Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.

Biology Centre Institute of Parasitology Czech Academy of Sciences České Budějovice Budweis Czech Republic

Faculty of Science Charles University Biocev Vestec Czech Republic

Faculty of Science University of Ostrava Ostrava Czech Republic

Faculty of Sciences University of South Bohemia České Budějovice Budweis Czech Republic

School of Life Sciences University of Dundee Dundee United Kingdom

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Visualisation of Euglena gracilis organelles and cytoskeleton using expansion microscopy

Lessons from the deep: mechanisms behind diversification of eukaryotic protein complexes

Heterotrophic euglenid Rhabdomonas costata resembles its phototrophic relatives in many aspects of molecular and cell biology

Single-cell genomics unveils a canonical origin of the diverse mitochondrial genomes of euglenozoans

Determinism and contingencies shaped the evolution of mitochondrial protein import

Inventory and Evolution of Mitochondrion-localized Family A DNA Polymerases in Euglenozoa