Clathrins are involved in the endocytosis of host cytosol in the malaria parasite

Abstract

In eukaryotic cells, clathrins interact with adaptor protein complexes to regulate key intracellular trafficking events. Specifically, they are associated with the adaptor protein (AP) complex-2 (AP2) to facilitate endocytosis and AP1 to mediate secretion and trafficking between endosome and Golgi. In Plasmodium falciparum, recent studies revealed that the Kelch domain-containing protein 13 and AP2 participate in hemoglobin uptake via cytostomes. However, clathrins appear not to be involved in this process because they primarily associate with AP1. To investigate the roles of clathrins in P. falciparum, we characterized the clathrin heavy chain (PfCHC), the clathrin light chain (PfCLC), and the AP1 {gamma} subunit (PfAP1 {gamma}). Extensive interactome analyses confirmed the major association of clathrins with AP1 components alongside proteins involved in cytostome formation. Live-cell imaging and protein colocalization studies showed that PfCHC, PfCLC, and PfAP1 {gamma} 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. Consistent with these observations, the knockdown of PfCHC led to the formation of abnormally long cytostome tubes and impaired hemoglobin digestion. This study demonstrates that clathrins are essential for proper cytostome formation in P. falciparum, highlighting their critical role in the parasites hemoglobin uptake and digestion processes. IMPORTANCEMalaria is still one of the most severe public health problems worldwide and understanding how malaria parasites obtain hemoglobin from their host cells, red blood cells (RBCs), is critical for identifying targets for antimalarials. In this study, affinity purification revealed that clathrins (PfCHC and PfCLC) were not only mainly associated with AP1 complex but also marginally interacted with proteins that participated in hemoglobin uptake. Localization analysis demonstrated that clathrins and AP1 coated the clusters of vesicles at the parasite periphery and at the neck of the cytostome, an organelle for hemoglobin uptake. Consistently, the knockdown of PfCHC caused the formation of abnormally long cytostome tubes and reduced hemoglobin digestion. Collectively, this study demonstrates that clathrins play a critical role in the parasites hemoglobin uptake.