Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. We demonstrate that the highly divergent β-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in β-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages.

Department of Cell Biology Faculty of Science Charles University Prague Czech Republic

Department of Parasitology Faculty of Science Charles University BIOCEV Prague Czech Republic

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University Prague Czech Republic

Interfaculty Institute of Biochemistry University of Tübingen Tübingen Germany

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A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis

The divergent ER-mitochondria encounter structures (ERMES) are conserved in parabasalids but lost in several anaerobic lineages with hydrogenosomes

A mitochondrion-free eukaryote contains proteins capable of import into an exogenous mitochondrion-related organelle

Reduced mitochondria provide an essential function for the cytosolic methionine cycle

Anaerobic derivates of mitochondria and peroxisomes in the free-living amoeba Pelomyxa schiedti revealed by single-cell genomics

Fates of Sec, Tat, and YidC Translocases in Mitochondria and Other Eukaryotic Compartments

Anaerobic peroxisomes in Entamoeba histolytica metabolize myo-inositol

The Mastigamoeba balamuthi Genome and the Nature of the Free-Living Ancestor of Entamoeba

Determinism and contingencies shaped the evolution of mitochondrial protein import

Anaerobic peroxisomes in Mastigamoeba balamuthi