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...