Molecular analysis of the hydrogenosomal ferredoxin of the anaerobic protist Trichomonas vaginalis.

Abstract

We have determined the primary structure of The presence of mitochondria is regarded as a universal hallmark ofeukaryotic cells. Certain protists are an exception to this rule. Although some have no membrane-bounded organelles of energy metabolism, others contain hydrogenosomes, unusual organelles surrounded by two membranes (1, 2). These organelles are the site of oxidative decarboxylation of pyruvate with the formation of acetyl-CoA. This process is mediated by pyruvate:ferredoxin oxidoreductase and is coupled to anaerobic formation of H2 catalyzed by hydrogenase. The electron carrier linking these reactions is an iron-sulfur protein, ferredoxin. In subsequent reactions the energy of the thioester bond of acetyl-CoA is conserved by substrate-level phosphorylation of ADP. This pathway and its enzymes render the hydrogenosomes markedly different from mitochondria (1, 2). Hydrogenosomes were originally described from trichomonad flagellates, among them Trichomonas vaginalis, a parasite of humans (1, 2). Subsequently they have been detected in a number of free-living anaerobic ciliates (3, 4) and in ciliates and a fungus of the rumen (2). The biological nature and origin of hydrogenosomes remain enigmatic but an endosymbiotic origin is favored. The main difference in opinion centers on whether hydrogenosomes arose by conversion from mitochondria (4, 5) or through independent endosymbiosis involving anaerobic bacteria (1, 2, 6).A MATERIALS AND METHODS Organism. T. vaginalis strain C1 (ATCC 30001) was cultured and harvested as described earlier (11).Polyclonal Antiserum and Protein Sequencing. T. vaginalis ferredoxin was purified as described (11). Polyclonal antibodies reacting with this protein were developed in New Zealand White rabbits. The sera were tested with enzymelinked immunoassay using either the purified protein or whole cell extracts on Western blots.For amino acid sequencing, apoferredoxin was prepared by precipitation ofthe native protein with trichloroacetic acid (11). The apoprotein was fragmented with CNBr and, after propylation (14), by treatment with trypsin. The peptides were separated by HPLC. The intact protein and the peptides were carboxymethylated before sequencing. Gas-phase sequencing was performed on the whole protein, yielding results through residue 58, on a CNBr fragment, yielding results for residues 33-85, and a tryptic fragment, giving results for residues 76-93.Recombinant DNA Methods. A T. vaginalis cDNA library was prepared in Agtll by means of standard techniques (15 6097The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Identification and molecular cloning of four cysteine proteinase genes from the pathogenic protozoon Trichomonas vaginalis

“…The only other proteins of T. vaginalis whose targeting has been studied are ferredoxin and p-succinyl-coenzyme A synthetase. Both are nuclear-encoded and transported into hydrogenosomes, membrane-bound organelles involved in energy metabolism (Muller, 1993), and both have unusually short Nterminal pre-sequences when compared with those known to direct proteins to mitochondria and chloroplasts in other organisms (Johnson et al, 1990; Lahti e t al., 1992). , 1993), with the individual genes being identical or very closely related.…”

Transient and selectable transformation of the parasitic protist Trichomonas vaginalis

“…High molecular weight DNA was extracted from 100 ml cultures of transformed clones grown in the presence of 50 g͞ml paromomycin using DNAzol (GIBCO͞BRL). Southern blots were prepared and hybridized with a 32 P-labeled neo probe as previously described (17). To determine the copy number of the NEO construct in selectable transformants, the amount of the neo gene (800 bp) that would represent one copy per T. vaginalis genome was calculated using the genome size of 2.5 ϫ 10 7 bp (18).…”

Presence of a Member of the Mitochondrial Carrier Family in Hydrogenosomes: Conservation of Membrane-Targeting Pathways between Hydrogenosomes and Mitochondria

“…We have also investigated the effect of fusing a hydrogenosomal presequence from a matrix protein, ferredoxin, Fd (4,18,25), to the presequence-minus Hmp31 moiety by creating a transformant expressing Fd L ⌬Hmp31-(HA) 2 . Interestingly, this fusion protein was exclusively targeted to the organellar fraction, with no detectable trace in the soluble cellular fraction (Fig.…”