Unusual Polypeptide Synthesis in the Kinetoplast-Mitochondria from Leishmania tarentolae

Abstract

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...

The Effect of RNA Interference Down-regulation of RNA Editing 3′-Terminal Uridylyl Transferase (TUTase) 1 on Mitochondrial de Novo Protein Synthesis and Stability of Respiratory Complexes in Trypanosoma brucei

“…2) and insensitivity to chloramphenicol, tetracycline, hygromycin B, as was previously found in organello mitochondrial protein synthesis in L. tarentolae (22). This method for in vivo labeling and detection of the mitochondrial Cyb and COI proteins represents a valuable tool to study mitochondrial protein synthesis in these cells.…”

“…Polypeptide Synthesis in Whole Cells-The procedures were described previously (22). Cells (10 8 ) were pelleted by centrifugation at 16,000 ϫ g for 5 min and washed with SoTE buffer (0.6 M sorbitol, 20 mM Tris-HCl, pH 7.5, 2 mM EDTA).…”

Dual Targeting of a tRNAAsp Requires Two Different Aspartyl-tRNA Synthetases in Trypanosoma brucei

“…However, mitochondrial translation in trypanosomatids is notoriously difficult to measure (31). Most of the proteins encoded in the mitochondrion of T. brucei function either directly in oxidative phosphorylation or as components of the mitochondrial translation machinery that produces them (32).…”

The Importance of the 45 S Ribosomal Small Subunit-related Complex for Mitochondrial Translation in Trypanosoma brucei

“…Cytosolic translation was inhibited with cycloheximide, and the products of mitochondrial translation were labeled in vivo using [ 35 S] EasyTag Express Protein Labeling Mix (PerkinElmer Life Sciences) as described previously (28). Labeled products were resolved in two-dimensional denaturing gels and revealed by fluorography (47,48).…”

“…These polypeptides can be efficiently separated by a twodimensional (9 versus 14%) Tris-glycine-SDS-PAGE, and their synthesis can be investigated in vivo after inhibition of the cytosolic translation with cycloheximide (47,48). A similarly efficient approach still needs to be designed for analysis of the remaining proteins, which are often even more hydrophobic and prone to loss due to aggregation.…”