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.

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.

Pheromone 3 mRNA of the ciliate Euplotes octocarihatus contains three in-frame UGA codons that are translated as cysteines. This was revealed from cDNA sequencing and from plasma desorption mass spectrometry of cleaved pheromone 3 in connection with pyridylethylation of the fragments. N-terminal sequence analysis of carboxymethylated protein confirmed this conclusion for the first of the three UGA codons. Besides UGA the common cysteine codons UGU and UGC are also used to encode cysteine. UAA functions as a termination codon. Preparation of RNA. Total RNA was prepared by disruption of 1-3 x 107 cells in 8 M urea/4 M LiCl in a PotterElvehjem homogenizer, followed by precipitation on ice overnight. RNA was collected by centrifugation, dissolved in 10 mM Mops, pH 7.5/0.5% SDS, and extracted three times with phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1). Total RNA was then precipitated by addition of 0.1 volume of 4 M LiCl and 2.5 volumes of absolute ethanol.Poly(A)+ RNA was prepared by affinity chromatography on oligo(dT)-cellulose (Bethesda Research Laboratories) as recommended by the supplier with the exception that Mops was used as the buffer instead of Tris. Poly(A)+ RNA was precipitated by the addition of LiCI and ethanol as described above and redissolved in water. Quantity and quality were determined spectrophotometrically by measuring absorption at 260 and 280 nm (14).cDNA Synthesis and Cloning. The cDNA library was constructed (15) in the vector AgtlO. The cDNA was treated with S1 nuclease and ligated with EcoRI linkers prior to its insertion into the EcoRI site of the vector. The pheromone 3 gene was identified by plaque hybridization with the synthetic oligodeoxynucleotide 5'-GTRTANGGYTCYTCCCA-3', corresponding to the N terminus of the secreted pheromone, and was isolated by standard techniques (14).DNA Sequencing. Eight positively hybridizing plaques were obtained from 105 transformants. Five of them were further subcloned for sequencing by the dideoxy chain-termination method. Their nucleotide sequences were determined from double-stranded and single-stranded templates (pUC12, pT7T3, M13mpl8, and M13mp19 as sequencing vectors) according to the sequencing strategy outlined in Fig. 1 1To whom reprint requests should be addressed.

An abundant integral membrane protein, Hmp35, has been isolated from hydrogenosomes of Trichomonas vaginalis. This protein has no known homologue and exists as a stable 300-kDa complex, termed HMP35, in membranes of the hydrogenosome. By using blue native gel electrophoresis, we found the HMP35 complex to be stable in 2 M NaCl and up to 5 M urea. The endogenous Hmp35 protein was largely protease-resistant. The protein has a predominantly ␤-sheet structure and predicted transmembrane domains that may form a pore. Interestingly, the protein has a high number of cysteine residues, some of which are arranged in motifs that resemble the RING finger, suggesting that they could be coordinating zinc or another divalent cation. Our data show that Hmp35 forms one intramolecular but no intermolecular disulfide bonds. We have isolated the HMP35 complex by expressing a His-tagged Hmp35 protein in vivo followed by purification with nickel-agarose beads. The purified 300-kDa complex consists of mostly Hmp35 with lesser amounts of 12-, 25-27-, and 32-kDa proteins. The stoichiometry of proteins in the complex indicates that Hmp35 exists as an oligomer. Hmp35 can be targeted heterologously into yeast mitochondria, despite the lack of homology with any yeast protein, demonstrating the compatibility of mitochondrial and hydrogenosomal protein translocation machineries.Trichomonas vaginalis is a deep-branching protist that lacks archetypal eukaryotic "aerobic" organelles, specifically mitochondria and peroxisomes. This microaerophilic human-infective parasite carries out fermentative carbohydrate metabolism within hydrogenosomes. Hydrogenosomes are bounded by double membranes and produce ATP by substrate level phosphorylation (1). Hydrogenosomes are also found in certain chytrids, ciliates, and fungi, in lineages that are phylogenetically distant to the Parabasalian lineage to which trichomonads belong (1-4). Currently, several lines of evidence support a common endosymbiotic ancestry for hydrogenosomes and mitochondria, despite their distinct metabolic pathways (3,5,6). Although the origin of hydrogenosomes within ciliate and fungi lineages is debated, these lineages branch with mitochondria-containing groups and the hydrogenosomes confined therein exhibit strong similarity to ciliate and fungal mitochondria (7,8).Trichomonad hydrogenosomes, on the other hand, are markedly less similar to mitochondria. These organelles lack a genome (9) which would have provided a means to investigate their endosymbiotic origin as has been elegantly and convincingly done for mitochondria (10). In lieu of this, we and others have attempted to define the relationship between trichomonad hydrogenosomes and mitochondria by examining the origin of their chaperonins, metabolic enzymes, and membrane proteins. Chaperonin genes, specifically heat shock protein (Hsp) 1 70, cpn60,, and the IscS enzyme, involved in FeS cluster formation (15) appear to have a mitochondrial origin. However, analyses of metabolic enzymes such as hydrogenase (16,17), which is typi...

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