Elucidation of iron homeostasis in Acanthamoeba castellanii

•Acanthamoeba castellanii employs ferric reductase and the divalent iron transporter for iron assimilation.•Expression levels of these proteins are significantly higher under iron limitation.•Both proteins are localized to the membranes of acidified digestive vacuoles.•Iron uptake is induced under iron depletion while the pinocytosis rate is reduced.•Excessive iron is removed from the cytosol into the vacuoles by the VIT family protein.