Detection of the Mitochondrially Encoded Cytochrome cOxidase Subunit I in the Trypanosomatid Protozoan Leishmania tarentolae

Horváth, Anton; Kingan, Timothy G.; Maslov, Dmitri

doi:10.1074/jbc.m907246199

Cited by 48 publications

(47 citation statements)

References 27 publications

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“…It is difficult to directly measure mitochondrial translation in trypanosomatids (30,31). Thus, as an alternative, we decided to measure the consequences that ablation of IF2 has on mitochondrial ATP production (Fig.…”

Section: Resultsmentioning

confidence: 99%

Mitochondrial Initiation Factor 2 of Trypanosoma brucei Binds Imported Formylated Elongator-type tRNAMet

Charrière

Tan

Schneider

2005

Journal of Biological Chemistry

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The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of nucleus-encoded tRNAs. Thus, except for the cytosol-specific initiator tRNA Met , all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The only tRNA Met present in T. brucei mitochondria is therefore the one which, in the cytosol, is involved in translation elongation. Mitochondrial translation initiation depends on an initiator tRNA Met carrying a formylated methionine. This tRNA is then recognized by initiation factor 2, which brings it to the ribosome. To guarantee mitochondrial translation initiation, T. brucei has an unusual methionyl-tRNA formyltransferase that formylates elongator tRNA Met . In the present study, we have identified initiation factor 2 of T. brucei and shown that its carboxyl-terminal domain specifically binds formylated trypanosomal elongator tRNA Met . Furthermore, the protein also recognizes the structurally very different Escherichia coli initiator tRNA Met , suggesting that the main determinant recognized is the formylated methionine. In vivo studies using stable RNA interference cell lines showed that knockdown of initiation factor 2, depending on which construct was used, causes slow growth or even growth arrest. Moreover, concomitantly with ablation of the protein, a loss of oxidative phosphorylation was observed. Finally, although ablation of the methionyltRNA formyltransferase on its own did not impair growth, a complete growth arrest was observed when it was combined with the initiation factor 2 RNA interference cell line showing the slow growth phenotype. Thus, these experiments illustrate the importance of mitochondrial translation initiation for growth of procyclic T. brucei.

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Section: Resultsmentioning

confidence: 99%

Mitochondrial Initiation Factor 2 of Trypanosoma brucei Binds Imported Formylated Elongator-type tRNAMet

Charrière

Tan

Schneider

2005

Journal of Biological Chemistry

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“…The resolved polypeptides were transferred onto nitrocellulose membranes by semidry blotting, as described previously (19). After electrophoresis, the gels were stained either with Coomassie Brilliant Blue R250 (Sigma) or SYPRO Ruby (Molecular Probes).…”

Section: Methodsmentioning

confidence: 99%

“…The complexes were isolated according to our earlier procedure (26). The rabbit antisera against subunits I and IV of cytochrome c oxidase were described earlier (19,27). The antisera against F 1 and S9 of F 1 ⅐F 0 -ATPase (28) were provided by N. Williams.…”

Section: Methodsmentioning

confidence: 99%

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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

Neboháčová

Maslov

Falick

et al. 2004

Journal of Biological Chemistry

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Inhibition of RNA editing by down-regulation of expression of the mitochondrial RNA editing TUTase 1 by RNA interference had profound effects on kinetoplast biogenesis in Trypanosoma brucei procyclic cells. De novo synthesis of the apocytochrome b and cytochrome oxidase subunit I proteins was no longer detectable after 3 days of RNAi. The effect on protein synthesis correlated with a decline in the levels of the assembled mitochondrial respiratory complexes III and IV, and also cyanide-sensitive oxygen uptake. The steady-state levels of nuclear-encoded subunits of complexes III and IV were also significantly decreased. Because the levels of the corresponding mRNAs were not affected, the observed effect was likely due to an increased turnover of these imported mitochondrial proteins. This induced protein degradation was selective for components of complexes III and IV, because little effect was observed on components of the F 1 ⅐F 0 -ATPase complex and on several other mitochondrial proteins.Gene expression in the kinetoplast-mitochondrion of trypanosomatid protists involves a unique post-transcriptional mRNA maturation process, termed RNA editing, whereby Uresidues are inserted to or deleted from a pre-edited transcript, and a translatable reading frame is thus created (1-5). 12 of the 18 protein-coding genes in the maxicircle component of the mitochondrial or kinetoplast DNA in Trypanosoma brucei or Leishmania tarentolae encode transcripts that require varying degrees of editing for translation competence (6). These include mRNAs for apocytochrome b (Cyb), 1 subunits II and III of cytochrome c oxidase (COII and COIII, respectively), subunit 6 of ATPase (6), several subunits of NADH dehydrogenase, and a few others. Some transcripts, such as the cytochrome oxidase subunit I (COI) mRNA, do not require editing for translation. The mechanism of editing includes cleavage of RNA at specific editing sites, and the addition or deletion of a defined number of U-residues followed by religation (7,8). The reactions are performed by large protein complexes which include a 3Ј terminal uridylyl transferase (TUTase), an RNA ligase, endo-and exonuclease activities, as well as several additional auxiliary factors of undefined function (9 -12). Subcomplexes exist that contain subsets of proteins and which may specialize in Uaddition or U-deletion (13). The information for selection of editing sites resides in small "guide" RNAs that are mainly encoded in the kinetoplast DNA minicircles (7, 14). These RNA molecules have 3Ј oligo-U tails (15), which are added posttranscriptionally by the RET1 TUTase (16, 17). Down-regulation of RET1 expression by RNA interference (RNAi) results in a decrease in the steady-state abundance of edited transcripts (16) because of an effect on the 3Ј oligo-U tail of the guide RNAs (37).Although editing provides translatable transcripts, inhibition of editing should affect protein synthesis and, consequently, the assembly of respiratory complexes in the kinetoplast. The de novo synthesis of COI and Cyb po...

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“…Upon the addition of complex poly-U/A tails 27, 28 ( Figure 4), the fully edited transcripts are translated on protein-rich and rRNA-poor ribosomes 39 , but the role of the additional 45S ribosomal subunit of unique protein composition is still unclear 40 . Likely owing to their extreme hydrophobicity, only a few of the de novo synthesized mt proteins have been observed 41, 42 .…”

Section: Mechanisms Of Mitochondrial Gene Expressionmentioning

confidence: 99%

From simple to supercomplex: mitochondrial genomes of euglenozoan protists

et al. 2016

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Mitochondria are double membrane organelles of endosymbiotic origin, best known for constituting the centre of energetics of a eukaryotic cell. They contain their own mitochondrial genome, which as a consequence of gradual reduction during evolution typically contains less than two dozens of genes. In this review, we highlight the extremely diverse architecture of mitochondrial genomes and mechanisms of gene expression between the three sister groups constituting the phylum Euglenozoa - Euglenida, Diplonemea and Kinetoplastea. The earliest diverging euglenids possess a simplified mitochondrial genome and a conventional gene expression, whereas both are highly complex in the two other groups. The expression of their mitochondrial-encoded proteins requires extensive post-transcriptional modifications guided by complex protein machineries and multiple small RNA molecules. Moreover, the least studied diplonemids, which have been recently discovered as a highly abundant component of the world ocean plankton, possess one of the most complicated mitochondrial genome organisations known to date.

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Detection of the Mitochondrially Encoded Cytochrome cOxidase Subunit I in the Trypanosomatid Protozoan Leishmania tarentolae

Cited by 48 publications

References 27 publications

Mitochondrial Initiation Factor 2 of Trypanosoma brucei Binds Imported Formylated Elongator-type tRNAMet

Mitochondrial Initiation Factor 2 of Trypanosoma brucei Binds Imported Formylated Elongator-type tRNAMet

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

From simple to supercomplex: mitochondrial genomes of euglenozoan protists

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