A Rieske monooxygenase highly conserved in animals is the sterol-C7 desaturase of Tetrahymena thermophila

SEBASTIÁN R. NAJLE; ALEJANDRO D. NUSBLAT; CLAUDIO H. SLAMOVITS; CLARA B. NUDEL; ANTONIO D. UTTARO

Oslo

Conferencia; Protist2012; 2012

International Society of Protistologists (ISOP) - International Society of Evolutionary Protistology (ISEP)

Tetrahymena thermophila does not require sterols for living, but when sterols are present in the growth media they are incorporated by the cells, inhibiting the synthesis of the sterol surrogate tetrahymanol, and modified by desaturation at positions C5(6), C7(8) and C22(23). This work reports the identification of a Rieske-type monooxygenase, highly conserved in protostome animals, as the sterol C7-desaturase (Des7p). We used RNA interference and knock-out mutagenesis to demonstrate the activity of Des7p in vivo in T. thermophila cells. GC-MS analysis of lipid extracts from interfered or knocked-out cultures supplemented with different sterols exhibited, respectively, a significant decrease or the complete abolition of C7-desaturase activity, as compared with controls. Confocal microscopy analysis of a strain expressing a Des7p:GFP fusion shows that the protein is localized in microsomal and phagosomal compartments and its expression seems to be induced in the presence of sterols. A bioinformatic analysis revealed the existence of orthologous proteins in the species T. malaccensis, T. elliotti and T. borealis, as well as the presence of 6 paralogs in the genome of Paramecium tetraurelia. This data raise the hypothesis that the Rieske sterol C7-desaturase is an ancient character, present in ciliates before the divergence of the clade Oligohymenophorea.