The endobiotic flagellate Monocercomonoides exilis is the only known eukaryote to have lost mitochondria and all its associated proteins in its evolutionary past. This final stage of the mitochondrial evolutionary pathway may serve as a model to explain events at their very beginning such as the initiation of protein import. We have assessed the capability of proteins from this eukaryote to enter emerging mitochondria using a specifically designed in vitro assay. Hydrogenosomes (reduced mitochondria) of Trichomonas vaginalis were incubated with a soluble protein pool derived from a cytosolic fraction of M. exilis, and proteins entering hydrogenosomes were subsequently detected by mass spectrometry. The assay detected 19 specifically and reproducibly imported proteins, and in 14 cases the import was confirmed by the overexpression of their tagged version in T. vaginalis. In most cases, only a small portion of the signal reached the hydrogenosomes, suggesting specific but inefficient transport. Most of these proteins represent enzymes of carbon metabolism, and none exhibited clear signatures of proteins targeted to hydrogenosomes or mitochondria, which is consistent with their inefficient import. The observed phenomenon may resemble a primaeval type of protein import which might play a role in the establishment of the organelle and shaping of its proteome in the initial stages of endosymbiosis.

• Klíčová slova
• evolution of protein targeting, hydrogenosome, mitochondrion-free eukaryote, protein import,
• MeSH
• Eukaryota * metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• organely chemie   metabolismus   MeSH
• protozoální proteiny * metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny * MeSH

Monocercomonoides exilis is considered the first known eukaryote to completely lack mitochondria. This conclusion is based primarily on a genomic and transcriptomic study which failed to identify any mitochondrial hallmark proteins. However, the available genome assembly has limited contiguity and around 1.5 % of the genome sequence is represented by unknown bases. To improve the contiguity, we re-sequenced the genome and transcriptome of M. exilis using Oxford Nanopore Technology (ONT). The resulting draft genome is assembled in 101 contigs with an N50 value of 1.38 Mbp, almost 20 times higher than the previously published assembly. Using a newly generated ONT transcriptome, we further improve the gene prediction and add high quality untranslated region (UTR) annotations, in which we identify two putative polyadenylation signals present in the 3'UTR regions and characterise the Kozak sequence in the 5'UTR regions. All these improvements are reflected by higher BUSCO genome completeness values. Regardless of an overall more complete genome assembly without missing bases and a better gene prediction, we still failed to identify any mitochondrial hallmark genes, thus further supporting the hypothesis on the absence of mitochondrion.

• Klíčová slova
• Monocercomonoides, amitochondriate, genome, nanopore,
• MeSH
• anotace sekvence MeSH
• délka genomu MeSH
• nanoporové sekvenování MeSH
• Oxymonadida klasifikace   genetika   MeSH
• protozoální proteiny genetika   MeSH
• regulace genové exprese MeSH
• sekvenování celého genomu metody   MeSH
• stanovení celkové genové exprese metody   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

BACKGROUND: Multiple prokaryotic lineages use the arginine deiminase (ADI) pathway for anaerobic energy production by arginine degradation. The distribution of this pathway among eukaryotes has been thought to be very limited, with only two specialized groups living in low oxygen environments (Parabasalia and Diplomonadida) known to possess the complete set of all three enzymes. We have performed an extensive survey of available sequence data in order to map the distribution of these enzymes among eukaryotes and to reconstruct their phylogenies. RESULTS: We have found genes for the complete pathway in almost all examined representatives of Metamonada, the anaerobic protist group that includes parabasalids and diplomonads. Phylogenetic analyses indicate the presence of the complete pathway in the last common ancestor of metamonads and heterologous transformation experiments suggest its cytosolic localization in the metamonad ancestor. Outside Metamonada, the complete pathway occurs rarely, nevertheless, it was found in representatives of most major eukaryotic clades. CONCLUSIONS: Phylogenetic relationships of complete pathways are consistent with the presence of the Archaea-derived ADI pathway in the last common ancestor of all eukaryotes, although other evolutionary scenarios remain possible. The presence of the incomplete set of enzymes is relatively common among eukaryotes and it may be related to the fact that these enzymes are involved in other cellular processes, such as the ornithine-urea cycle. Single protein phylogenies suggest that the evolutionary history of all three enzymes has been shaped by frequent gene losses and horizontal transfers, which may sometimes be connected with their diverse roles in cellular metabolism.

• Klíčová slova
• Arginine deiminase, Carbamate kinase, Metamonada, Ornithine transcarbamylase, Phylogeny, Preaxostyla, Protists,
• MeSH
• Archaea metabolismus   MeSH
• arginin metabolismus   MeSH
• Diplomonadida enzymologie   MeSH
• Eukaryota klasifikace   genetika   metabolismus   MeSH
• fylogeneze MeSH
• hydrolasy metabolismus   MeSH
• metabolické sítě a dráhy * MeSH
• molekulární evoluce * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• arginin MeSH
• arginine deiminase MeSH Prohlížeč
• hydrolasy MeSH

Trichomonas vaginalis is one of a few eukaryotes that have been found to encode several homologues of flavodiiron proteins (FDPs). Widespread among anaerobic prokaryotes, these proteins are believed to function as oxygen and/or nitric oxide reductases to provide protection against oxidative/nitrosative stresses and host immune responses. One of the T. vaginalis FDP homologues is equipped with a hydrogenosomal targeting sequence and is expressed in the hydrogenosomes, oxygen-sensitive organelles that participate in carbohydrate metabolism and assemble iron-sulfur clusters. The bacterial homologues characterized thus far have been dimers or tetramers; the trichomonad protein is a dimer of identical 45-kDa subunits, each noncovalently binding one flavin mononucleotide. The protein reduces dioxygen to water but is unable to utilize nitric oxide as a substrate, similarly to its closest homologue from another human parasite Giardia intestinalis and related archaebacterial proteins. T. vaginalis FDP is able to accept electrons derived from pyruvate or NADH via ferredoxin and is proposed to play a role in the protection of hydrogenosomes against oxygen.

• MeSH
• ferredoxiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• flavinmononukleotid metabolismus   MeSH
• kyslík metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• organely chemie   enzymologie   genetika   MeSH
• oxidoreduktasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• protozoální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• sekvence aminokyselin MeSH
• substrátová specifita MeSH
• Trichomonas vaginalis chemie   enzymologie   genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ferredoxiny MeSH
• flavinmononukleotid MeSH
• kyslík MeSH
• oxidoreduktasy MeSH
• protozoální proteiny MeSH