BACKGROUND: Hydrogenosomes are a specific type of mitochondria that have adapted for life under anaerobiosis. Limited availability of oxygen has resulted in the loss of the membrane-associated respiratory chain, and consequently in the generation of minimal inner membrane potential (Δψ), and inefficient ATP synthesis via substrate-level phosphorylation. The changes in energy metabolism are directly linked with the organelle biogenesis. In mitochondria, proteins are imported across the outer membrane via the Translocase of the Outer Membrane (TOM complex), while two Translocases of the Inner Membrane, TIM22, and TIM23, facilitate import to the inner membrane and matrix. TIM23-mediated steps are entirely dependent on Δψ and ATP hydrolysis, while TIM22 requires only Δψ. The character of the hydrogenosomal inner membrane translocase and the mechanism of translocation is currently unknown. RESULTS: We report unprecedented modification of TIM in hydrogenosomes of the human parasite Trichomonas vaginalis (TvTIM). We show that the import of the presequence-containing protein into the hydrogenosomal matrix is mediated by the hybrid TIM22-TIM23 complex that includes three highly divergent core components, TvTim22, TvTim23, and TvTim17-like proteins. The hybrid character of the TvTIM is underlined by the presence of both TvTim22 and TvTim17/23, association with small Tim chaperones (Tim9-10), which in mitochondria are known to facilitate the transfer of substrates to the TIM22 complex, and the coupling with TIM23-specific ATP-dependent presequence translocase-associated motor (PAM). Interactome reconstruction based on co-immunoprecipitation (coIP) and mass spectrometry revealed that hybrid TvTIM is formed with the compositional variations of paralogs. Single-particle electron microscopy for the 132-kDa purified TvTIM revealed the presence of a single ring of small Tims complex, while mitochondrial TIM22 complex bears twin small Tims hexamer. TvTIM is currently the only TIM visualized outside of Opisthokonta, which raised the question of which form is prevailing across eukaryotes. The tight association of the hybrid TvTIM with ADP/ATP carriers (AAC) suggests that AAC may directly supply ATP for the protein import since ATP synthesis is limited in hydrogenosomes. CONCLUSIONS: The hybrid TvTIM in hydrogenosomes represents an original structural solution that evolved for protein import when Δψ is negligible and remarkable example of evolutionary adaptation to an anaerobic lifestyle.

• Klíčová slova
• Trichomonas vaginalis, Hydrogenosomes, Mitochondria, Parasite, Presequence translocase-associated motor, Protein import machinery, TIM22 complex, TIM23 complex,
• MeSH
• mitochondriální importní komplex MeSH
• mitochondrie metabolismus   MeSH
• organely metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• transport proteinů * MeSH
• Trichomonas vaginalis * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mitochondriální importní komplex MeSH
• protozoální proteiny MeSH

BACKGROUND: The Tim17 family of proteins plays a fundamental role in the biogenesis of mitochondria. Three Tim17 family proteins, Tim17, Tim22, and Tim23, are the central components of the widely conserved multi-subunit protein translocases, TIM23 and TIM22, which mediate protein transport across and into the inner mitochondrial membrane, respectively. In addition, several Tim17 family proteins occupy the inner and outer membranes of plastids. RESULTS: We have performed comprehensive sequence analyses on 5631 proteomes from all domains of life deposited in the Uniprot database. The analyses showed that the Tim17 family of proteins is much more diverse than previously thought and involves at least ten functionally and phylogenetically distinct groups of proteins. As previously shown, mitochondrial inner membrane accommodates prototypical Tim17, Tim22 and Tim23 and two Tim17 proteins, TIMMDC1 and NDUFA11, which participate in the assembly of complex I of the respiratory chain. In addition, we have identified Romo1/Mgr2 as Tim17 family member. The protein has been shown to control lateral release of substrates fromTIM23 complex in yeast and to participate in the production of reactive oxygen species in mammalian cells. Two peroxisomal proteins, Pmp24 and Tmem135, of so far unknown function also belong to Tim17 protein family. Additionally, a new group of Tim17 family proteins carrying a C-terminal coiled-coil domain has been identified predominantly in fungi. CONCLUSIONS: We have mapped the distribution of Tim17 family members in the eukaryotic supergroups and found that the mitochondrial Tim17, Tim22 and Tim23 proteins, as well as the peroxisomal Tim17 family proteins, were all likely to be present in the last eukaryotic common ancestor (LECA). Thus, kinetoplastid mitochondria previously identified as carrying a single Tim17protein family homologue are likely to be the outcome of a secondary reduction. The eukaryotic cell has modified mitochondrial Tim17 family proteins to mediate different functions in multiple cellular compartments including mitochondria, plastids and peroxisomes. Concerning the origin of Tim17 protein family, our analyses do not support the affiliation of the protein family and the component of bacterial amino acid permease. Thus, it is likely that Tim17 protein family is exclusive to eukaryotes. REVIEWERS: The article was reviewed by Michael Gray, Martijn Huynen and Kira Makarova.

• Klíčová slova
• Evolution, Mitochondria, NDUFA11, Oep16, Peroxisome, Plastid, Pmp24, Protein import, Romo1, TIMMDC1, Tim17, Tim22, Tim23, Tmem135,
• MeSH
• fylogeneze MeSH
• molekulární evoluce * MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• transportní proteiny mitochondriální membrány chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• transportní proteiny mitochondriální membrány MeSH

Mitochondria have evolved diverse forms across eukaryotic diversity in adaptation to anoxia. Mitosomes are the simplest and the least well-studied type of anaerobic mitochondria. Transport of proteins via TIM complexes, composed of three proteins of the Tim17 protein family (Tim17/22/23), is one of the key unifying aspects of mitochondria and mitochondria-derived organelles. However, multiple experimental and bioinformatic attempts have so far failed to identify the nature of TIM in mitosomes of the anaerobic metamonad protist, Giardia intestinalis, one of the few experimental models for mitosome biology. Here, we present the identification of a single G. intestinalis Tim17 protein (GiTim17), made possible only by the implementation of a metamonad-specific hidden Markov model. While very divergent in primary sequence and in predicted membrane topology, experimental data suggest that GiTim17 is an inner membrane mitosomal protein, forming a disulphide-linked dimer. We suggest that the peculiar GiTim17 sequence reflects adaptation to the unusual, detergent resistant, inner mitosomal membrane. Specific pull-down experiments indicate interaction of GiTim17 with mitosomal Tim44, the tethering component of the import motor complex. Analysis of TIM complexes across eukaryote diversity suggests that a "single Tim" translocase is a convergent adaptation of mitosomes in anaerobic protists, with Tim22 and Tim17 (but not Tim23), providing the protein backbone.

• MeSH
• anaerobióza MeSH
• Giardia lamblia enzymologie   MeSH
• mitochondrie enzymologie   MeSH
• molekulární evoluce * MeSH
• sekvence aminokyselin MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• transportní proteiny mitochondriální membrány MeSH