Characterization of two novel proteins involved in mitochondrial DNA anchoring in Trypanosoma brucei.

Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC.

The Trypanosome UDP-Glucose Pyrophosphorylase Is Imported by Piggybacking into Glycosomes, Where Unconventional Sugar Nucleotide Synthesis Takes Place.

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways, such as glycolysis and biosynthesis of sugar nucleotides, usually present in the cytosol of other eukaryotes. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localized in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisome-targeting signal (PTS1 and PTS2). The questions that we address here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotides functional and (ii) how is the PTS-free UGP imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3 to 10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for the growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knockdown UGP mutant cell line (RNAiUGP, where RNAi indicates RNA interference) was rescued by expressing a recoded UGP (rUGP) in the organelle (RNAiUGP/EXPrUGP-GPDH, where GPDH is glycerol-3-phosphate dehydrogenase). Our conclusion was supported by targeted metabolomic analyses (ion chromatography-high-resolution mass spectrometry [IC-HRMS]) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. IMPORTANCE Unusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localization of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here, we showed that the cytosolic and glycosomal pathways are functional. As in all other eukaryotes, the cytosolic pathways feed glycosylation reactions; however, the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK); they are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase.

The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δpepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δpepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δpepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this process.

A comparison of three approaches for the discovery of novel tripartite attachment complex proteins in Trypanosoma brucei.

Trypanosoma brucei is a single celled eukaryotic parasite and the causative agent of human African trypanosomiasis and nagana in cattle. Aside from its medical relevance, T. brucei has also been key to the discovery of several general biological principles including GPI-anchoring, RNA-editing and trans-splicing. The parasite contains a single mitochondrion with a singular genome. Recent studies have identified several molecular components of the mitochondrial genome segregation machinery (tripartite attachment complex, TAC), which connects the basal body of the flagellum to the mitochondrial DNA of T. brucei. The TAC component in closest proximity to the mitochondrial DNA is TAC102. Here we apply and compare three different approaches (proximity labelling, immunoprecipitation and yeast two-hybrid) to identify novel interactors of TAC102 and subsequently verify their localisation. Furthermore, we establish the direct interaction of TAC102 and p166 in the unilateral filaments of the TAC.