ABSTRACT: 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.
Abhijith Makki 
