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.

Biology Centre Institute of Parasitology Czech Academy of Sciences Budweis Czech Republic

Department of Crystallography Birkbeck University of London London United Kingdom

Department of Infection Biology Faculty of Infectious Diseases London School of Hygiene and Tropical Medicine London United Kingdom

Department of Molecular Microbiology Washington University School of Medicine St Louis Missouri USA

Department of Pathogen Molecular Biology Faculty of Infectious Diseases London School of Hygiene and Tropical Medicine London United Kingdom

Department of Pediatrics Washington University School of Medicine St Louis Missouri USA

PHE Malaria Reference Laboratory London School of Hygiene and Tropical Medicine London United Kingdom

School of Life Sciences University of Dundee Dundee United Kingdom

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Stable endocytic structures navigate the complex pellicle of apicomplexan parasites

A Phosphoinositide-Binding Protein Acts in the Trafficking Pathway of Hemoglobin in the Malaria Parasite Plasmodium falciparum

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13