A two‐component ortholog of the Niemann‐Pick C 1 protein is essential for normal growth and sterol trafficking and signaling in Tetrahymena thermophila

The free‐living ciliate Tetrahymena thermophila avidly acquires exogenous sterols through phagocytosis and pinocytosis. We have previously shown that the aminosteroid U18666A, known to bind the mammalian Niemann‐Pick C1 protein (NPC1), completely inhibited both uptake processes, resulting in cholesterol accumulation in phagosome‐like vesicles and severe inhibition of sterol esterification. This suggests a blockage of sterol transfer between phagolysosomes and the endoplasmic reticulum (ER). The ciliate possesses a 50% similar ortholog of NPC1 (NPC, TTHERM_00672270), previously localized to the phagosomes. Since U18666A binds to other cholesterol‐related proteins, an off‐target effect in T. thermophila could not be ruled out. Therefore, we analyzed the role of Tt‐NPC in cholesterol transfer using reverse genetics. Tt‐NPC lacks the equivalent N‐terminal domain of NPC1 (NTD). Using NTD as a query, a protein with 38% similarity (TTHERM_00336030) was retrieved and subsequently named NTD. Knockdown of either the Tt‐NPC or Tt‐NTD genes produced similar phenotypes. The cell growth was arrested after cholesterol supplementation, with cholesterol accumulating in phagosome‐like vesicles. Additionally, the ER processes, sterol esterification and bioconversion, were severely impaired. Transcriptional regulation of two well‐characterized sterol‐responsive genes was lost in Tt‐NTD and significantly delayed in Tt‐NPC. This evidence suggests the presence of a functional ortholog of NPC1 in T. thermophila, composed of two peptides and a partial conservation of sterol trafficking across eukaryotic lineages.