Clathrins Are Involved in the Endocytosis of Host Cytosol in the Malaria Parasite

In eukaryotic cells, clathrins interact with the adaptor protein (AP) complex-2 (AP-2) to facilitate endocytosis and AP-1 to mediate secretion and trafficking between the endosome and Golgi. In Plasmodium falciparum, recent studies revealed that the Kelch domain-containing protein 13 and AP-2 participate in hemoglobin uptake via cytostomes. However, clathrins appear not to be involved in this process because they primarily associate with AP-1. To investigate the roles of clathrins in P. falciparum, we characterized the clathrin heavy chain (PfCHC), the clathrin light chain (PfCLC), and the AP-1 γ subunit (PfAP-1 γ). Extensive interactome analyses confirmed the major association of clathrins with AP-1 components alongside proteins involved in cytostome formation. Live-cell imaging and protein colocalization studies showed that PfCHC, PfCLC, and PfAP-1 γ are localized in the parasite cytoplasm, predominantly at the parasite periphery and near the cis-Golgi. Ultrastructural studies using ascorbate peroxidase 2-based electron microscopy confirmed their presence at coated vesicle-like structures at the parasite periphery and, unexpectedly, at the collars of cytostomes. Knockdown of PfCHC led to the formation of abnormally long cytostome tubes and impaired hemoglobin uptake, suggesting that clathrins are involved in cytostome processes in P. falciparum.