Detection of the mitochondrially encoded cytochrome c oxidase subunit I in the trypanosomatid protozoan Leishmania tarentolae -: Evidence for translation of unedited mRNA in the kinetoplast

With the aim of identification of kinetoplast-encoded proteins we investigated the subunit composition of cytochrome c oxidase (respiratory complex IV) from kinetoplast mitochondria of the trypanosomatid protozoan Leishmania tarentolae. Eleven stoichiometric subunits were visible in Coomassie-stained, two-dimensional Blue Native/Tricine-SDS electrophoretic gels. Their partial amino acid sequences indicated that these polypeptides are nuclear-encoded. The mitochondrial subunit I was detected with the polyclonal antibodies against an internal region of this polypeptide. In two-dimensional (9 versus 14%) polyacrylamide glycine-SDS gels this subunit is found as a series of spots located off the main diagonal, a property that can be explained by abnormal electrophoretic migration and aggregation. In gels loaded with high amounts of the purified, enzymatically active oxidase, the subunit I spots could be visualized by staining. The determined N-terminal amino acid sequence of the putative monomeric subunit I (MFXLCLVCLSVS) matched with the predicted sequence, thus indicating that the corresponding kinetoplast unedited mRNA is translated into a functional protein.