Retromer and Retriever systems are conserved and differentially expanded in Parabasalids.

Early Endosomes sort transmembrane cargo whether for lysosomal degradation or retrieval to the plasma membrane or the Golgi complex. Endosomal retrieval in eukaryotes is governed by the anciently homologous Retromer or Retriever complexes. Each comprises a core tri-protein subcomplex, membrane-deformation proteins, and interacting partner complexes, together retrieving a variety of known cargo proteins. Trichomonas vaginalis; a sexually transmitted human parasite uses the endomembrane system for pathogenesis. It has massively and selectively expanded its endomembrane protein complement, the evolutionary path of which has been largely unexplored. Our molecular evolutionary study of Retromer, Retriever and associated machinery in parabasalids and its free-living sister lineage of Anaeramoeba, demonstrates specific expansion of the Retromer machinery, contrasting with the Retriever components. We also observe partial loss of Commander complex and Sorting Nexins in Parabasalia but complete retention in Anaeramoeba. Notably, we identify putative parabasalid Sorting Nexin analogues. Finally, we report the first Retriever protein localization in a non-metazoan group along with Retromer protein localization in T. vaginalis.

A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis.

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.

Varroa destructor parasitism and Deformed wing virus infection in honey bees are linked to peroxisome-induced pathways.

The ectoparasitic mite Varroa destructor transmits and triggers viral infections that have deleterious effects on honey bee colonies worldwide. We performed a manipulative experiment in which worker bees collected at emergence were exposed to Varroa for 72 h, and their proteomes were compared with those of untreated control bees. Label-free quantitative proteomics identified 77 differentially expressed A. mellifera proteins (DEPs). In addition, viral proteins were identified by orthogonal analysis, and most importantly, Deformed wing virus (DWV) was found at high levels/intensity in Varroa-exposed bees. Pathway enrichment analysis suggested that the main pathways affected included peroxisomal metabolism, cyto-/exoskeleton reorganization, and cuticular proteins. Detailed examination of individual DEPs revealed that additional changes in DEPs were associated with peroxisomal function. In addition, the proteome data support the importance of TGF-ß signaling in Varroa-DWV interaction and the involvement of the mTORC1 and Hippo pathways. These results suggest that the effect of DWV on bees associated with Varroa feeding results in aberrant autophagy. In particular, autophagy is selectively modulated by peroxisomes, to which the observed proteome changes strongly corresponded. This study complements previous research with different study designs and suggests the importance of the peroxisome, which plays a key role in viral infections.

The protist cultural renaissance.

Protists are key players in the biosphere. Here, we provide a perspective on integrating protist culturing with omics approaches, imaging, and high-throughput single-cell manipulation strategies, concluding with actions required for a successful return of the golden age of protist culturing.

The divergent ER-mitochondria encounter structures (ERMES) are conserved in parabasalids but lost in several anaerobic lineages with hydrogenosomes.

BACKGROUND: The endoplasmic reticulum (ER)-mitochondria membrane contact sites (MCS) are extensively studied in aerobic eukaryotes; however, little is known about MCS in anaerobes with reduced forms of mitochondria named hydrogenosomes. In several eukaryotic lineages, the direct physical tether between ER and the outer mitochondrial membrane is formed by ER-mitochondria encounter structure (ERMES). The complex consists of four core proteins (Mmm1, Mmm2, Mdm12, and Mdm10) which are involved in phospholipid trafficking. Here we investigated ERMES distribution in organisms bearing hydrogenosomes and employed Trichomonas vaginalis as a model to estimate ERMES cellular localization, structure, and function. RESULTS: Homology searches revealed that Parabasalia-Anaeramoebae, anaerobic jakobids, and anaerobic fungi are lineages with hydrogenosomes that retain ERMES, while ERMES components were gradually lost in Fornicata, and are absent in Preaxostyla and Archamoebae. In T. vaginalis and other parabasalids, three ERMES components were found with the expansion of Mmm1. Immunofluorescence microscopy confirmed that Mmm1 localized in ER, while Mdm12 and Mmm2 were partially localized in hydrogenosomes. Pull-down assays and mass spectrometry of the ERMES components identified a parabasalid-specific Porin2 as a substitute for the Mdm10. ERMES modeling predicted a formation of a continuous hydrophobic tunnel of TvMmm1-TvMdm12-TvMmm2 that is anchored via Porin2 to the hydrogenosomal outer membrane. Phospholipid-ERMES docking and Mdm12-phospholipid dot-blot indicated that ERMES is involved in the transport of phosphatidylinositol phosphates. The absence of enzymes involved in hydrogenosomal phospholipid metabolism implies that ERMES is not involved in the exchange of substrates between ER and hydrogenosomes but in the unidirectional import of phospholipids into hydrogenosomal membranes. CONCLUSIONS: Our investigation demonstrated that ERMES mediates ER-hydrogenosome interactions in parabasalid T. vaginalis, while the complex was lost in several other lineages with hydrogenosomes.

Comparative analysis of mitochondrion-related organelles in anaerobic amoebozoans.

Archamoebae comprises free-living or endobiotic amoebiform protists that inhabit anaerobic or microaerophilic environments and possess mitochondrion-related organelles (MROs) adapted to function anaerobically. We compared in silico reconstructed MRO proteomes of eight species (six genera) and found that the common ancestor of Archamoebae possessed very few typical components of the protein translocation machinery, electron transport chain and tricarboxylic acid cycle. On the other hand, it contained a sulphate activation pathway and bacterial iron-sulphur (Fe-S) assembly system of MIS-type. The metabolic capacity of the MROs, however, varies markedly within this clade. The glycine cleavage system is widely conserved among Archamoebae, except in Entamoeba, probably owing to its role in catabolic function or one-carbon metabolism. MRO-based pyruvate metabolism was dispensed within subgroups Entamoebidae and Rhizomastixidae, whereas sulphate activation could have been lost in isolated cases of Rhizomastix libera, Mastigamoeba abducta and Endolimax sp. The MIS (Fe-S) assembly system was duplicated in the common ancestor of Mastigamoebidae and Pelomyxidae, and one of the copies took over Fe-S assembly in their MRO. In Entamoebidae and Rhizomastixidae, we hypothesize that Fe-S cluster assembly in both compartments may be facilitated by dual localization of the single system. We could not find evidence for changes in metabolic functions of the MRO in response to changes in habitat; it appears that such environmental drivers do not strongly affect MRO reduction in this group of eukaryotes.

Evolutionary analysis of cellular reduction and anaerobicity in the hyper-prevalent gut microbe Blastocystis.

Blastocystis is the most prevalent microbial eukaryote in the human and animal gut, yet its role as commensal or parasite is still under debate. Blastocystis has clearly undergone evolutionary adaptation to the gut environment and possesses minimal cellular compartmentalization, reduced anaerobic mitochondria, no flagella, and no reported peroxisomes. To address this poorly understood evolutionary transition, we have taken a multi-disciplinary approach to characterize Proteromonas lacertae, the closest canonical stramenopile relative of Blastocystis. Genomic data reveal an abundance of unique genes in P. lacertae but also reductive evolution of the genomic complement in Blastocystis. Comparative genomic analysis sheds light on flagellar evolution, including 37 new candidate components implicated with mastigonemes, the stramenopile morphological hallmark. The P. lacertae membrane-trafficking system (MTS) complement is only slightly more canonical than that of Blastocystis, but notably, we identified that both organisms encode the complete enigmatic endocytic TSET complex, a first for the entire stramenopile lineage. Investigation also details the modulation of mitochondrial composition and metabolism in both P. lacertae and Blastocystis. Unexpectedly, we identify in P. lacertae the most reduced peroxisome-derived organelle reported to date, which leads us to speculate on a mechanism of constraint guiding the dynamics of peroxisome-mitochondrion reductive evolution on the path to anaerobiosis. Overall, these analyses provide a launching point to investigate organellar evolution and reveal in detail the evolutionary path that Blastocystis has taken from a canonical flagellated protist to the hyper-divergent and hyper-prevalent animal and human gut microbe.

Double-Stranded RNA Viruses Are Released From Trichomonas vaginalis Inside Small Extracellular Vesicles and Modulate the Exosomal Cargo.

Trichomonas vaginalis is a parasitic protist that infects the human urogenital tract. During the infection, trichomonads adhere to the host mucosa, acquire nutrients from the vaginal/prostate environment, and release small extracellular vesicles (sEVs) that contribute to the trichomonad adherence and modulate the host-parasite communication. Approximately 40-70% of T. vaginalis strains harbor a double-stranded RNA virus called Trichomonasvirus (TVV). Naked TVV particles have the potential to stimulate a proinflammatory response in human cells, however, the mode of TVV release from trichomonads to the environment is not clear. In this report, we showed for the first time that TVV particles are released from T. vaginalis cells within sEVs. The sEVs loaded with TVV stimulated a higher proinflammatory response of human HaCaT cells in comparison to sEVs from TVV negative parasites. Moreover, a comparison of T. vaginalis isogenic TVV plus and TVV minus clones revealed a significant impact of TVV infection on the sEV proteome and RNA cargo. Small EVs from TVV positive trichomonads contained 12 enriched and 8 unique proteins including membrane-associated BspA adhesine, and about a 2.5-fold increase in the content of small regulatory tsRNA. As T. vaginalis isolates are frequently infected with TVV, the release of TVV via sEVs to the environment represents an important factor with the potential to enhance inflammation-related pathogenesis during trichomoniasis.

The hydrogenosome of Trichomonas vaginalis.

This review is dedicated to the 50th anniversary of the discovery of hydrogenosomes by Miklós Müller and Donald Lindmark, which we will celebrate the following year. It was a long journey from the first observation of enigmatic rows of granules in trichomonads at the end of the 19th century to their first biochemical characterization in 1973. The key experiments by Müller and Lindmark revealed that the isolated granules contain hydrogen-producing hydrogenase, similar to some anaerobic bacteria-a discovery that gave birth to the field of hydrogenosomes. It is also important to acknowledge the parallel work of the team of Apolena Cerkasovová, Jirí Cerkasov, and Jaroslav Kulda, who demonstrated that these granules, similar to mitochondria, produce ATP. However, the evolutionary origin of hydrogenosomes remained enigmatic until the turn of the millennium, when it was finally accepted that hydrogenosomes and mitochondria evolved from a common ancestor. After a historical introduction, the review provides an overview of hydrogenosome biogenesis, hydrogenosomal protein import, and the relationship between the peculiar structure of membrane translocases and its low inner membrane potential due to the lack of respiratory complexes. Next, it summarizes the current state of knowledge on energy metabolism, the oxygen defense system, and iron/sulfur cluster assembly.

Anaerobic derivates of mitochondria and peroxisomes in the free-living amoeba Pelomyxa schiedti revealed by single-cell genomics.

BACKGROUND: Mitochondria and peroxisomes are the two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform into anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, collectively called mitochondrion-related organelles (MROs), which vary in enzymatic capacity. Anaerobic peroxisomes were identified only recently, and their putatively most conserved function seems to be the metabolism of inositol. The group Archamoebae includes anaerobes bearing both anaerobic peroxisomes and MROs, specifically hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica, while the organelles within the third lineage represented by Pelomyxa remain uncharacterized. RESULTS: We generated high-quality genome and transcriptome drafts from Pelomyxa schiedti using single-cell omics. These data provided clear evidence for anaerobic derivates of mitochondria and peroxisomes in this species, and corresponding vesicles were tentatively identified in electron micrographs. In silico reconstructed MRO metabolism harbors respiratory complex II, electron-transferring flavoprotein, a partial TCA cycle running presumably in the reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, a glycine cleavage system, a sulfate activation pathway, and an expanded set of NIF enzymes for iron-sulfur cluster assembly. When expressed in the heterologous system of yeast, some of these candidates localized into mitochondria, supporting their involvement in the MRO metabolism. The putative functions of P. schiedti peroxisomes could be pyridoxal 5'-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Unexpectedly, out of 67 predicted peroxisomal enzymes, only four were also reported in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and D-lactate dehydrogenase. Localizations in yeast corroborated peroxisomal functions of the latter two. CONCLUSIONS: This study revealed the presence and partially annotated the function of anaerobic derivates of mitochondria and peroxisomes in P. schiedti using single-cell genomics, localizations in yeast heterologous systems, and transmission electron microscopy. The MRO metabolism resembles that of M. balamuthi and most likely reflects the state in the common ancestor of Archamoebae. The peroxisomal metabolism is strikingly richer in P. schiedti. The presence of myo-inositol 2-dehydrogenase in the predicted peroxisomal proteome corroborates the situation in other Archamoebae, but future experimental evidence is needed to verify additional functions of this organelle.

Identification of Endosymbiotic Virus in Small Extracellular Vesicles Derived from Trichomonas vaginalis.

Accumulated evidence suggests that the endosymbiotic Trichomonasvirus (TVV) may play a role in the pathogenesis and drug susceptibility of Trichomonas vaginalis. Several reports have shown that extracellular vesicles (EVs) released from TVV-positive (TVV+) trichomonads can modulate the immune response in human vaginal epithelial cells and animal models. These results prompted us to examine whether EVs released from TVV+ isolates contained TVV. We isolated small extracellular vesicles (sEVs) from six T. vaginalis isolates that were either TVV free (ATCC 50143), harbored a single (ATCC 30236, ATCC 30238, T1), two (ATCC PRA-98), or three TVV subspecies (ATCC 50148). The presence of TVV subspecies in the six isolates was observed using reverse transcription-polymerase chain reaction (RT-PCR). Transmission electron microscopy (TEM) confirmed the presence of cup-shaped sEVs with a size range from 30-150 nm. Trichomonas vaginalis tetraspanin (TvTSP1; TVAG_019180), the classical exosome marker, was identified in all the sEV preparations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that all the sEVs isolated from TVV+ isolates contain viral capsid proteins derived from the same TVV subspecies in that isolate as demonstrated by RT-PCR. To provide more comprehensive information on the TVV subspecies population in other T. vaginalis isolates, we investigated the distribution of TVV subspecies in twenty-four isolates by mining the New-Generation Sequencing (NGS) RNAseq datasets. Our results should be beneficial for future studies investigating the role of TVV on the pathogenicity of T. vaginalis and the possible transmission of virus subspecies among different isolates via sEVs.

Cytidine nucleoside analog is an effective antiviral drug against Trichomonasvirus.

BACKGROUND: Trichomonas vaginalis is the causative agent of a sexually transmitted disease in humans. The virulence of the parasite depends on multiple factors, including the presence of endosymbiotic dsRNA viruses. The presence of Trichomonasviruses (TVV) was associated with more severe genital symptoms, increased proinflammatory host reactions, and modulated parasite sensitivity to metronidazole. However, no efficient antiviral drugs are available against TVV to derive isogenic TVV-positive and TVV-negative cell lines that are essential for investigations of the TVV impact on T. vaginalis biology. METHODS: 7-Deaza-2'-C-methyladenosine (7d2CMA) and 2'-C-methylcytidine (2CMC) were used for TVV inhibitory assay. TVV replication was monitored using quantitative reverse transcription PCR (RT qPCR) and western blotting. Modeling of TVV1 RNA-dependent RNA polymerase (RdRp) was performed to visualize the inhibitor-RdRp interaction. Susceptibility to metronidazole was performed under aerobic and anaerobic conditions. RESULTS: We demonstrated that 2CMC but not 7d2CMA is a potent inhibitor of TVV replication. Molecular modeling suggested that the RdRp active site can accommodate 2CMC in the active triphosphate nucleotide form. The effect of 2CMC was shown on strains infected with a single and multiple TVV species. The optimal 2CMC concentration (10 µM) demonstrated strong selectivity for TVVs over trichomonad growth. The presence of TVV has no effect on T. vaginalis metronidazole susceptibility in derived isogenic cell lines. CONCLUSIONS: 2CMC acts against TVVs and represents a new inhibitor against Totiviridae viruses. Our isogenic clones are now available for further studies of various aspects of T. vaginalis biology related to TVV infection.

A cytosolic ferredoxin-independent hydrogenase possibly mediates hydrogen uptake in Trichomonas vaginalis.

Trichomonads, represented by the highly prevalent sexually transmitted human parasite Trichomonas vaginalis, are anaerobic eukaryotes with hydrogenosomes in the place of the standard mitochondria. Hydrogenosomes form indispensable FeS-clusters, synthesize ATP, and release molecular hydrogen as a waste product. Hydrogen formation is catalyzed by [FeFe] hydrogenase, the hallmark enzyme of all hydrogenosomes found in various eukaryotic anaerobes. Eukaryotic hydrogenases were originally thought to be exclusively localized within organelles, but today few eukaryotic anaerobes are known that possess hydrogenase in their cytosol. We identified a thus-far unknown hydrogenase in T. vaginalis cytosol that cannot use ferredoxin as a redox partner but can use cytochrome b5 as an electron acceptor. Trichomonads overexpressing the cytosolic hydrogenase, while maintaining the carbon flux through hydrogenosomes, show decreased excretion of hydrogen and increased excretion of methylated alcohols, suggesting that the cytosolic hydrogenase uses the hydrogen gas as a source of reducing power for the reactions occurring in the cytoplasm and thus accounts for the overall redox balance. This is the first evidence of hydrogen uptake in a eukaryote, although further work is needed to confirm it. Assembly of the catalytic center of [FeFe] hydrogenases (H-cluster) requires the activity of three dedicated maturases, and these proteins in T. vaginalis are exclusively localized in hydrogenosomes, where they participate in the maturation of organellar hydrogenases. Despite the different subcellular localization of cytosolic hydrogenase and maturases, the H-cluster is present in the cytosolic enzyme, suggesting the existence of an alternative mechanism of H-cluster assembly.

Proteomic Analysis of Trichomonas vaginalis Phagolysosome, Lysosomal Targeting, and Unconventional Secretion of Cysteine Peptidases.

The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted ß-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.

Anaerobic peroxisomes in Entamoeba histolytica metabolize myo-inositol.

Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90-100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0.044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.

The iron-sulfur scaffold protein HCF101 unveils the complexity of organellar evolution in SAR, Haptista and Cryptista.

BACKGROUND: Nbp35-like proteins (Nbp35, Cfd1, HCF101, Ind1, and AbpC) are P-loop NTPases that serve as components of iron-sulfur cluster (FeS) assembly machineries. In eukaryotes, Ind1 is present in mitochondria, and its function is associated with the assembly of FeS clusters in subunits of respiratory Complex I, Nbp35 and Cfd1 are the components of the cytosolic FeS assembly (CIA) pathway, and HCF101 is involved in FeS assembly of photosystem I in plastids of plants (chHCF101). The AbpC protein operates in Bacteria and Archaea. To date, the cellular distribution of these proteins is considered to be highly conserved with only a few exceptions. RESULTS: We searched for the genes of all members of the Nbp35-like protein family and analyzed their targeting sequences. Nbp35 and Cfd1 were predicted to reside in the cytoplasm with some exceptions of Nbp35 localization to the mitochondria; Ind1was found in the mitochondria, and HCF101 was predicted to reside in plastids (chHCF101) of all photosynthetically active eukaryotes. Surprisingly, we found a second HCF101 paralog in all members of Cryptista, Haptista, and SAR that was predicted to predominantly target mitochondria (mHCF101), whereas Ind1 appeared to be absent in these organisms. We also identified a few exceptions, as apicomplexans possess mHCF101 predicted to localize in the cytosol and Nbp35 in the mitochondria. Our predictions were experimentally confirmed in selected representatives of Apicomplexa (Toxoplasma gondii), Stramenopila (Phaeodactylum tricornutum, Thalassiosira pseudonana), and Ciliophora (Tetrahymena thermophila) by tagging proteins with a transgenic reporter. Phylogenetic analysis suggested that chHCF101 and mHCF101 evolved from a common ancestral HCF101 independently of the Nbp35/Cfd1 and Ind1 proteins. Interestingly, phylogenetic analysis supports rather a lateral gene transfer of ancestral HCF101 from bacteria than its acquisition being associated with either α-proteobacterial or cyanobacterial endosymbionts. CONCLUSION: Our searches for Nbp35-like proteins across eukaryotic lineages revealed that SAR, Haptista, and Cryptista possess mitochondrial HCF101. Because plastid localization of HCF101 was only known thus far, the discovery of its mitochondrial paralog explains confusion regarding the presence of HCF101 in organisms that possibly lost secondary plastids (e.g., ciliates, Cryptosporidium) or possess reduced nonphotosynthetic plastids (apicomplexans).

The Mastigamoeba balamuthi Genome and the Nature of the Free-Living Ancestor of Entamoeba.

The transition of free-living organisms to parasitic organisms is a mysterious process that occurs in all major eukaryotic lineages. Parasites display seemingly unique features associated with their pathogenicity; however, it is important to distinguish ancestral preconditions to parasitism from truly new parasite-specific functions. Here, we sequenced the genome and transcriptome of anaerobic free-living Mastigamoeba balamuthi and performed phylogenomic analysis of four related members of the Archamoebae, including Entamoeba histolytica, an important intestinal pathogen of humans. We aimed to trace gene histories throughout the adaptation of the aerobic ancestor of Archamoebae to anaerobiosis and throughout the transition from a free-living to a parasitic lifestyle. These events were associated with massive gene losses that, in parasitic lineages, resulted in a reduction in structural features, complete losses of some metabolic pathways, and a reduction in metabolic complexity. By reconstructing the features of the common ancestor of Archamoebae, we estimated preconditions for the evolution of parasitism in this lineage. The ancestor could apparently form chitinous cysts, possessed proteolytic enzyme machinery, compartmentalized the sulfate activation pathway in mitochondrion-related organelles, and possessed the components for anaerobic energy metabolism. After the split of Entamoebidae, this lineage gained genes encoding surface membrane proteins that are involved in host-parasite interactions. In contrast, gene gains identified in the M. balamuthi lineage were predominantly associated with polysaccharide catabolic processes. A phylogenetic analysis of acquired genes suggested an essential role of lateral gene transfer in parasite evolution (Entamoeba) and in adaptation to anaerobic aquatic sediments (Mastigamoeba).

Determinism and contingencies shaped the evolution of mitochondrial protein import.

Mitochondrial protein import requires outer membrane receptors that evolved independently in different lineages. Here we used quantitative proteomics and in vitro binding assays to investigate the substrate preferences of ATOM46 and ATOM69, the two mitochondrial import receptors of Trypanosoma brucei The results show that ATOM46 prefers presequence-containing, hydrophilic proteins that lack transmembrane domains (TMDs), whereas ATOM69 prefers presequence-lacking, hydrophobic substrates that have TMDs. Thus, the ATOM46/yeast Tom20 and the ATOM69/yeast Tom70 pairs have similar substrate preferences. However, ATOM46 mainly uses electrostatic, and Tom20 hydrophobic, interactions for substrate binding. In vivo replacement of T. brucei ATOM46 by yeast Tom20 did not restore import. However, replacement of ATOM69 by the recently discovered Tom36 receptor of Trichomonas hydrogenosomes, while not allowing for growth, restored import of a large subset of trypanosomal proteins that lack TMDs. Thus, even though ATOM69 and Tom36 share the same domain structure and topology, they have different substrate preferences. The study establishes complementation experiments, combined with quantitative proteomics, as a highly versatile and sensitive method to compare in vivo preferences of protein import receptors. Moreover, it illustrates the role determinism and contingencies played in the evolution of mitochondrial protein import receptors.

Anaerobic peroxisomes in Mastigamoeba balamuthi.

The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, hydrogenosome-bearing protist Mastigamoeba balamuthi We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs (MbPex3, MbPex11, and MbPex14) were visualized in numerous vesicles distinct from hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either ß-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named "anaerobic" peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.

Investigation of the Secretory Pathway in Trichomonas vaginalis Argues against a Moonlighting Function of Hydrogenosomal Enzymes.

The enzymes pyruvate ferredoxin oxidoreductase (PFO), malic enzyme (ME), and the α- and ß-subunits of succinyl-CoA synthetase (SCS) catalyze key steps of energy metabolism in Trichomonas vaginalis hydrogenosomes. These proteins have also been characterized as the adhesins AP120 (PFO), AP65 (ME), AP33, and AP51 (α- and ß-SCS), which are localized on the cell surface and mediate the T. vaginalis cytoadherence. However, the mechanisms that facilitate the targeting of these proteins to the cell surface via the secretory pathway and/or to hydrogenosomes are not known. Here we adapted an in vivo biotinylation system to perform highly sensitive tracing of protein trafficking in T. vaginalis. We showed that α- and ß-SCS are biotinylated in the cytosol and imported exclusively into the hydrogenosomes. Neither α- nor ß-SCS is biotinylated in the endoplasmic reticulum and delivered to the cell surface via the secretory pathway. In contrast, two surface proteins, tetratricopeptide domain-containing membrane-associated protein and tetraspanin family surface protein, as well as soluble-secreted ß-amylase-1 are biotinylated in the endoplasmic reticulum and delivered through the secretory pathway to their final destinations. Taken together, these results demonstrate that the α- and ß-SCS subunits are targeted only to the hydrogenosomes, which argues against their putative moonlighting function.