The nuclear mRNA export receptor Mex67-Mtr2 of Trypanosoma brucei contains a unique and essential zinc finger motif.

Trypanosoma brucei is the causative agent of Human African Trypanosomiasis. Trypanosomes are early diverged protozoan parasites and show significant differences in their gene expression compared with higher eukaryotes. Due to a lack of individual gene promoters, large polycistronic transcripts are produced and individual mRNAs mature by trans-splicing and polyadenylation. In the absence of transcriptional control, regulation of gene expression occurs post-transcriptionally mainly by control of transcript stability and translation. Regulation of mRNA export from the nucleus to the cytoplasm might be an additional post-transcriptional event involved in gene regulation. However, our knowledge about mRNA export in trypanosomes is very limited. Although export factors of higher eukaryotes are reported to be conserved, only a few orthologues can be readily identified in the genome of T. brucei. Hence, biochemical approaches are needed to identify the export machinery of trypanosomes. Here, we report the functional characterization of the essential mRNA export factor TbMex67. TbMex67 contains a unique and essential N-terminal zinc finger motif. Furthermore, we could identify two interacting export factors namely TbMtr2 and the karyopherin TbIMP1. Our data show that the general heterodimeric export receptor Mex67-Mtr2 is conserved throughout the eukaryotic kingdom albeit exhibiting parasite-specific features.

Trypanosomal TAC40 constitutes a novel subclass of mitochondrial -barrel proteins specialized in mitochondrial genome inheritance

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum–mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA–cytoskeleton linkage that is essential for mitochondrial DNA inheritance.

Erythrina abyssinica prevents meningoencephalitis in chronic Trypanosoma brucei brucei mouse model

Human African trypanosomiasis is prevalent in Sub-sahara African countries that lie between 14° North and 29° south of the equator. Sixty million people are at risk of infection. Trypanosoma brucei gambesience occurs in West and Central Africa while Trypanosoma brucei rhodesience occurs in East and Southern Africa. The neurological stage of the disease is characterized by neuroinflammation. About 10% of patients treated with the recommended drug, melarsoprol develop post treatment reactive encephalopathy, which is fatal in 50% of these patients, thus melarsoprol is fatal in 5% of all treated patients. This study was aimed at establishing the potential activity of Erythrina abyssinica in reducing neuroinflammation following infection with Trypanosoma brucei brucei. Swiss white mice were divided into ten groups, two control groups and eight infected groups. Infected mice received either methanol or water extract of Erythrina abyssinica at 12.5, 25, 50 or 100 mg/kg body weight. Parasite counts were monitored in peripheral circulation from the third day post infection up to the end of the study. Brains were processed for histology, immunohistochemistry scanning and transmission electron microscopy. Following infection, trypanosomes were observed in circulation 3 days post-infection, with the parasitaemia occurring in waves. In the cerebrum, typical brain pathology of chronic trypanosomiasis was reproduced. This was exhibited as astrocytosis, perivascular cuffing and infiltration of inflammatory cells into the neuropil. However, mice treated with Erythrina abyssinica water extract exhibited significant reduction in perivascular cuffing, lymphocytic infiltration and astrocytosis in the cerebrum. The methanol extract did not have a significant difference compared to the non-treated group. This study provides evidence of anti-inflammatory properties of Erythrina abyssinica and may support its wide use as a medicinal plant by various communities in Kenya.

A conserved mitochondrial outer membrane protein mediates kDNA maintenace in Trypanosoma brucei

Kinetoplastids are defined by the unique organization of their mitochondrial DNA (kDNA). It forms a highly concatenated DNA network that is linked to the basal body of the flagellum by the tripartite attachment complex (TAC). The TAC encompasses intra and extramitochondrial filaments and a highly differentiated region of the two mitochondrial membranes. Here we identify and characterize a mitochondrial outer membrane protein of Trypanosoma brucei that is predominantly localized in the TAC. The protein is essential for growth in both life cycle stages. Immunofluorescence shows that ablation of the protein does not affect kDNA replication but abolishes the segregation of the replicated kDNA network causing rapid loss of kDNA. Besides its role in kDNA maintenance in vivo and in vitro experiments show that the protein is involved in mitochondrial protein import and that it interacts with a recently discovered protein import factor. RNAi experiments in a T. brucei cell line in which the kDNA is dispensable suggest that the essential function is linked to kDNA maintenance. Bioinformatic analysis shows that the studied outer membrane protein has beta-barrel topology and that it belongs to the mitochondrial porin family comprising VDAC, Tom40 and Mdm10. Interestingly, Mdm10 has sofar only been found in yeast. Ist function in protein import and mitochondrial DNA maintenance suggests that the protein in our study is the functional homologue of Mdm10. Thus, the TAC – a defining structure of Kinetoplastids – contains a conserved protein which in yeast and trypanosomes performs the same function. Our study therefore provides an example that trypanosomal biology, rather than being unique, often simply represents a more extreme manifestation of a conserved biological concept.

What a unicellular parasite can tell us about mitochondrial biogenesis?

Most of what we know about mitochondrial biogenesis stems from work in yeast and mammals, which are quite closely related. To understand the conserved features of mitochondria and the evolutionary forces that shaped it, it is important to study a more diverse group of eukaryotes. The parasitic protozoan Trypanosoma brucei and its relatives are excellent systems to do so, since they appear to have diverged from other eukaryotes very early in evolution. This is reflected in a number of unique and extreme features in their mitochondrial biology, including a single continuous mitochondrion that contains a one unit mitochondrial genome that is physically connected across the two membranes with the basal body of the flagellum. Moreover, many mitochondrial transcripts have to be extensively edited in order to become functional mRNAs and organellar translation requires extensive import of cytosolic tRNAs. In my talk I will focus on the discovery and characterization of the elusive mitochondrial protein import system of the mitochondrial outer membrane of trypanosomes. In addition I will present data on a central outer membrane component of the mitochondrial genome inheritance system of T. brucei and compare it to the better characterized system of yeast. - I hope that I can convince you in my talk, that a better understanding of the mitochondrial biology in T. brucei will provide insights into both fundamentally conserved and fundamentally diverged aspects of mitochondrial biogenesis and thus of the evolutionary hstory of mitochondria in general.

Mitochondrial outer membrane proteome of T.brucei reveals novel factors required to maintain mitochondrial morphology

African trypanosomes, the causative agent of Human African Trypanosomiasis (HAT) are among the earliest diverging eukaryotes that have bona fide mitochondria capable of oxidative phosphorylation. The mitochondrial outer membrane (MOM) of T. brucei is essentially unchartered territory. The beta barrel membrane proteins VDAC, Sam50 and archaic TOM are the only MOM proteins that have been characterized so far. Using biochemical fractionation and correlated protein abundance-profiling we were able to raise the protein inventory of the MOM. Of the 82 candidate proteins two-thirds have never been associated with mitochondria before. The function of 42 proteins remains unknown. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three MOM candidate proteins of unknown function resulted in a collapse of the network-like mitochondrion of insect-stage parasites and therefore directly or indirectly are involved in the regulation of mitochondrial morphology in T. brucei.

Mitochondrial outer membrane proteome of Trypanosoma brucei reveals macromolecular transport machineries and novel factors required to maintain mitochondrial morphology

The mitochondrial outer membrane (MOM) separates the mitochondria from the cytoplasm, serving both as a barrier and as a gateway. Protein complexes — believed to be universally conserved in all eukaryotes — reside in the MOM to orchestrate and control metabolite exchange, lipid metabolism and uptake of biopolymers such as protein and RNA. African trypanosomes are the causative agent of the sleeping sickness in humans. The parasites are among the earliest diverging eukaryotes that have bona fide mitochondria capable of oxidative phosphorylation. Trypanosomes have unique mitochondrial biology that concerns their mitochondrial metabolism and their unusual mitochondrial morphology that differs to great extent between life stages. Another striking feature is the organization of the mitochondrial genome that does not encode any tRNA genes, thus all tRNAs needed for mitochondrial translation have to be imported. However, the MOM of T. brucei is essentially unchartered territory. It lacks a canonical protein import machinery and facilitation of tRNA translocation remains completely elusive. Using biochemical fractionation and label-free quantitative mass spectrometry for correlated protein abundance-profiling we were able to identify a cluster of 82 candidate proteins that can be localized to the trypanosomal MOM with high confidence. This enabled us to identify a highly unusual, potentially archaic protein import machinery that might also transport tRNAs. Moreover, two-thirds of the identified polypeptides present on the MOM have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the MOM of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of insect-stage parasites and therefore directly or indirectly are involved in the regulation of mitochondrial morphology.

Mitochondrial outer membrane proteome of T.brucei reveals novel factors required to maintain mitochondrial morphology.

The mitochondrial outer membrane (MOM) separates the mitochondria from the cytoplasm, serving both as a barrier and as a gateway. Protein complexes residing in the MOM orchestrate protein and tRNA import, metabolite exchange and lipid metabolism. African trypanosomes are among the earliest diverging eukaryotes that have bona fide mitochondria capable of oxidative phosphorylation. The MOM of T. brucei is essentially unchartered territory. It lacks a canonical TOM-complex and proteins are imported across the MOM using ATOM, which is related to both Tom40 and to the bacterial Omp85-protein family. The beta barrel membrane proteins ATOM, VDAC and Sam50 are the only MOM proteins that have been characterized in T. brucei so far. Using biochemical fractionation and correlated protein abundance-profiling we were able to identify a cluster of 82 candidate proteins that can be localized to the trypanosomal MOM with high confidence Two-thirds of these polypeptides have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the MOM of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and therefore directly or indirectly are involved in the regulation of mitochondrial morphology in T. brucei.

A conserved mitochondrial outer membrane protein mediates kDNA maintenace in Trypanosoma brucei

Kinetoplastids are defined by the unique organization of their mitochondrial DNA (kDNA). It forms a highly concatenated DNA network that is linked to the basal body of the flagellum by the tripartite attachment complex (TAC). The TAC encompasses intra and extramitochondrial filaments and a highly differentiated region of the two mitochondrial membranes. Here we identify and characterize a mitochondrial outer membrane protein of Trypanosoma brucei that is predominantly localized in the TAC. The protein is essential for growth in both life cycle stages. Immunofluorescence shows that ablation of the protein does not affect kDNA replication but abolishes the segregation of the replicated kDNA network causing rapid loss of kDNA. Besides its role in kDNA maintenance in vivo and in vitro experiments show that the protein is involved in mitochondrial protein import and that it interacts with a recently discovered protein import factor. RNAi experiments in a T. brucei cell line in which the kDNA is dispensable suggest that the essential function is linked to kDNA maintenance. Bioinformatic analysis shows that the studied outer membrane protein has beta-barrel topology and that it belongs to the mitochondrial porin family comprising VDAC, Tom40 and Mdm10. Interestingly, Mdm10 has so far only been found in yeast. Its function in protein import and mitochondrial DNA maintenance suggests that the protein in our study is the functional homologue of Mdm10. Thus, the TAC – a defining structure of Kinetoplastids – contains a conserved protein which in yeast and trypanosomes performs the same function. Our study therefore provides an example that trypanosomal biology, rather than being unique, often simply represents a more extreme manifestation of a conserved biological concept.

Trypanosoma brucei eflornithine transporter AAT6 is a low-affinity low-selective transporter for neutral amino acids

Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the uptake of exogenous amino acids. Amino acid transporters are also of high pharmacological relevance because they may mediate uptake of toxic amino acid analogues. In the present study we show that the eflornithine transporter AAT6 from Trypanosoma brucei (TbAAT6) mediates growth on neutral amino acids when expressed in Saccharomyces cerevisiae mutants. The transport was electrogenic and further analysed in Xenopus laevis oocytes. Neutral amino acids, proline analogues, eflornithine and acivicin induced inward currents. For proline, glycine and tryptophan the apparent affinities and maximal transport rates increased with more negative membrane potentials. Proline-induced currents were dependent on pH, but not on sodium. Although proline represents the primary energy source of T. brucei in the tsetse fly, down-regulation of TbAAT6-expression by RNAi showed that in culture TbAAT6 is not essential for growth of procyclic form trypanosomes in the presence of glucose or proline as energy source. TbAAT6-RNAi lines of both bloodstream and procyclic form trypanosomes showed reduced susceptibility to eflornithine, whereas the sensitivity to acivicin remained unchanged, indicating that acivicin enters the cell by more than one transporter

Characterization of choline uptake in Trypanosoma brucei and involvement of a mitochondrial carrier in resistance to choline analogs

Choline is an essential nutrient for eukaryotic cells, where it is used as precursor for the synthesis of choline-­containing phospholipids, such as phosphatidylcholine (PC). Our experiments showed – for the first time – that Trypanosoma brucei, the causative agent of human African sleeping sickness, is able to take up choline from the culture medium to use for PC synthesis, indicating that trypanosomes express a transporter for choline at the plasma membrane. Further characterization in procyclic and bloodstream forms revealed that choline uptake is saturable and can be inhibited by HC-3, a known inhibitor of choline uptake in mammalian cells. To obtain additional insights on choline uptake and metabolism, we investigated the effects of choline-analogs that were previously shown to be toxic for T. brucei parasites in culture. Interestingly, we found that all analogs tested effectively inhibited choline uptake into both bloodstream and procyclic form parasites. Subsequently, selected compounds were used to search for possible candidate genes encoding choline transporters in T. brucei, using an RNAi library in bloodstream forms. We identified a protein belonging to the mitochondrial carrier family, previously annotated as TbMCP14, as prime candidate. Down‐regulation of TbMCP14 by RNAi prevented drug-­induced loss of mitochondrial membrane potential and conferred 8­‐fold resistance of T. brucei bloodstream forms to choline analogs. Conversely, over‐expression of the carrier increased parasite susceptibility more than 13-­fold. However, subsequent experiments demonstrated that TbMCP14 was not involved in metabolism of choline. Instead, growth curves in glucose‐depleted medium using RNAi or knock‐out parasites suggested that TbMCP14 is involved in metabolism of amino acids for energy production. Together, our data demonstrate that the identified member of the mitochondrial carrier family is involved in drug uptake into the mitochondrion and has a vital function in energy production in T. brucei.

Functional proteomics and biochemsitry: working together to unravel mitochondrial phenomena of African trypansomes

Eukaryotic cells are compartmentalized into membrane-bound organelles in order to provide sheltered reaction rooms for various specific processes. Organelles are not randomly distributed in a cell or operate isolated from each other. At the contrary — some organelles are closely linked and their functions are tightly orchestrated. The most well-known example of two such organelles acting in concert are the ER and the mitochondrion that work together in order to coordinate cellular lipid biosynthesis, maintain Ca2+-homeostasis, regulate mitochondrial division and control mitochondrial/ER shape as well as to synchronize the movement of these organelles within a cell. To study the mitochondrion and its interface to the ER requires a simplified mitochondrial system. African trypanosomes represent such a system. The unicellular parasite that causes devastating diseases in humans and animals has only one large mitochondrion that does not undergo fission/fusion events except for the context of cell division. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the protozoan. Central to the understanding of how mitochondria control their morphology, communicate with their surroundings and manage exchange of metabolites and transport of biopolymers (proteins, RNAs) is the mitochondrial outer membrane (MOM), as the MOM defines the boundary of the organelle. Recently, we have purified the MOM of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal MOM proteome consists of 82 proteins, two thirds of which have never been associated with mitochondria before. Among these, we identified novel factors required to regulate mitochondrial morphology and the long-elusive protein import machinery of T. brucei. A comparison with the MOM proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. One of these is the Miro-GTPase Gem1. In yeast, this Ca2+-EF-Hand containing polypeptide is thought to be involved in a protein complex that physically tethers the mitochondrion to the ER. Interestingly, a putative tethering complex in mammalian cells was linked to the mitochondrial fusion/fission machinery. Thus, the concept of a protein complex-mediated connection seems to be a general and conserved feature. We are currently investigating, if such a protein complex exists in T. brucei and if the trypanosomal Gem1 protein is involved. This ER-subdomain associated with mitochondria has been termed mitochondria-associated ER-membranes or MAM. The MAM has recently been implicated to play a key role in Alzheimer’s disease. It is therefore of broad and general interest to establish other eukaryotic model systems in order to investigate the MAM-MOM connection in more detail.

What can a unicellular parasite tell us about mitochondrial biogenesis?

Most of what we know about mitochondrial biogenesis stems from work in yeast and mammals, which are quite closely related. To understand the conserved features of mitochondria and the evolutionary forces that shaped it, it is important to study a more diverse group of eukaryotes. The parasitic protozoan Trypanosoma brucei and its relatives are excellent systems to do so, since they appear to have diverged from other eukaryotes very early in evolution. This is reflected in a number of unique and extreme features in their mitochondrial biology, including a single continuous mitochondrion that contains a one unit mitochondrial genome that is physically connected across the two membranes with the basal body of the flagellum. Moreover, many mitochondrial transcripts have to be extensively edited in order to become functional mRNAs and organellar translation requires extensive import of cytosolic tRNAs. In my talk I will focus on the discovery and characterization of the elusive mitochondrial protein import system of the mitochondrial outer membrane of trypanosomes. In addition I will present data on a central outer membrane component of the mitochondrial genome inheritance system of T. brucei and compare it to the better characterized system of yeast. - I hope that I can convince you in my talk, that a better understanding of the mitochondrial biology in T. brucei will provide insights into both fundamentally conserved and fundamentally diverged aspects of mitochondrial biogenesis and thus of the evolutionary history of mitochondria in general.

Functional characterization of the trypanosome translational repressor SCD6

The storage of translationally inactive mRNAs in cytosolic granules enables cells to react flexibly to environmental changes. In eukaryotes, Scd6 (suppressor of clathrin deficiency 6)/Rap55 (RNA-associated protein 55), a member of the LSm14 (like-Sm14) family, is an important factor in the formation and activity of P-bodies, where mRNA decay factors accumulate, in stress granules that store mRNAs under adverse conditions and in granules that store developmentally regulated mRNAs. SCD6 from Trypanosoma brucei (TbSCD6) shares the same domain architecture as orthologous proteins in other organisms and is also present in cytosolic granules (equivalent to P-bodies). We show that TbSCD6 is a general repressor of translation and that its depletion by RNAi results in a global increase in protein synthesis. With few exceptions, the steady-state levels of proteins are unchanged. TbSCD6 is not required for the formation of starvation-induced granules in trypanosomes, and unlike Scd6 from yeast, Plasmodium and all multicellular organisms analysed to date, it does not form a complex with the helicase Dhh1 (DExD/H-box helicase 1). In common with Xenopus laevis RAP55, TbSCD6 co-purifies with two arginine methyltransferases; moreover, TbSCD6 itself is methylated on three arginine residues. Finally, a detailed analysis identified roles for the Lsm and N-rich domains in both protein localization and tr

Trypanosoma brucei RRM1 is a nuclear RNA-binding protein and modulator of chromatin structure

TbRRM1 of Trypanosoma brucei is a nucleoprotein that was previously identified in a search for splicing factors in T. brucei. We show that TbRRM1 associates with mRNAs and with the auxiliary splicing factor polypyrimidine tract-binding protein 2, but not with components of the core spliceosome. TbRRM1 also interacts with several retrotransposon hot spot (RHS) proteins and histones. RNA immunoprecipitation of a tagged form of TbRRM1 from procyclic (insect) form trypanosomes identified ca. 1,500 transcripts that were enriched and 3,000 transcripts that were underrepresented compared to cellular mRNA. Enriched transcripts encoded RNA-binding proteins, including TbRRM1 itself, several RHS transcripts, mRNAs with long coding regions, and a high proportion of stage-regulated mRNAs that are more highly expressed in bloodstream forms. Transcripts encoding ribosomal proteins, other factors involved in translation, and procyclic-specific transcripts were underrepresented. Knockdown of TbRRM1 by RNA interference caused widespread changes in mRNA abundance, but these changes did not correlate with the binding of the protein to transcripts, and most splice sites were unchanged, negating a general role for TbRRM1 in splice site selection. When changes in mRNA abundance were mapped across the genome, regions with many downregulated mRNAs were identified. Two regions were analyzed by chromatin immunoprecipitation, both of which exhibited increases in nucleosome occupancy upon TbRRM1 depletion. In addition, subjecting cells to heat shock resulted in translocation of TbRRM1 to the cytoplasm and compaction of chromatin, consistent with a second role for TbRRM1 in modulating chromatin structure. IMPORTANCE: Trypanosoma brucei, the parasite that causes human sleeping sickness, is transmitted by tsetse flies. The parasite progresses through different life cycle stages in its two hosts, altering its pattern of gene expression in the process. In trypanosomes, protein-coding genes are organized as polycistronic units that are processed into monocistronic mRNAs. Since genes in the same unit can be regulated independently of each other, it is believed that gene regulation is essentially posttranscriptional. In this study, we investigated the role of a nuclear RNA-binding protein, TbRRM1, in the insect stage of the parasite. We found that TbRRM1 binds nuclear mRNAs and also affects chromatin status. Reduction of nuclear TbRRM1 by RNA interference or heat shock resulted in chromatin compaction. We propose that TbRRM1 regulates RNA polymerase II-driven gene expression both cotranscriptionally, by facilitating transcription and efficient splicing, and posttranscriptionally, via its interaction with nuclear mRNAs.