Fig 4.

TbERV1 is essential in procyclic and bloodstream-form T. brucei. (A) Immunoblot analysis reveals constitutive expression of TbERV1 in procyclic and bloodstream stages of T. brucei. WT, wild-type parental cell lines from which RNAi mutants were derived; −Tet, RNAi mutants grown in the absence of tetracycline; +Tet, RNAi mutants 72 h postinduction of RNAi. Blots were labeled with antibodies recognizing either TbERV1 or enolase, which served as a loading control. (B) Subcellular fractionation of procyclic cells by digitonin extraction reveals TbERV1 to be a mitochondrial protein. Soluble fractions from the digitonin extraction were blotted and probed with antibodies detecting the cytosolic marker enolase, the glycosomal markers aldolase and triosephosphate isomerase, the mitochondrial markers MRP2 and prohibitin, or TbERV1. Arrowheads indicate the minimal concentration of digitonin required to release the designated protein. (C and D) Growth of procyclic (C) or bloodstream-form (D) TbERV1 RNAi mutants in the absence (dotted line) or presence (dashed line) of tetracycline (1 μg ml⁻¹); the solid line denotes the growth of the parental wild-type cell line from which the RNAi mutants were derived.