The de novo synthesis of cytochrome c oxidase subunits I, II (COI and COII), and apocytochrome b (Cyb) was investigated in kinetoplast-mitochondria of Leishmania. The organelles were isolated after breaking whole cells with nitrogen cavitation. Individual COI, COII, and Cyb polypeptides were identified by fractionation of the kinetoplast membranes, labeled with [ 35 S]methionine and cysteine, using two-dimensional (9 versus 14% and 20 versus 11%) denaturing gel electrophoresis. The reaction did not require exogenous energy sources or amino acids. On the contrary, the presence of amino acids other than methionine somewhat inhibited the labeling reaction probably by competing with the uptake of labeled amino acids. The synthesis reaction was insensitive to 100 g/ml chloramphenicol, gentamycin, paromomycin, lincomycin, hygromycin, and tetracycline, as well as cycloheximide. The process showed a linear increase in the amount of synthesized polypeptides during the first 2 h of incubation, followed by a slower accumulation of products for up to 4 h. The de novo synthesized polypeptides were stable for several additional hours. Their assembly into respiratory complexes, investigated using two-dimensional Blue Native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-SDS gels, began early during the incubation and continued throughout the course of the synthesis. This work represents the first unequivocal identification of the polypeptide synthesis in kinetoplasts.The kinetoplast-mitochondrial genetic system of trypanosomes has revealed features that clearly distinguish it from animal, fungal, or plant mitochondria. The maxicircle component of kinetoplast DNA networks contains cryptic genes whose expression depends on post-transcriptional mRNA editing by uridylate insertions and deletions (1-3). This process is mediated by guide RNAs encoded in the kinetoplast DNA minicircles, which account for the bulk of DNA in these mitochondria. After the discoveries of RNA editing in 1986 (4) and guide RNAs in 1990 (5), there has been a steady progress toward elucidating the mechanism of this process, which has now been demonstrated to involve several enzymes and protein factors (6 -10).At the same time very little is known about other aspects of kinetoplast gene expression, especially at the level of translation. The very existence of a functional translation system remained unproven for almost 3 decades (summarized in Ref. 11). This question is important because kinetoplast translation must utilize templates modified or created by editing, and it is interesting to investigate interactions of these two systems. Early works demonstrated that cycloheximide at low concentrations effectively inhibited cytosolic translation in Crithidia luciliae and Trypanosoma brucei in vivo. A small fraction of the total cell protein synthesis was resistant to cycloheximide and at the same time insensitive to D-chloramphenicol and other inhibitors at concentrations that inhibit mitochondrial translation in other organisms (12, 13). However, high conce...
