Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei.

To determine how microbody enzymes enter microbodies, we are studying the genes for cytosolic and glycosomal (microbody) isoenzymes in Trypanosoma brucei. We have found three genes (A, B and C) coding for phosphoglycerate kinase (PGK) in a tandem array in T. brucei. Gene B codes for the cytosolic and gene C for the glycosomal isoenzyme. Genes B and C are 95% homologous, and the predicted protein sequences share approximately 45% amino acid homology with other eukaryote PGKs. The microbody isoenzyme differs from the cytosolic form and other PGKs in two respects: a high positive charge and a carboxy-terminal extension of 20 amino acids. Our results show that few alterations are required to redirect a protein from cytosol to microbody. From a comparison of our results with the unpublished data for three other glycosomal glycolytic enzymes we infer that the high positive charge represents the major topogenic signal for uptake of proteins into glycosomes.     

The topogenic signal of the glycosomal (microbody) phosphoglycerate kinase of Crithidia fasciculata resides in a carboxy-terminal extension.

To determine how microbody proteins enter microbodies, we have previously compared the genes for the cytosolic and glycosomal (microbody) phosphoglycerate kinases (PGKs) of Trypanosoma brucei and found the microbody enzyme to differ from other PGKs and the cytosolic form in two respects: a high net positive charge and a C-terminal extension of 20 amino acids (Osinga et al., 1985). Here we present the comparison of the genes for the cytosolic and glycosomal PGKs of Crithidia fasciculata, another kinetoplastid organism. The amino acid sequences of the two Crithidia isoenzymes are virtually identical, except for a C-terminal extension of 38 amino acids. We conclude that this extension must direct the glycosomal PGK to the glycosome. The extensions of the Crithidia and Trypanosoma enzymes are both rich in small hydrophobic and hydroxyl amino acids.     

The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.     

Differential gene expression of chloroplast and cytosolic phosphoglycerate kinase in tobacco

Northern blot analysis of RNA extracted from leaves of increasing age and different organs, indicates that genes encoding both isoenzymes of tobacco phosphoglycerate kinase (PGK, EC 2.7.2.3) are differentially expressed in a developmental and tissue-specific manner. The genes for both chloroplast PGK (chl-PGK) and cytosolic PGK (cyt-PGK) also show light-modulated gene expression in vivo. In dark-grown developing cotyledonary leaves of tobacco both PGK mRNAs are present, but only the concentration of the chl-PGK mRNA increased on illumination. In contrast, on transfer to darkness, the concentration of both mRNAs decreased in light-grown seedlings and then increased again on resumption of illumination.     

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.     

Six related nucleoside/nucleobase transporters from Trypanosoma brucei exhibit distinct biochemical functions

Purine nucleoside and nucleobase transporters are of fundamental importance for Trypanosoma brucei and related kinetoplastid parasites because these protozoa are not able to synthesize purines de novo and must salvage the compounds from their hosts. In the studies reported here, we have identified a family of six clustered genes in T. brucei that encode nucleoside/nucleobase transporters. These genes, TbNT2/927, TbNT3, TbNT4, TbNT5, TbNT6, and TbNT7, have predicted amino acid sequences that show high identity to each other and to TbNT2, a P1 type nucleoside transporter recently identified in our laboratory. Expression in Xenopus laevis oocytes revealed that TbNT2/927, TbNT5, TbNT6, and TbNT7 are high affinity adenosine/inosine transporters with K(m) values of <5 microm. In addition, TbNT5, and to a limited degree TbNT6 and TbNT7, also mediate the uptake of the nucleobase hypoxanthine. Ribonuclease protection assays showed that mRNA from all of the six members of this gene family are expressed in the bloodstream stage of the T. brucei life cycle but that TbNT2/927 and TbNT5 mRNAs are also expressed in the insect stage of the life cycle. These results demonstrate that T. brucei expresses multiple purine transporters with distinct substrate specificities and different patterns of expression during the parasite life cycle.     

Probing the role of compartmentation of glycolysis in procyclic form Trypanosoma brucei: RNA interference studies of PEX14, hexokinase, and phosphofructokinase

Trypanosoma brucei and related organisms contain an organelle evolutionarily related to peroxisomes that sequesters glycolysis, among other pathways. We have shown previously that disruption of protein import into this organelle, the glycosome, can be accomplished through RNA interference (RNAi)-mediated knockdown of the peroxin PEX14. Decreased PEX14 in turn leads to cell death, which, at least in the procyclic stage, can be triggered by the presence of glucose. Here we show that fructose, which is taken up and metabolized by procyclic form T. brucei, and glycerol, which interfaces with the glycosomal glycolytic pathway, are also toxic during PEX14 RNAi. Earlier computer modeling studies predicted that glycolysis would be toxic to T. brucei in the absence of glycosomal compartmentation because of the intrinsic lack of feedback regulation of the parasite hexokinase and phosphofructokinase. To further test this hypothesis, we performed double RNAi, targeting hexokinase and PEX14. Knockdown of hexokinase rescued PEX14 knockdown cells from glucose toxicity, even though glycosomal proteins continue to be mislocalized to the cytosol. Knockdown of phosphofructokinase was benign in the absence of glucose but toxic in the presence of glucose. When PEX14 and phosphofructokinase mRNAs were jointly targeted for RNAi, glycerol remained toxic to the parasites. Taken together, these data indicate that the glycosome provides significant, but not complete, protection of trypanosomes from the dangerous design of glycolysis.     

Common elements on the surface of glycolytic enzymes from Trypanosoma brucei may serve as topogenic signals for import into glycosomes.

In Trypanosoma brucei, a major pathogenic protozoan parasite of Central Africa, a number of glycolytic enzymes present in the cytosol of other organisms are uniquely segregated in a microbody-like organelle, the glycosome, which they are believed to reach post-translationally after being synthesized by free ribosomes in the cytosol. In a search for possible topogenic signals responsible for import into glycosomes we have compared the amino acid sequences of four glycosomal enzymes: triosephosphate isomerase (TIM), glyceraldehyde-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and aldolase (ALDO), with each other and with their cytosolic counterparts. Each of these enzymes contains a marked excess of positive charges, distributed in two or more clusters along the polypeptide chain. Modelling of the three-dimensional structures of TIM, PGK and GAPDH using the known structural coordinates of homologous enzymes from other organisms indicates that all three may have in common two 'hot spots' about 40 A apart, which themselves include a pair of basic amino acid residues separated by a distance of about 7 A. The sequence of glycosomal ALDO, for which no three-dimensional information is available, is compatible with the presence of the same configuration on the surface of this enzyme. We propose that this feature plays an essential role in the import of enzymes into glycosomes.     

Tubulin mRNAs of Trypanosoma brucei

The tubulin genes of Trypanosoma brucei are located in a single, tightly packed cluster of ten tandemly arranged alternating alpha and beta-genes. No tubulin genes are detected outside the clustered array. Therefore, the cluster can be assumed to be the locus of tubulin gene expression. Single bands of alpha and beta-tubulin mRNAs are observed in cultured procyclic as well as in bloodstream trypanosomes. Both alpha and beta-tubulin mRNAs have distinct 5' termini, which carry a 35-nucleotide mini-exon sequence. The 3' termini of both mRNA populations are heterogeneous.     

Higher-plant chloroplast and cytosolic 3-phosphoglycerate kinases: a case of endosymbiotic gene replacement

Previous studies indicated that plant nuclear genes for chloroplast and cytosolic isoenzymes of 3-phosphoglycerate kinase (PGK) arose through recombination between a preexisting gene of the eukaryotic host nucleus for the cytosolic enzyme and an endosymbiont-derived gene for the chloroplast enzyme. We readdressed the evolution of eukaryotic pgk genes through isolation and characterisation of a pgk gene from the extreme halophilic, photosynthetic archaebacterium Haloarcula vallismortis and analysis of PGK sequences from the three urkingdoms. A very high calculated net negative charge of 63 for PGK from H. vallismortis was found which is suggested to result from selection for enzyme solubility in this extremely halophilic cytosol. We refute the recombination hypothesis proposed for the origin of plant PGK isoenzymes. The data indicate that the ancestral gene from which contemporary homologues for the Calvin cycle/glycolytic isoenzymes in higher plants derive was acquired by the nucleus from (endosymbiotic) eubacteria. Gene duplication subsequent to separation of Chlamydomonas and land plant lineages gave rise to the contemporary genes for chloroplast and cytosolic PGK isoenzymes in higher plants, and resulted in replacement of the preexisting gene for PGK of the eukaryotic cytosol. Evidence suggesting a eubacterial origin of plant genes for PGK via endosymbiotic gene replacement indicates that plant nuclear genomes are more highly chimaeric, i.e. contain more genes of eubacterial origin, than is generally assumed.Abbreviations PGK 3-phosphoglycerate kinase - FBA fructose-1,6-bisphosphate aldolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - TPI triosephosphate isomerase