Abstract
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.
Highlights
Mitochondria originated from proteobacterial endosymbionts [1], and over time, massive endosymbiotic gene transfer to the host nucleus or gene deletion forged the development of a mechanism for retargeting of nuclear-encoded proteins to the evolving organelle [2]
Embedded in the mitochondrial outer membrane is a molecular machine, known as the translocase of the outer membrane (TOM) complex, that plays a key role in protein import and biogenesis of the organelle
The T. vaginalis TOM (TvTOM) complex has a conserved triplet-pore structure but with a unique skull-like shape suggesting that the TOM in the early mitochondrion could have formed three pores
Summary
Mitochondria originated from proteobacterial endosymbionts [1], and over time, massive endosymbiotic gene transfer to the host nucleus or gene deletion forged the development of a mechanism for retargeting of nuclear-encoded proteins to the evolving organelle [2]. The TOM complex in yeast consists of Tom and six α-helical proteins: two that are anchored to the outer mitochondrial membrane (OMM) by an N-terminal transmembrane domain (TMD; Tom and Tom70) and four that are anchored by a C-terminal TMD (Tom, Tom, Tom, and Tom). A prominent feature of the TOM complex is the variation in receptors across different eukaryotic lineages. A signal-anchored Tom is present in animals and fungi, whereas plant Tom evolved independently with a C-terminal anchor [7]. In the excavate Trypanosoma brucei, the TOM complex (named the archaic translocase of the outer membrane [ATOM]) has only two orthologues, a highly divergent Tom (ATOM40) and a Tom22-like protein (ATOM14) [11,13]. Instead of Tom and Tom, two unique receptors were identified, a TA protein ATOM69 and a signal-anchored ATOM46 [11]
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