Updated project metadata. The formation of mitochondria has enabled eukaryotes to thrive and evolve into multitude of unicellular and multicellular species. The nature of the early mitochondria and its relationship to the ancestral host cell is still unknown. Using a phylogenomic approach we have discovered the presence of core subunits of type II secretion pathway (T2SS) in several unicellular eukaryotes (protists) belonging to jakobids, hetereloboseans and malawimonads. We show that the proteins carry mitochondrial signal sequences and are localized in the protist mitochondria.The mitochondria of Nagleria gruberi were isolated by differential centrifugation and analysed by mass spectrometry. The data analyses revealed about 1000 proteins enriched in the mitochondrial fraction including the components of T2SS.