Organelles in Blastocystis that blur the distinction between mitochondria and hydrogenosomes

Blastocystis is a unicellular stramenopile of controversial pathogenicity in humans. Although it is a strict anaerobe, Blastocystis has mitochondrion-like organelles with cristae, a transmembrane potential and DNA. An apparent lack of several typical mitochondrial pathways has led some to suggest that these organelles might be hydrogenosomes, anaerobic organelles related to mitochondria. We generated 12,767 expressed sequence tags (ESTs) from Blastocystis and identified 115 clusters that encode putative mitochondrial and hydrogenosomal proteins. Among these is the canonical hydrogenosomal protein iron-only [FeFe] hydrogenase that we show localizes to the organelles. The organelles also have mitochondrial characteristics, including pathways for amino acid metabolism, iron-sulfur cluster biogenesis, and an incomplete tricarboxylic acid cycle as well as a mitochondrial genome. Although complexes I and II of the electron transport chain (ETC) are present, we found no evidence for complexes III and IV or F1Fo ATPases. The Blastocystis organelles have metabolic properties of aerobic and anaerobic mitochondria and of hydrogenosomes. They are convergently similar to organelles recently described in the unrelated ciliate Nyctotherus ovalis. These findings blur the boundaries between mitochondria, hydrogenosomes, and mitosomes, as currently defined, underscoring the disparate selective forces that shape these organelles in eukaryotes.     

Targeting and translocation of proteins into the hydrogenosome of the protist Trichomonas: similarities with mitochondrial protein import.

Trichomonads are early-diverging eukaryotes that lack both mitochondria and peroxisomes. They do contain a double membrane-bound organelle, called the hydrogenosome, that metabolizes pyruvate and produces ATP. To address the origin and biological nature of hydrogenosomes, we have established an in vitro protein import assay. Using purified hydrogenosomes and radiolabeled hydrogenosomal precursor ferredoxin (pFd), we demonstrate that protein import requires intact organelles, ATP and N-ethylmaleimide-sensitive cytosolic factors. Protein import is also affected by high concentrations of the protonophore, m-chlorophenylhydrazone (CCCP). Binding and translocation of pFd into hydrogenosomes requires the presence of an eight amino acid N-terminal presequence that is similar to presequences found on all examined hydrogenosomal proteins. Upon import, pFd is processed to a size consistent with cleavage of the presequence. Mutation of a conserved leucine at position 2 in the presequence to a glycine disrupts import of pFd into the organelle. Interestingly, a comparison of hydrogenosomal and mitochondrial protein presequences reveals striking similarities. These data indicate that mechanisms underlying protein targeting and biogenesis of hydrogenosomes and mitochondria are similar, consistent with the notion that these two organelles arose from a common endosymbiont.     

The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria

Peroxisomes and mitochondria are multifunctional eukaryotic organelles that are not only interconnected metabolically but also share proteins in division. Two evolutionarily conserved division factors, dynamin-related protein (DRP) and its organelle anchor FISSION1 (FIS1), mediate the fission of both peroxisomes and mitochondria. Here, we identified and characterized a plant-specific protein shared by these two types of organelles. The Arabidopsis thaliana PEROXISOMAL and MITOCHONDRIAL DIVISION FACTOR1 (PMD1) is a coiled-coil protein tethered to the membranes of peroxisomes and mitochondria by its C terminus. Null mutants of PMD1 contain enlarged peroxisomes and elongated mitochondria, and plants overexpressing PMD1 have an increased number of these organelles that are smaller in size and often aggregated. PMD1 lacks physical interaction with the known division proteins DRP3 and FIS1; it is also not required for DRP3's organelle targeting. Affinity purifications pulled down PMD1's homolog, PMD2, which exclusively targets to mitochondria and plays a specific role in mitochondrial morphogenesis. PMD1 and PMD2 can form homo- and heterocomplexes. Organelle targeting signals reside in the C termini of these proteins. Our results suggest that PMD1 facilitates peroxisomal and mitochondrial proliferation in a FIS1/DRP3-independent manner and that the homologous proteins PMD1 and PMD2 perform nonredundant functions in organelle morphogenesis.     

Diversity and reductive evolution of mitochondria among microbial eukaryotes

All extant eukaryotes are now considered to possess mitochondria in one form or another. Many parasites or anaerobic protists have highly reduced versions of mitochondria, which have generally lost their genome and the capacity to generate ATP through oxidative phosphorylation. These organelles have been called hydrogenosomes, when they make hydrogen, or remnant mitochondria or mitosomes when their functions were cryptic. More recently, organelles with features blurring the distinction between mitochondria, hydrogenosomes and mitosomes have been identified. These organelles have retained a mitochondrial genome and include the mitochondrial-like organelle of Blastocystis and the hydrogenosome of the anaerobic ciliate Nyctotherus. Studying eukaryotic diversity from the perspective of their mitochondrial variants has yielded important insights into eukaryote molecular cell biology and evolution. These investigations are contributing to understanding the essential functions of mitochondria, defined in the broadest sense, and the limits to which reductive evolution can proceed while maintaining a viable organelle.     

Novel mitochondrion-related organelles in the anaerobic amoeba Mastigamoeba balamuthi

Unicellular eukaryotes that lack mitochondria typically contain related organelles such as hydrogenosomes or mitosomes. To characterize the evolutionary diversity of these organelles, we conducted an expressed sequence tag (EST) survey on the free-living amoeba Mastigamoeba balamuthi, a relative of the human parasite Entamoeba histolytica. From 19 182 ESTs, we identified 21 putative mitochondrial proteins implicated in protein import, amino acid interconversion and carbohydrate metabolism, two components of the iron-sulphur cluster (Fe-S) assembly apparatus as well as two enzymes characteristic of hydrogenosomes. By immunofluorescence microscopy and subcellular fractionation, we show that mitochondrial chaperonin 60 is targeted to small abundant organelles within Mastigamoeba. In transmission electron micrographs, we identified double-membraned compartments that likely correspond to these mitochondrion-derived organelles, The predicted organellar proteome of the Mastigamoeba organelle indicates a unique spectrum of functions that collectively have never been observed in mitochondrion-related organelles. However, like Entamoeba, the Fe-S cluster assembly proteins in Mastigamoeba were acquired by lateral gene transfer from epsilon-proteobacteria and do not possess obvious organellar targeting peptides. These data indicate that the loss of classical aerobic mitochondrial functions and acquisition of anaerobic enzymes and Fe-S cluster assembly proteins occurred in a free-living member of the eukaryote super-kingdom Amoebozoa.     

Presence of a latent mitochondrial targeting signal in gene on mitochondrial genome

Organelles, such as mitochondria and chloroplasts, are derived from endosymbionts. Gene transfer events from organelles to the nucleus have occurred over evolutionary time. In the case that a transferred gene in the nucleus needs to go back to the original organelle, it must obtain targeting information for sorting its protein to that organelle. Here, we reveal that the genes for the ribosomal proteins L2 and S4 in the Arabidopsis thaliana mitochondrial (mt) genome contain information for protein targeting into the mitochondria. Similarly, the genes for the ribosomal proteins L2 and S19 in the Oryza sativa mt genome contain information for protein targeting into mitochondria. These results suggest that targeting information already existed in each gene in the plant mt genome before the transfer event to the nucleus occurred. We provide new insights into the timing of the appearance of targeting signals in evolution.     

Genetic evidence for a mitochondriate ancestry in the 'amitochondriate' flagellate Trimastix pyriformis

Most modern eukaryotes diverged from a common ancestor that contained the alpha-proteobacterial endosymbiont that gave rise to mitochondria. The 'amitochondriate' anaerobic protist parasites that have been studied to date, such as Giardia and Trichomonas harbor mitochondrion-related organelles, such as mitosomes or hydrogenosomes. Yet there is one remaining group of mitochondrion-lacking flagellates known as the Preaxostyla that could represent a primitive 'pre-mitochondrial' lineage of eukaryotes. To test this hypothesis, we conducted an expressed sequence tag (EST) survey on the preaxostylid flagellate Trimastix pyriformis, a poorly-studied free-living anaerobe. Among the ESTs we detected 19 proteins that, in other eukaryotes, typically function in mitochondria, hydrogenosomes or mitosomes, 12 of which are found exclusively within these organelles. Interestingly, one of the proteins, aconitase, functions in the tricarboxylic acid cycle typical of aerobic mitochondria, whereas others, such as pyruvate:ferredoxin oxidoreductase and [FeFe] hydrogenase, are characteristic of anaerobic hydrogenosomes. Since Trimastix retains genetic evidence of a mitochondriate ancestry, we can now say definitively that all known living eukaryote lineages descend from a common ancestor that had mitochondria.     

Trypanosome Alternative Oxidase Possesses both an N-Terminal and Internal Mitochondrial Targeting Signal

Recognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form of Trypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondria in vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to a T. brucei mitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.     

Mitochondrial involvement in cell death of non-mammalian eukaryotes

Although mitochondria are essential organelles for long-term survival of eukaryotic cells, recent discoveries in biochemistry and genetics have advanced our understanding of the requirements for mitochondria in cell death. Much of what we understand about cell death is based on the identification of conserved cell death genes in Drosophila melanogaster and Caenorhabditis elegans. However, the role of mitochondria in cell death in these models has been much less clear. Considering the active role that mitochondria play in apoptosis in mammalian cells, the mitochondrial contribution to cell death in non-mammalian systems has been an area of active investigation. In this article, we review the current research on this topic in three non-mammalian models, C. elegans, Drosophila, and Saccharomyces cerevisiae. In addition, we discuss how non-mammalian models have provided important insight into the mechanisms of human disease as they relate to the mitochondrial pathway of cell death. The unique perspective derived from each of these model systems provides a more complete understanding of mitochondria in programmed cell death. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Defining the Determinants for Dual Targeting of Amino Acyl-tRNA Synthetases to Mitochondria and Chloroplasts

Most of the organellar amino acyl-tRNA synthetases (aaRSs) are dually targeted to both mitochondria and chloroplasts using dual targeting peptides (dTPs). We have investigated the targeting properties and domain structure of dTPs of seven aaRSs by studying the in vitro and in vivo import of N-terminal deleted constructs of dTPs fused to green fluorescent protein. The deletion constructs were designed based on prediction programs, TargetP and Predotar, as well as LogoPlots derived from organellar proteomes in Arabidopsis thaliana. In vitro import was performed either into a single isolated organelle or as dual import (i.e., into a mixture of isolated mitochondria and chloroplasts followed by reisolation of the organelles). In vivo import was investigated as transient expression of the green fluorescent protein constructs in Nicotiana benthamiana protoplasts. Characterization of recognition determinants showed that the N-terminal portions of TyrRS-, ValRS- and ThrRS-dTPs (27, 22 and 23 amino acids, respectively) are required for targeting into both mitochondria and chloroplasts. Surprisingly, these N-terminal portions contain no or very few arginines (or lysines) but very high number of hydroxylated residues (26-51%). For two aaRSs, a domain structure of the dTP became evident. Removal of 20 residues from the dTP of ProRS abolished chloroplastic import, indicating that the N-terminal region was required for chloroplast targeting, whereas deletion of 16 N-terminal amino acids from AspRS-dTP inhibited the mitochondrial import, showing that in this case, the N-terminal portion was required for the mitochondrial import. Finally, deletion of N-terminal regions of dTPs for IleRS and LysRS did not affect dual targeting. In summary, it can be concluded that there is no general rule for how the determinants for dual targeting are distributed within dTPs; in most cases, the N-terminal portion is essential for import into both organelles, but in a few cases, a domain structure was observed.     

Presence of a member of the mitochondrial carrier family in hydrogenosomes: conservation of membrane-targeting pathways between hydrogenosomes and mitochondria

A number of microaerophilic eukaryotes lack mitochondria but possess another organelle involved in energy metabolism, the hydrogenosome. Limited phylogenetic analyses of nuclear genes support a common origin for these two organelles. We have identified a protein of the mitochondrial carrier family in the hydrogenosome of Trichomonas vaginalis and have shown that this protein, Hmp31, is phylogenetically related to the mitochondrial ADP-ATP carrier (AAC). We demonstrate that the hydrogenosomal AAC can be targeted to the inner membrane of mitochondria isolated from Saccharomyces cerevisiae through the Tim9-Tim10 import pathway used for the assembly of mitochondrial carrier proteins. Conversely, yeast mitochondrial AAC can be targeted into the membranes of hydrogenosomes. The hydrogenosomal AAC contains a cleavable, N-terminal presequence; however, this sequence is not necessary for targeting the protein to the organelle. These data indicate that the membrane-targeting signal(s) for hydrogenosomal AAC is internal, similar to that found for mitochondrial carrier proteins. Our findings indicate that the membrane carriers and membrane protein-targeting machinery of hydrogenosomes and mitochondria have a common evolutionary origin. Together, they provide strong evidence that a single endosymbiont evolved into a progenitor organelle in early eukaryotic cells that ultimately give rise to these two distinct organelles and support the hydrogen hypothesis for the origin of the eukaryotic cell.     

Hydrogenosomes, mitochondria and early eukaryotic evolution

Available data suggest that unusual organelles called hydrogenosomes, that make ATP and hydrogen, and which are found in diverse anaerobic eukaryotes, were once mitochondria. The evolutionary origins of the enzymes used to make hydrogen, pyruvate:ferredoxin oxidoreductase (PFO) and hydrogenase, are unresolved, but it seems likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobes, these proteins are found in diverse eukaryotic cells, including our own, where they are targeted to different cell compartments. Organelles related to mitochondria and hydrogenosomes have now been found in species of anaerobic and parasitic protozoa that were previously thought to have separated from other eukaryotes before the mitochondrial endosymbiosis. Thus it is possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, bearing testimony to the important role that the mitochondrial endosymbiosis has played in eukaryotic evolution. It remains to be seen if members of this family of organelles share a common function essential to the eukaryotic cell, that provides the underlying selection pressure for organelle retention under different living conditions.     

Dual targeting to mitochondria and chloroplasts.

Plant cells contain two organelles originally derived from endosymbiotic bacteria: mitochondria and plastids. Their endosymbiotic origin explains why these organelles contain their own DNA, nonetheless only a few dozens of genes are actually encoded by these genomes. Many of the other genes originally present have been transferred to the nuclear genome of the host, the product of their expression being targeted back to the corresponding organelle. Although targeting of proteins to mitochondria and chloroplasts is generally highly specific, an increasing number of examples have been discovered where the same protein is imported into both organelles. The object of this review is to compare and discuss these examples in order to try and identify common features of dual-targeted proteins. The study helps throw some light on the factors determining organelle targeting specificity, and suggests that dual-targeted proteins may well be far more common than once thought.