An internal sequence targets Trypanosoma brucei triosephosphate isomerase to glycosomes.

In kinetoplastid protists, glycolysis is compartmentalized in glycosomes, organelles belonging to the peroxisome family. The Trypanosoma brucei glycosomal enzyme triosephosphate isomerase (TPI) does not contain either of the two established peroxisome-targeting signals, but we identified a 22 amino acids long fragment, present at an internal position of the polypeptide, that has the capacity to route a reporter protein to glycosomes in transfected trypanosomes, as demonstrated by cell-fractionation experiments and corroborating immunofluorescence studies. This polypeptide-internal routing information seems to be unique for the sequence of the trypanosome enzyme: a reporter protein fused to a Saccharomyces cerevisiae peptide containing the sequence corresponding to the 22-residue fragment of the T. brucei enzyme, was not targeted to glycosomes. In yeasts, as in most other organisms, TPI is indeed exclusively present in the cytosol. These results suggest that it may be possible to develop new trypanocidal drugs by targeting specifically the glycosome import mechanism of TPI.

Characterization of Trypanosoma brucei PEX14 and its role in the import of glycosomal matrix proteins.

It has been shown previously in various organisms that the peroxin PEX14 is a component of a docking complex at the peroxisomal membrane, where it is involved in the import of matrix proteins into the organelle after their synthesis in the cytosol and recognition by a receptor. Here we present a characterization of the Trypanosoma brucei homologue of PEX14. It is shown that the protein is associated with glycosomes, the peroxisome-like organelles of trypanosomatids in which most glycolytic enzymes are compartmentalized. The N-terminal part of the protein binds specifically to TbPEX5, the cytosolic receptor for glycosomal matrix proteins with a peroxisome-targeting signal type 1 (PTS-1). TbPEX14 mRNA depletion by RNA interference results, in both bloodstream-form and procyclic, insect-stage T. brucei, in mislocalization of glycosomal proteins to the cytosol. The mislocalization was observed for different classes of matrix proteins: proteins with a C-terminal PTS-1, a N-terminal PTS-2 and a polypeptide internal I-PTS. The RNA interference experiments also showed that TbPEX14 is essential for the survival of bloodstream-form and procyclic trypanosomes. These data indicate the protein's great potential as a target for selective trypanocidal drugs.

Functional integration of mitochondrial and hydrogenosomal ADP/ATP carriers in the Escherichia coli membrane reveals different biochemical characteristics for plants, mammals and anaerobic chytrids.

The expression of mitochondrial and hydrogenosomal ADP/ATP carriers (AACs) from plants, rat and the anaerobic chytridiomycete fungus Neocallimastix spec. L2 in Escherichia coli allows a functional integration of the recombinant proteins into the bacterial cytoplasmic membrane. For AAC1 and AAC2 from rat, apparent Km values of about 40 microm for ADP, and 105 microm or 140 microm, respectively, for ATP have been determined, similar to the data reported for isolated rat mitochondria. The apparent Km for ATP decreased up to 10-fold in the presence of the protonophore m-chlorocarbonylcyanide phenylhydrazone (CCCP). The hydrogenosomal AAC isolated from the chytrid fungus Neocallimastix spec. L2 exhibited the same characteristics, but the affinities for ADP (165 microm) and ATP (2.33 mm) were significantly lower. Notably, AAC1-3 from Arabidopsis thaliana and AAC1 from Solanum tuberosum (potato) showed significantly higher external affinities for both nucleotides (10-22 microm); they were only slightly influenced by CCCP. Studies on intact plant mitochondria confirmed these observations. Back exchange experiments with preloaded E. coli cells expressing AACs indicate a preferential export of ATP for all AACs tested. This is the first report of a functional integration of proteins belonging to the mitochondrial carrier family (MCF) into a bacterial cytoplasmic membrane. The technique described here provides a relatively simple and highly reproducible method for functional studies of individual mitochondrial-type carrier proteins from organisms that do not allow the application of sophisticated genetic techniques.

A hydrogenosomal [Fe]-hydrogenase from the anaerobic chytrid Neocallimastix sp. L2.

The presence of a [Fe]-hydrogenase in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2 has been demonstrated by immunocytochemistry, subcellular fractionation, Western-blotting and measurements of hydrogenase activity in the presence of various concentrations of carbon monoxide (CO). Since the hydrogenosomal hydrogenase activity can be inhibited nearly completely by low concentrations of CO, it is likely that the [Fe]-hydrogenase is responsible for at least 90% of the hydrogen production in isolated hydrogenosomes. Most likely, this hydrogenase is encoded by the gene hydL2 that exhibits all the motifs that are characteristic of [Fe]-hydrogenases. The open reading frame starts with an N-terminal extension of 38 amino acids that has the potential to function as a hydrogenosomal targeting signal. The downstream sequences encode an enzyme of a calculated molecular mass of 66.4 kDa that perfectly matches the molecular mass of the mature hydrogenase in the hydrogenosome. Phylogenetic analysis revealed that the hydrogenase of Neocallimastix sp. L2. clusters together with similar ('long-type') [Fe]-hydrogenases from Trichomonas vaginalis, Nyctotherus ovalis, Desulfovibrio vulgaris and Thermotoga maritima. Phylogenetic analysis based on the H-cluster - the only module of [Fe]-hydrogenases that is shared by all types of [Fe]-hydrogenases and hydrogenase-like proteins - revealed a monophyly of all hydrogenase-like proteins of the aerobic eukaryotes. Our analysis suggests that the evolution of the various [Fe]-hydrogenases and hydrogenase-like proteins occurred by a differential loss of Fe-S clusters in the N-terminal part of the [Fe]-hydrogenase.