ATP synthase epsilon subunit: An unconventional adaptor of clathrin-mediated endocytosis of hemoglobin in Leishmania.

In clathrin-mediated endocytosis, cytoplasmic domain of the receptor binds with AP2-adaptor which recruits clathrin to mediate endocytosis. Classical AP2-adaptor Leishmania is not yet characterized. Here, we have identified ATP Synthase epsilon (LdATPSɛ) subunit as a novel adaptor in Leishmania using yeast two-hybrid screening. Subsequently, we have cloned and expressed LdATPSɛ from Leishmania and have shown that LdATPSɛ co-localizes with LdClathrin and hemoglobin receptor in Leishmania. We have found that LdATPSɛ directly binds with cargo-binding motif 'YLAP' in the cytoplasmic domain of LdHbR, whereas it interacts with LdClathrin terminal domain via clathrin-binding motif 'LSELD'. Consequently, we have shown that mutated clathrin-binding box LdATPSɛL133A/L136A/D137A does not bind with clathrin, fails to localize in the flagellar pocket and its overexpression completely blocks the hemoglobin internalization in Leishmania. LdATPSɛ-/- knock out parasites are not viable indicating its essential function. However, hemoglobin internalization in LdATPSɛ+/- parasites is significantly blocked and LdATPSɛ+/- parasites fails to grow in macrophages as parasite in unable internalize hemoglobin. Our results have demonstrated that LdATPSɛ is a novel adaptor for clathrin in hemoglobin endocytosis in Leishmania.