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.

• MeSH
• fylogeneze MeSH
• membránové proteiny metabolismus   MeSH
• membránové transportní proteiny metabolismus   MeSH
• mitochondriální importní komplex MeSH
• mitochondrie metabolismus   MeSH
• organely MeSH
• transport proteinů fyziologie   MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• transportní proteiny genetika   metabolismus   fyziologie   MeSH
• Trichomonas vaginalis metabolismus   patogenita   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• membránové transportní proteiny MeSH
• mitochondriální importní komplex MeSH
• transportní proteiny mitochondriální membrány MeSH
• transportní proteiny MeSH

The origin of protein import was a key step in the endosymbiotic acquisition of mitochondria. Though the main translocon of the mitochondrial outer membrane, TOM40, is ubiquitous among organelles of mitochondrial ancestry, the transit peptides, or N-terminal targeting sequences (NTSs), recognised by the TOM complex, are not. To better understand the nature of evolutionary conservation in mitochondrial protein import, we investigated the targeting behavior of Trichomonas vaginalis hydrogenosomal proteins in Saccharomyces cerevisiae and vice versa. Hydrogenosomes import yeast mitochondrial proteins even in the absence of their native NTSs, but do not import yeast cytosolic proteins. Conversely, yeast mitochondria import hydrogenosomal proteins with and without their short NTSs. Conservation of an NTS-independent mitochondrial import route from excavates to opisthokonts indicates its presence in the eukaryote common ancestor. Mitochondrial protein import is known to entail electrophoresis of positively charged NTSs across the electrochemical gradient of the inner mitochondrial membrane. Our present findings indicate that mitochondrial transit peptides, which readily arise from random sequences, were initially selected as a signal for charge-dependent protein targeting specifically to the mitochondrial matrix. Evolutionary loss of the electron transport chain in hydrogenosomes and mitosomes lifted the selective constraints that maintain positive charge in NTSs, allowing first the NTS charge, and subsequently the NTS itself, to be lost. This resulted in NTS-independent matrix targeting, which is conserved across the evolutionary divide separating trichomonads and yeast, and which we propose is the ancestral state of mitochondrial protein import.

• Klíčová slova
• TOM/TIM, hydrogenosomes, mitochondria, mitosomes, protein import,
• MeSH
• mitochondriální proteiny chemie   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• molekulární evoluce * MeSH
• proteiny - lokalizační signály * MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• transport proteinů MeSH
• Trichomonas vaginalis metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• proteiny - lokalizační signály * MeSH
• Saccharomyces cerevisiae - proteiny MeSH