Paulus Michels

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 From a comparison of our results with the unpublished data for three other glycosomal glycolytic enzymes we infer that the high positive charge represents the major topogenic signal for uptake of proteins into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.

Two tandemly linked identical genes code for the glycosomal glyceraldehyde-phosphate dehydrogenase in Trypanosoma brucei.

Trypanosoma brucei contains two isoenzymes for glyceraldehyde‐phosphate dehydrogenase (GAPDH); one enzyme resides in a microbody‐like organelle, the glycosome, the other one is found in the cytosol. We show here that the glycosomal enzyme is encoded by two tandemly linked genes of identical sequence. These genes code for a protein of 358 amino acids, with a mol. wt of 38.9 kd. This is considerably larger than all other GAPDH proteins studied so far, including the enzyme that is located in the cytosol of the trypanosome. The glycosomal enzyme shows 52‐57% homology with known sequences of GAPDH proteins from 10 other organisms, both prokaryotes and eukaryotes. The residues that are involved in NAD+ binding, catalysis and subunit contacts are well conserved between all these GAPDH molecules, including the trypanosomal one. However, the glycosomal protein of T. brucei has some distinct features. Firstly, it contains a number of insertions, 1‐8 amino acids long, which are responsible for the high mol. wt of the protein. Secondly, an unusually high number of positively charged amino acids confer a high isoelectric point (pI 9.3) to the protein. Part of the additional basic residues are present in the insertions. We discuss the genomic organization of the genes for the glycosomal GAPDH and the possibility that the particular features of the protein are involved in its transfer from the cytoplasm, where it is synthesized, into the glycosome.

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.

The inactivation and reactivation of an expression-linked gene copy for a variant surface glycoprotein in Trypanosoma brucei.

We have previously shown that the gene for variant surface glycoprotein 118 of Trypanosoma brucei (strain 427) is activated by a duplicative transposition to a telomeric expression site. In chronically‐infected animals, this expression‐linked copy is lost when the 118 gene is replaced at the expression site by another variant surface glycoprotein gene. We show here that expression of the 118 gene can also be switched off without loss of the extra expression‐linked copy. In two variants, called 1.8b and 1.8c, we find expression of the variant surface glycoprotein 1.8 gene, notwithstanding the continued presence of the 118 expression‐linked copy. The 1.8 gene activated has a telomeric location, like the 118 expression‐linked copy. In variant 1.8b, activation is accompanied by duplication of the 1.8 gene, resulting in an extra telomeric gene copy; in variant 1.8c it is not. Variants 1.8b and 1.8c both switch back preferentially to expression of the 118 gene. The 5′‐flanking regions of the active, inactive and reactivated versions of the 118 expression‐linked copy are indistinguishable by restriction mapping up to 28 kb. We conclude that there are at least two separate telomeric expression sites in our T. brucei strain. How these are switched on and off is unclear. The ability to retain expression‐linked copies in inactive form may allow the trypanosome to re‐programme the order in which variant surface glycoprotein genes are expressed.

Molecular analysis of glyceraldehyde-3-phosphate dehydrogenase in Trypanoplasma borelli: an evolutionary scenario of subcellular compartmentation in kinetoplastida.

In Trypanoplasma borelli, a representative of the Bodonina within the Kinetoplastida, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in both the cytosol and glycosomes. This situation is similar to that previously found in Trypanosomatidae, belonging to a different Kinetoplastida suborder. In Trypanosomatidae different isoenzymes, only distantly related, are responsible for the activity in the two cell compartments. In contrast, immunoblot analysis indicated that the GAPDH activity in cytosol and glycosomes of T. borelli should be attributed to identical or at least very similar proteins related to the glycosomal GAPDH of Trypanosomatidae. Moreover, only genes related to the glycosomal GAPDH genes of Trypanosomatidae could be detected. All attempts to identify a gene related to the one coding for the trypanosomatid cytosolic GAPDH remained unsuccessful. Two tandemly arranged genes were found which are 95% identical. The two encoded polypeptides differ in 17 residues. Their sequences are 72–77% identical to the glycosomal GAPDH of the other Kinetoplastida and share with them some characteristic features: an excess of positively charged residues, specific insertions, and a small carboxy-terminal extension containing the sequence -AKL. This tripeptide conforms to the consensus signal for targeting of proteins to glycosomes. One of the two gene copies has undergone some mutations at positions coding for highly conserved residues of the active site and the NAD+-binding domain of GAPDH. Modeling of the proteins three-dimensional structure suggested that several of the substitutions compensate each other, retaining the functional coenzyme-binding capacity, although this binding may be less tight. The presented analysis of GAPDH in T. borelli gives further support to the assertion that one isoenzyme, the cytosolic one, was acquired by horizontal gene transfer during the evolution of the Kinetoplastida, in the lineage leading to the suborder Trypanosomatina (Trypanosome, Leishmania), after the divergence from the Bodonina (Trypanoplasma). Furthermore, the data clearly suggest that the original GAPDH of the Kinetoplastida has been compartmentalized during evolution.

The evolutionary origin of glycosomes

The glycolytic pathway of the Kinetoplastida is organized in a unique manner: the majority of its enzymes are contained in organelles called glycosomes. In this article Paul Michels and Fred Opperdoes argue that the glycosomes are equivalent to the microbodies and peroxisomes identified in other eukaryotic cells. They explore the possible evolutionary origin of the glycosome by comparing many of its structural and functional properties with those of other members of the microbody family and with some features of other organelles, the mitochondria and chloroplasts, which have been studied in much more detail.

The glycosomes of the Kinetoplastida.

Glycosomes are the microbodies of the organisms belonging to the order of the Kinetoplastida, comprising trypanosomes and leishmanias, both pathogens to man. The organelles sequester a number of glycolytic enzymes that are normally located in the cytosol in other eukaryotic organisms, and share some enzymes with peroxisomes and glyoxysomes of other protists, plants and animals. Proteins enter the glycosome by a mechanism of post-translational translocation which involves in some, but not all, cases a C-terminal oligopeptide sequence.

Pyruvate transport across the plasma membrane of the bloodstream form of Trypanosoma brucei is mediated by a facilitated diffusion carrier.

The characteristics of pyruvate transport across the plasma membrane in the bloodstream form of Trypanosoma brucei were studied using [14C]pyruvate in combination with the silicone-oil centrifugation technique. We present evidence for the existence of a facilitated diffusion carrier in the plasma membrane of T. brucei which specifically mediates the translocation of pyruvate. The uptake of pyruvate followed saturation kinetics (Km 1.96 +/- 0.28 mM; Cmax 36.61 +/- 1.15 nmol pyruvate/30 sec.mg protein), after correction of the data for a nonsaturable diffusion component. The uptake of pyruvate was competitively inhibited by a number of (oxo)monocarboxylic acids, including pyruvate analogs and metabolically related substances, but not by L-lactate. The transport exhibited the phenomenon of transacceleration, indicative for the involvement of a facilitated diffusion carrier. The carrier is highly specific for pyruvate and differs from other known monocarboxylate carriers present in the mitochondrial and/or plasma membrane of other eukaryotic cells in that it does not transport L-lactate.

The electrochemical proton gradient in the bloodstream form of Trypanosoma brucei is dependent on the temperature

The membrane potential and pH gradient over the plasma membrane of the protozoan parasite Trypanosoma brucei were measured with radioactive indicators in combination with the silicone oil centrifugation technique over a range of temperatures. At 37 degrees C a small membrane potential and pH gradient of similar magnitude, but of opposite polarity, were measured. The resulting electrochemical proton gradient was almost zero. However, when the temperature was lowered from 37 degrees C to 22 degrees C, the internal pH was kept constant independent of the external pH and a membrane potential of between -100 and -150 mV was measured, depending on the external pH. Measurements at various temperatures between 15 degrees C and 37 degrees C revealed that above 26 degrees C the membrane potential collapsed and that this collapse correlated with a sudden increase in membrane fluidity. The uptake of 2-deoxy-D-glucose and of pyruvate, which are both mediated by facilitated diffusion carriers in the plasma membrane of the trypanosome, were also affected by this sudden increase in fluidity of the membrane. The overall rate of the conversion of glucose into its metabolites, which is independent of the plasma membrane, varied only gradually. We conclude (i) that major changes occur in the plasma membrane of T. brucei around 26 degrees C, that affect all membrane related processes; (ii) that the electrochemical proton gradient plays a minor role in the energy metabolism of T. brucei when it resides in the bloodstream of the mammalian host at 37 degrees C; and (iii) that below 26 degrees C an electrochemical proton gradient is maintained over the plasma membrane.

Trypanosoma brucei glycosomal glyceraldehyde-3-phosphate dehydrogenase genes are stage-regulated at the transcriptional level.

Regions 5′ of the glycosomal glyceraldehyde‐3‐phosphate dehydrogenase (gGAPDH) gene from Trypanosoma brucei were tested for their ability to promote chloramphenicol acetyl‐transferase (CAT) expression on reintroduction by electroporation into the parasite. Deletion analysis mapped the gGAPDH promoter to within 403 nts of the start of translation. A transcription initiation site was mapped at around −190 nts from the ATG start codon by RNase protection and by primer extension. The higher expression of gGAPDH in bloodstream T. brucei, compared to procyclic (insect) forms, was largely attributed to differences in promoter activity. The gGAPDH promoter gave rise to relatively high CAT signals upon transfection into bloodstream T. brucei and relatively low signals in procyclic T. brucei, compared with levels resulting from transfection with the procyclic acidic repetitive protein (PARP) promoter. In addition, RNase protection data showed a higher level of gGAPDH primary transcripts in bloodstream. T. brucei. The PARP mini‐exon addition region abolished transient CAT expression directed by either the gGAPDH or PARP promoters in bloodstream T. brucei implying that transplicing can be a point of stage‐specific gene regulation.