Plasmodium falciparum Atg18 localization to the food vacuole is phosphatidylinositol 3-phosphate-dependent and quinoline-sensitive

Abstract

Autophagy is a lysosome-dependent degradative process involving over 35 Atg proteins. The autophagy repertoire in malaria parasites is limited and does not appear to be a major degradative process. To better understand the autophagy process, we investigated Plasmodium falciparum Atg18 (PfAtg18), a PROPPIN family protein, whose members like S. cerevisiae Atg18 (ScAtg18) and human WIPI2 are essential for autophagy. Wild type and mutant PfAtg18 were expressed in P. falciparum and assessed for localization, the effect of various inhibitors and antimalarials on PfAtg18 localization, and identification of PfAtg18-interacting proteins. PfAtg18 is expressed in asexual erythrocytic stages and localized to the food vacuole, which was also observed with other Plasmodium Atg18 proteins, indicating that food vacuole localization is a conserved feature. Interaction of PfAtg18 with the food vacuole-associated PI3P is essential for localization, as PfAtg18 mutants of PI3P-binding motifs neither bound PI3P nor localized to the food vacuole. Interestingly, ScAtg18 showed complete cytoplasmic localization despite binding with PI3P, indicating additional requirement for PfAtg18 localization. The food vacuole multi-drug resistance protein 1 (MDR1) was consistently identified in the PfAtg18 immunoprecipitate, and also interacted with PfAtg18. In contrast to PfAtg18, ScAtg18 did not interact with the MDR1, which, in addition to PI3P, could play a critical role in localization of PfAtg18. Chloroquine and amodiaquine greatly affected PfAtg18 localization, suggesting that these quinolines target PfAtg18 or the proteins that might be involved in its localization. Thus, PI3P and MDR1are critical mediators of PfAtg18 localization, and PfAtg18 may modulate MDR1 activity.