PfATG18 links V-ATPase assembly to endocytic membrane dynamics in malaria parasites

Malaria parasites replicate inside red blood cells, degrading hemoglobin within a specialized digestive vacuole. Efficient hemoglobin processing is essential for parasite survival and influences antimalarial drug susceptibility. The vacuole constantly fuses with incoming hemoglobin-filled vesicles, yet the mechanisms that balance cargo influx with membrane homeostasis remain unclear. Here, using conditional reverse genetics, quantitative live-cell imaging, and 3D electron microscopy, we characterize the autophagy-related protein 18 of Plasmodium falciparum ( Pf ATG18) as a key regulator of vacuolar membrane dynamics. Loss of Pf ATG18 caused vacuole fragmentation, accumulation of hemoglobin-filled vesicles, and parasite death. These defects were preceded by broad architectural destabilization of the parasite’s V-ATPase, a proton pump controlling organelle acidification and the vacuole’s fusion–fission equilibrium. Direct interference with its membrane sector phenocopied Pf ATG18 deficiency. We found that Pf ATG18 does not interact directly with the proton pump but instead associates with a putative V-ATPase assembly factor and with complexes regulating phosphoinositide balance and vesicle trafficking. The breadth of these interactions indicates a multifaceted role at the vacuolar membrane and a regulatory influence on V-ATPase mediated through associated protein machinery. Although a point mutation in Pf ATG18 has been linked to artemisinin resistance, its complete knockout did not decrease sensitivity. Instead, it rendered ring-stage parasites hypersensitive to dihydroartemisinin, while leaving their susceptibility to other antimalarial drugs unchanged. Together, these findings establish Pf ATG18 as a central regulator of endocytic membrane homeostasis, essential for V-ATPase function and asexual parasite proliferation in the human blood.