ATP synthase epsilon subunit is an unconventional adaptor in clathrin-mediated endocytosis of hemoglobin in Leishmania.

In clathrin-mediated endocytosis, the cytoplasmic domain of the receptor binds to the AP2 adaptor, which recruits clathrin to mediate endocytosis. The classical AP2 adaptor in Leishmania has not yet been characterized. Here, we identified ATP synthase epsilon (LdATPSε) subunit as a novel adaptor in Leishmania using yeast two-hybrid screening. Subsequently, we cloned and expressed LdATPSε from Leishmania and showed that LdATPSε colocalizes with LdClathrin and the hemoglobin receptor in Leishmania. We found that LdATPSε directly binds to a cargo-binding motif, 'YLAP', in the cytoplasmic domain of the high-affinity hemoglobin receptor, whereas it interacts with the LdClathrin terminal domain via a clathrin-binding motif, 'LSELD'. Consequently, we showed that mutated clathrin binding box LdATPSεL133A/L136A/D137A does not bind to clathrin and fails to localize in the flagellar pocket, and its overexpression completely blocks hemoglobin internalization in Leishmania. LdATPSε-/- parasites are not viable, indicating the essential function of LdATPSε. However, hemoglobin internalization in LdATPSε+/- parasites is significantly blocked, and LdATPSε+/- parasites fail to grow in macrophages as the parasites are unable to internalize hemoglobin. Our results demonstrate that LdATPSε is a novel adaptor for clathrin in hemoglobin endocytosis in Leishmania.