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The core components of organelle biogenesis and membrane transport in the hydrogenosomes of Trichomonas vaginalis.

ABSTRACT: Trichomonas vaginalis is a parasitic protist of the Excavata group. It contains an anaerobic form of mitochondria called hydrogenosomes, which produce hydrogen and ATP; the majority of mitochondrial pathways and the organellar genome were lost during the mitochondrion-to-hydrogenosome transition. Consequently, all hydrogenosomal proteins are encoded in the nucleus and imported into the organelles. However, little is known about the membrane machineries required for biogenesis of the organelle and metabolite exchange. Using a combination of mass spectrometry, immunofluorescence microscopy, in vitro import assays and reverse genetics, we characterized the membrane proteins of the hydrogenosome. We identified components of the outer membrane (TOM) and inner membrane (TIM) protein translocases include multiple paralogs of the core Tom40-type porins and Tim17/22/23 channel proteins, respectively, and uniquely modified small Tim chaperones. The inner membrane proteins TvTim17/22/23-1 and Pam18 were shown to possess conserved information for targeting to mitochondrial inner membranes, but too divergent in sequence to support the growth of yeast strains lacking Tim17, Tim22, Tim23 or Pam18. Full complementation was seen only when the J-domain of hydrogenosomal Pam18 was fused with N-terminal region and transmembrane segment of the yeast homolog. Candidates for metabolite exchange across the outer membrane were identified including multiple isoforms of the ?-barrel proteins, Hmp35 and Hmp36; inner membrane MCF-type metabolite carriers were limited to five homologs of the ATP/ADP carrier, Hmp31. Lastly, hydrogenosomes possess a pathway for the assembly of C-tail-anchored proteins into their outer membrane with several new tail-anchored proteins being identified. These results show that hydrogenosomes and mitochondria share common core membrane components required for protein import and metabolite exchange; however, they also reveal remarkable differences that reflect the functional adaptation of hydrogenosomes to anaerobic conditions and the peculiar evolutionary history of the Excavata group.

SUBMITTER: Rada P

PROVIDER: S-EPMC3174187 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Mitochondria originated from α-proteobacterial endosymbionts, and the endosymbiont-to-organelle transition is tightly linked to the establishment of 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, recent studies have shown diversity of TOM subunits between main eukaryotic lineages. However, experimental knowledge is currently limited to a few model organisms. Here, we analysed the TvTOM complex in hydrogenosomes, an anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. Using the pore-forming TvTom40-2 as bait, we immunoprecipitated the high molecular weight TvTOM complex both under crosslinking and native conditions and performed label-free quantitative mass spectrometry (LFQ-MS) to identify the components of the TvTOM complex. Identification of the components of the hydrogenosomal TOM complex To identify interaction partners for TvTom40-2 in the TOM complex, we performed co-immunoprecipitations (coIPs) of HA-tagged TvTom40-2 under protein crosslinking and under native conditions, and the eluted proteins were analyzed using label-free quantitative (LFQ) mass spectrometry. CoIPs using anti-HA antibody (Ab) were performed with hydrogenosomes isolated from both the strain expressing HA-tagged TvTom40-2 and the wild type strain, as a negative control. The analysis revealed 50 and 36 proteins that were enriched with HA-tagged TvTom40-2 under crosslinking and native conditions respectively. As TOM proteins are embedded in the hydrogenosomal outer membrane, we searched for proteins with TMDs in the datasets using TMHMM and found 19 and 13 proteins for crosslinking and native coIPs respectively. The intersection between the two datasets and the hydrogenosomal membrane proteome (Rada et al 2011) contained five TvTom40 isoforms, two TA proteins named Homp36 and Homp19 (Homp, Hydrogenosomal outer membrane protein), Sam50 and its paralogue Sam50p, and Hmp35. In addition, the intersection between the coIP dataset under crosslinking conditions and the membrane proteome contained two more TA proteins, Homp38 and Homp46. Based on these results, we selected Homp36 which was previously shown to reside in the hydrogenosomal outer membrane (Rada et al 2011) for the reciprocal coIPs. Proteins enriched in the HA-tagged Homp36 coIPs under crosslinking conditions included multiple isoforms of TvTom40, Sam50, Hmp35, Homp38, Homp46 and Homp19, and multiple isoforms of TvTom40, Sam50 and Hmp35 were enriched under native conditions. Altogether, the coIP and MS data indicated four TA candidate proteins, Homp19, Homp36, Homp38 and Homp46, and two β-barrel proteins, Sam50 and Hmp35. InterProScan predicted that Homp36, Homp38 and Homp46 carry an N-terminal Hsp20-like chaperone domain, three TPR-like domains and a C-terminal transmembrane helix. This domain architecture resembles the recently reported ATOM69 in T. brucei (Mani et al 2015). Indeed, HHpred searches using Homp36 and Homp38 as queries against the T. brucei proteome revealed ATOM69 as the first hit, with E values of 4.9e-17 and 2.3e-11 respectively. HHpred searches with Homp46 recognized various proteins with TPR domains, whereas no significant homology was observed for Homp19.

Project description:The parabasalian flagellate Trichomonas vaginalis harbors mitochondrion-related and H(2)-producing organelles of anaerobic ATP synthesis, called hydrogenosomes, which harbor oxygen-sensitive enzymes essential to its pyruvate metabolism. In the human urogenital tract, however, T. vaginalis is regularly exposed to low oxygen concentrations and therefore must possess antioxidant systems protecting the organellar environment against the detrimental effects of molecular oxygen and reactive oxygen species. We have identified two closely related hydrogenosomal thioredoxin reductases (TrxRs), the hitherto-missing component of a thioredoxin-linked hydrogenosomal antioxidant system. One of the two hydrogenosomal TrxR isoforms, TrxRh1, carried an N-terminal extension resembling known hydrogenosomal targeting signals. Expression of hemagglutinin-tagged TrxRh1 in transfected T. vaginalis cells revealed that its N-terminal extension was necessary to import the protein into the organelles. The second hydrogenosomal TrxR isoform, TrxRh2, had no N-terminal targeting signal but was nonetheless efficiently targeted to hydrogenosomes. N-terminal presequences from hydrogenosomal proteins with known processing sites, i.e., the alpha subunit of succinyl coenzyme A synthetase (SCSalpha) and pyruvate:ferredoxin oxidoreductase A, were investigated for their ability to direct mature TrxRh1 to hydrogenosomes. Neither presequence directed TrxRh1 to hydrogenosomes, indicating that neither extension is, by itself, sufficient for hydrogenosomal targeting. Moreover, SCSalpha lacking its N-terminal extension was efficiently imported into hydrogenosomes, indicating that this extension is not required for import of this major hydrogenosomal protein. The finding that some hydrogenosomal enzymes require N-terminal signals for import but that in others the N-terminal extension is not necessary for targeting indicates the presence of additional targeting signals within the mature subunits of several hydrogenosome-localized proteins.

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The core components of organelle biogenesis and membrane transport in the hydrogenosomes of Trichomonas vaginalis.

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