In kinetoplastid protists, all mitochondrial tRNAs are encoded in the nucleus and imported from the cytoplasm to maintain organellar translation. This also applies to the tryptophanyl tRNA (tRNATrp) encoded by a single-copy nuclear gene, with a CCA anticodon to read UGG codon used in the cytosolic translation. Yet, in the mitochondrion it is unable to decode the UGA codon specifying tryptophan. Following mitochondrial import of tRNATrp, this problem is solved at the RNA level by a single C34 to U34 editing event that creates the UCA anticodon, recognizing UGA. To identify the enzyme responsible for this critical editing activity, we scrutinized the genome of Trypanosoma brucei for putative cytidine deaminases as the most likely candidates. Using RNAi silencing and poisoned primer extension, we have identified a novel deaminase enzyme, named here TbmCDAT for mitochondrial Cytidine Deaminase Acting on tRNA, which is responsible for this organelle-specific activity in T. brucei. The ablation of TbmCDAT led to the downregulation of mitochondrial protein synthesis, supporting its role in decoding the UGA tryptophan codon.

• Keywords
• Mitochondrion, cytidine deaminase, editing, trypanosoma, tryptophanyl tRNA,
• MeSH
• Cytidine chemistry   genetics   MeSH
• Cytidine Deaminase genetics   metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Mitochondria enzymology   genetics   MeSH
• RNA, Mitochondrial analysis   genetics   MeSH
• RNA, Protozoan analysis   genetics   MeSH
• RNA, Transfer, Trp MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Homology MeSH
• Codon, Terminator * MeSH
• Trypanosoma brucei brucei genetics   growth & development   metabolism   MeSH
• Uridine chemistry   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytidine MeSH
• Cytidine Deaminase MeSH
• RNA, Mitochondrial MeSH
• RNA, Protozoan MeSH
• RNA, Transfer, Trp MeSH
• Codon, Terminator * MeSH
• Uridine MeSH

ZapE/Afg1 is a component of the inner cell membrane of some eubacteria and the inner mitochondrial membrane of eukaryotes. This protein is involved in FtsZ-dependent division of eubacteria. In the yeast and human mitochondrion, ZapE/Afg1 likely interacts with Oxa1 and facilitates the degradation of mitochondrion-encoded subunits of respiratory complexes. Furthermore, the depletion of ZapE increases resistance to apoptosis, decreases oxidative stress tolerance, and impacts mitochondrial protein homeostasis. It remains unclear whether ZapE is a multifunctional protein, or whether some of the described effects are just secondary phenotypes. Here, we have analyzed the functions of ZapE in Trypanosoma brucei, a parasitic protist, and an important model organism. Using a newly developed proximity-dependent biotinylation approach (BioID2), we have identified the inner mitochondrial membrane insertase Oxa1 among three putative interacting partners of ZapE, which is present in two paralogs. RNAi-mediated depletion of both ZapE paralogs likely affected the function of respiratory complexes I and IV. Consistently, we show that the distribution of mitochondrial ZapE is restricted only to organisms with Oxa1, respiratory complexes, and a mitochondrial genome. We propose that the evolutionarily conserved interaction of ZapE with Oxa1, which is required for proper insertion of many inner mitochondrial membrane proteins, is behind the multifaceted phenotype caused by the ablation of ZapE.

• MeSH
• Biotinylation MeSH
• Gene Deletion * MeSH
• Down-Regulation MeSH
• Eukaryota genetics   MeSH
• Phenotype MeSH
• Phylogeny MeSH
• Genome, Mitochondrial MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria metabolism   MeSH
• Protozoan Proteins metabolism   MeSH
• Electron Transport Complex I metabolism   MeSH
• Electron Transport Complex IV metabolism   MeSH
• Trypanosoma brucei brucei metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Mitochondrial Proteins MeSH
• Protozoan Proteins MeSH
• Electron Transport Complex I MeSH
• Electron Transport Complex IV MeSH

The majority of established model organisms belong to the supergroup Opisthokonta, which includes yeasts and animals. While enlightening, this focus has neglected protists, organisms that represent the bulk of eukaryotic diversity and are often regarded as primitive eukaryotes. One of these is the "supergroup" Excavata, which comprises unicellular flagellates of diverse lifestyles and contains species of medical importance, such as Trichomonas, Giardia, Naegleria, Trypanosoma and Leishmania. Excavata exhibits a continuum in mitochondrial forms, ranging from classical aerobic, cristae-bearing mitochondria to mitochondria-related organelles, such as hydrogenosomes and mitosomes, to the extreme case of a complete absence of the organelle. All forms of mitochondria house a machinery for the assembly of Fe-S clusters, ancient cofactors required in various biochemical activities needed to sustain every extant cell. In this review, we survey what is known about the Fe-S cluster assembly in the supergroup Excavata. We aim to bring attention to the diversity found in this group, reflected in gene losses and gains that have shaped the Fe-S cluster biogenesis pathways.

• Keywords
• Evolution, Excavata, Fe–S cluster, Mitochondria,
• MeSH
• Eukaryota cytology   metabolism   MeSH
• Mitochondria metabolism   MeSH
• Iron-Sulfur Proteins metabolism   MeSH
• Iron metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Iron-Sulfur Proteins MeSH
• Iron MeSH

Establishment of the early genetic code likely required strategies to ensure translational accuracy and inevitably involved tRNA post-transcriptional modifications. One such modification, wybutosine/wyosine is crucial for translational fidelity in Archaea and Eukarya; yet it does not occur in Bacteria and has never been described in mitochondria. Here, we present genetic, molecular and mass spectromery data demonstrating the first example of wyosine in mitochondria, a situation thus far unique to kinetoplastids. We also show that these modifications are important for mitochondrial function, underscoring their biological significance. This work focuses on TyW1, the enzyme required for the most critical step of wyosine biosynthesis. Based on molecular phylogeny, we suggest that the kinetoplastids pathways evolved via gene duplication and acquisition of an FMN-binding domain now prevalent in TyW1 of most eukaryotes. These findings are discussed in the context of the extensive U-insertion RNA editing in trypanosome mitochondria, which may have provided selective pressure for maintenance of mitochondrial wyosine in this lineage.

• MeSH
• Guanosine analogs & derivatives   biosynthesis   chemistry   metabolism   MeSH
• Mitochondria enzymology   MeSH
• RNA Processing, Post-Transcriptional MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• RNA, Transfer chemistry   metabolism   MeSH
• Trypanosoma brucei brucei enzymology   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Guanosine MeSH
• Protozoan Proteins MeSH
• RNA, Transfer MeSH
• wyosine MeSH Browser

UNLABELLED: Mitochondrial chaperones have multiple functions that are essential for proper functioning of mitochondria. In the human-pathogenic protist Trypanosoma brucei, we demonstrate a novel function of the highly conserved machinery composed of mitochondrial heat shock proteins 70 and 40 (mtHsp70/mtHsp40) and the ATP exchange factor Mge1. The mitochondrial DNA of T. brucei, also known as kinetoplast DNA (kDNA), is represented by a single catenated network composed of thousands of minicircles and dozens of maxicircles packed into an electron-dense kDNA disk. The chaperones mtHsp70 and mtHsp40 and their cofactor Mge1 are uniformly distributed throughout the single mitochondrial network and are all essential for the parasite. Following RNA interference (RNAi)-mediated depletion of each of these proteins, the kDNA network shrinks and eventually disappears. Ultrastructural analysis of cells depleted for mtHsp70 or mtHsp40 revealed that the otherwise compact kDNA network becomes severely compromised, a consequence of decreased maxicircle and minicircle copy numbers. Moreover, we show that the replication of minicircles is impaired, although the lack of these proteins has a bigger impact on the less abundant maxicircles. We provide additional evidence that these chaperones are indispensable for the maintenance and replication of kDNA, in addition to their already known functions in Fe-S cluster synthesis and protein import. IMPORTANCE: Impairment or loss of mitochondrial DNA is associated with mitochondrial dysfunction and a wide range of neural, muscular, and other diseases. We present the first evidence showing that the entire mtHsp70/mtHsp40 machinery plays an important role in mitochondrial DNA replication and maintenance, a function likely retained from prokaryotes. These abundant, ubiquitous, and multifunctional chaperones share phenotypes with enzymes engaged in the initial stages of replication of the mitochondrial DNA in T. brucei.

• MeSH
• DNA, Kinetoplast genetics   metabolism   MeSH
• Humans MeSH
• DNA, Mitochondrial genetics   metabolism   MeSH
• Mitochondria genetics   metabolism   MeSH
• HSP40 Heat-Shock Proteins genetics   metabolism   MeSH
• HSP70 Heat-Shock Proteins genetics   metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• DNA Replication * MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Trypanosomiasis, African parasitology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DNA, Kinetoplast MeSH
• DNA, Mitochondrial MeSH
• HSP40 Heat-Shock Proteins MeSH
• HSP70 Heat-Shock Proteins MeSH
• Protozoan Proteins MeSH

Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

• MeSH
• Active Transport, Cell Nucleus MeSH
• Cell Nucleus metabolism   MeSH
• Ferredoxins metabolism   MeSH
• Frataxin MeSH
• Nuclear Localization Signals MeSH
• Carbon-Sulfur Lyases chemistry   genetics   metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria metabolism   MeSH
• Molecular Sequence Data MeSH
• Protein Multimerization MeSH
• Nuclear Matrix-Associated Proteins chemistry   genetics   metabolism   MeSH
• Iron-Binding Proteins metabolism   MeSH
• Protozoan Proteins chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Trypanosoma brucei brucei enzymology   genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• cysteine desulfurase MeSH Browser
• Ferredoxins MeSH
• Nuclear Localization Signals MeSH
• Carbon-Sulfur Lyases MeSH
• Mitochondrial Proteins MeSH
• Nuclear Matrix-Associated Proteins MeSH
• Iron-Binding Proteins MeSH
• Protozoan Proteins MeSH