Fe-hydrogenase maturases in the hydrogenosomes of Trichomonas vaginalis

Abstract

Assembly of active Fe-hydrogenase in the chloroplasts of the green alga Chlamydomonas reinhardtii requires auxiliary maturases, the S-adenosylmethionine-dependent enzymes HydG and HydE and the GTPase HydF. Genes encoding homologous maturases had been found in the genomes of all eubacteria that contain Fe-hydrogenase genes but not yet in any other eukaryote. By means of proteomic analysis, we identified a homologue of HydG in the hydrogenosomes, mitochondrion-related organelles that produce hydrogen under anaerobiosis by the activity of Fe-hydrogenase, in the pathogenic protist Trichomonas vaginalis. Genes encoding two other components of the Hyd system, HydE and HydF, were found in the T. vaginalis genome database. Overexpression of HydG, HydE, and HydF in trichomonads showed that all three proteins are specifically targeted to the hydrogenosomes, the site of Fe-hydrogenase maturation. The results of Neighbor-Net analyses of sequence similarities are consistent with a common eubacterial ancestor of HydG, HydE, and HydF in T. vaginalis and C. reinhardtii, supporting a monophyletic origin of Fe-hydrogenase maturases in the two eukaryotes. Although Fe-hydrogenases exist in only a few eukaryotes, related Narf proteins with different cellular functions are widely distributed. Thus, we propose that the acquisition of Fe-hydrogenases, together with Hyd maturases, occurred once in eukaryotic evolution, followed by the appearance of Narf through gene duplication of the Fe-hydrogenase gene and subsequent loss of the Hyd proteins in eukaryotes in which Fe-hydrogenase function was lost.

FIG. 1.

Alignment of HydG sequences from T. vaginalis (Triva), C. reinhardtii (Chlre), Clostridium thermocellum (Cloth), and Bacteroides thetaiotaomicron (Bacth). A putative hydrogenosomal targeting sequence is shown in bold, and peptide sequences from hydrogenosomal protein spot S29 are shaded in gray. ▿, cysteine residues of the CXXXCXXC motif conserved in SAM-dependent proteins (30); *, conserved sites.

FIG. 2.

Detection of Hyd gene transcripts by RT-PCR. 1, HydG (88811.m00081); 2, HydF (87122.m00057); 3, HydE (81202.m00098).

FIG. 3.

Intracellular localization of Hyd proteins in T. vaginalis. (A) Comparison of the N-terminal targeting sequences of the hydrogenosomal proteins Hsp60 and α-SCS with the N-terminal sequences of Hyd proteins. ▾, protease processing site; ▿, putative protease processing site. The typical arginine residue (R) at position −2 of the protease processing site of hydrogenosomal targeting sequences is shown in red. (B) Localization of HA-tagged HydE, HydF, and HydG overexpressed in T. vaginalis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of HydF-overexpressing cells (top) and Western blots of cellular fractions from cells expressing tagged HydE, HydF, or HydG (bottom) are shown. ME was used as a hydrogenosomal marker protein. L, cell lysate; C, cytosolic fraction; H, hydrogenosomal fraction. (C) Immunolocalization of HydG in transfected cells shows colocalization of HA-tagged HydG with hydrogenosomal ME. DIC, differential interference contrast image; DAPI, 4′,6′-diamidino-2-phenylindole-stained image.

FIG. 4.

Neighbor-Net analysis of sequence similarities of HydG and HydE and related proteins from prokaryotes. HydG sequences from T. vaginalis and C. reinhardtii form a monophyletic cluster within a subset of prokaryotic ThiH sequences. HydE sequences from both eukaryotes appear on a common branch among prokaryotic biotin synthases.

FIG. 5.

Neighbor-Net analysis of HydF and related GTPases. Sequences from T. vaginalis and C. reinhardtii appear in the same subset of GTPases but do not form a monophyletic group.