Characterization of the respiratory chain from cultured Crithidia fasciculata

Abstract

Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplstids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F 0F 1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F 0F 1-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two F 0 subunits (b and c) and all of the F 1 subunits. The F 0 b subunit has a low degree of similarity to subunit b from other organisms. The F 1 α subunit is extremely small making the β subunit the largest F 1 subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex III) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V.