Protein Import into Hydrogenosomes ofTrichomonas vaginalisInvolves both N-Terminal and Internal Targeting Signals: a Case Study of Thioredoxin Reductases

Abstract

The parabasalian flagellate Trichomonas vaginalis harbors mitochondrion-related and H 2 -producing organelles of anaerobic ATP synthesis, called hydrogenosomes, which harbor oxygen-sensitive enzymes essential to its pyruvate metabolism. In the human urogenital tract, however, T. vaginalis is regularly exposed to low oxygen concentrations and therefore must possess antioxidant systems protecting the organellar environment against the detrimental effects of molecular oxygen and reactive oxygen species. We have identified two closely related hydrogenosomal thioredoxin reductases (TrxRs), the hitherto-missing component of a thioredoxinlinked hydrogenosomal antioxidant system. One of the two hydrogenosomal TrxR isoforms, TrxRh1, carried an N-terminal extension resembling known hydrogenosomal targeting signals. Expression of hemagglutinintagged TrxRh1 in transfected T. vaginalis cells revealed that its N-terminal extension was necessary to import the protein into the organelles. The second hydrogenosomal TrxR isoform, TrxRh2, had no N-terminal targeting signal but was nonetheless efficiently targeted to hydrogenosomes. N-terminal presequences from hydrogenosomal proteins with known processing sites, i.e., the alpha subunit of succinyl coenzyme A synthetase (SCS␣) and pyruvate:ferredoxin oxidoreductase A, were investigated for their ability to direct mature TrxRh1 to hydrogenosomes. Neither presequence directed TrxRh1 to hydrogenosomes, indicating that neither extension is, by itself, sufficient for hydrogenosomal targeting. Moreover, SCS␣ lacking its N-terminal extension was efficiently imported into hydrogenosomes, indicating that this extension is not required for import of this major hydrogenosomal protein. The finding that some hydrogenosomal enzymes require N-terminal signals for import but that in others the N-terminal extension is not necessary for targeting indicates the presence of additional targeting signals within the mature subunits of several hydrogenosome-localized proteins.The parabasalian flagellate Trichomonas vaginalis is the causative agent of the most prevalent nonviral sexually transmitted disease in humans (34) and possesses hydrogenosomes, which are anaerobic forms of mitochondria (30, 34). Hydrogenosomes, like the mitochondria of aerobic eukaryotes, are involved in energy metabolism, but ATP is synthesized through substrate-level phosphorylation instead of oxidative phosphorylation (35). Although this fermentative energy metabolism in T. vaginalis hydrogenosomes relies on the highly oxygen-sensitive enzymes pyruvate:ferredoxin oxidoreductase (PFO) (53) and [Fe]-hydrogenase (39), T. vaginalis is frequently exposed to oxygen concentrations of up to 60 M in its natural habitat on the vaginal surface (52). Furthermore, exposure to low oxygen concentrations on the order of 0.25 M yields its maximum growth rates (40), owing to the ability of the organism to more rapidly establish a redox balance with NAD(P)H-oxidases. These enzymes have high activities and rapidly reduce O 2 to water, thereby afford...

A Machine Learning Approach To Identify Hydrogenosomal Proteins in Trichomonas vaginalis

“…Many hydrogenosomal proteins are equipped with a short N-terminal hydrogenosomal targeting signal (HTS), which directs the preprotein to the organelle (11). Recently, though, the first proteins were identified that are apparently imported based on internal targeting signals (53). This could help to explain the discrepancy between the number of proteins estimated to be present in the hydrogenosome (about 500) (73) and those that harbor an HTS (about 220) (11).…”

“…The current estimate is significantly lower than the ϳ500 proteins expected to be found in the hydrogenosome (73). This is compounded by the finding that some HTS-lacking proteins are imported into hydrogenosomes, the alpha subunit of succinyl-coenzyme A (CoA) synthetase (TVAG_165340), and a thioredoxin reductase isoform (TVAG_125360) (53). Thus, protein properties in addition to an HTS are likely to serve as potential targeting precursors to the hydrogenosomes.…”

“…The hydrogenosomal localization of only 30 proteins has been verified experimentally (11,53,63,64,79). The current estimate is significantly lower than the ϳ500 proteins expected to be found in the hydrogenosome (73).…”

“…If the situation in Trichomonas mirrors that in yeast, hydrogenosomal membrane proteins are most likely targeted and integrated into the membrane by a process different from that of the matrix proteins (42,45,81). The latter are either recognized by their N-terminal motifs or by an alternative internal signal that replaced the N-terminal motif (53), whereas the membrane proteins in yeast insert auton- omously via a mechanism involving the Sam50 complex (42,45,81); in the hydrogenosomal membrane the mechanism could be similar. In either case, this will affect the prediction algorithm through the quality of the feature selection.…”

“…4). Intriguingly, the only HTS to our knowledge essential for import is that of a hydrogenosomal thioredoxin reductase (TrxRh1, TVAG_ 281360) (53), and that is found only once in the genome-on the TrxRh1 protein itself.…”