂殖体成熟至关重要。

当前抗疟药的疗效受到恶性疟原虫对青蒿素敏感性降低的威胁，这与 pfkelch13 突变有关 另一种具有已知可调节对青蒿素反应的变体的基因编码 AP-2 适应素转运复合物的 μ 亚基。为了阐明 AP-2μ 在恶性疟原虫中的细胞作用，我们进行了条件基因敲除，这严重破坏了裂殖体的组织和成熟，导致关键裂殖子蛋白的错误定位。因此，AP-2μ 对于血液阶段复制至关重要。我们生成了表达血凝素标记的 AP-2μ 的转基因恶性疟原虫寄生虫，并通过荧光和电子显微镜检查了细胞定位。连同共免疫沉淀蛋白的质谱分析，这些研究确定了 AP-2μ 相互作用伙伴，包括其他 AP-2 亚基、K10 kelch 结构域蛋白和 PfEHD，一种内吞作用和脂质动员的效应物，但没有发现与网格蛋白相互作用的证据，网格蛋白是 AP-2 囊泡的预期外壳蛋白。在网格蛋白纳米抗体的反向免疫沉淀实验中，其他异四聚体 AP 复合物显示与网格蛋白相互作用，但 AP-2 复合物亚基不存在。重要我们详细检查了来自疟疾寄生虫恶性疟原虫的 AP-2 适应素复合物在大多数研究中在生物体中，AP-2 参与将物质从外部带入细胞，这一过程称为内吞作用。以前的工作表明，AP-2 的 μ 亚基的变化会导致耐药性。我们的实验表明 AP-2 对于血液中寄生虫的发育至关重要，但在网格蛋白介导的内吞作用中没有任何作用。这表明 AP-2 的特殊功能已在疟疾寄生虫中形成，这对于了解其对耐药性的影响可能很重要。