Procyclic Forms Research Articles

TbSmee1 regulates hook complex morphology and the rate of flagellar pocket uptake in Trypanosoma brucei.

Trypanosoma brucei uses multiple mechanisms to evade detection by its insect and mammalian hosts. The flagellar pocket (FP) is the exclusive site of uptake from the environment in trypanosomes and shields receptors from exposure to the host. The FP neck is tightly associated with the flagellum via a series of cytoskeletal structures that include the hook complex (HC) and the centrin arm. These structures are implicated in facilitating macromolecule entry into the FP and nucleating the flagellum attachment zone (FAZ), which adheres the flagellum to the cell surface. TbSmee1 (Tb927.10.8820) is a component of the HC and a putative substrate of polo-like kinase (TbPLK), which is essential for centrin arm and FAZ duplication. We show that depletion of TbSmee1 in the insect-resident (procyclic) form of the parasite causes a 40% growth decrease and the appearance of multinucleated cells that result from defective cytokinesis. Cells lacking TbSmee1 contain HCs with aberrant morphology and show delayed uptake of both fluid-phase and membrane markers. TbPLK localization to the tip of the new FAZ is also blocked. These results argue that TbSmee1 is necessary for maintaining HC morphology, which is important for the parasite's ability to take up molecules from its environment.

Identification and characterization of the three members of the CLC family of anion transport proteins in Trypanosoma brucei.

CLC type anion transport proteins are homo-dimeric or hetero-dimeric with an integrated transport function in each subunit. We have identified and partially characterized three members of this family named TbVCL1, TbVCL2 and TbVCL3 in Trypanosoma brucei. Among the human CLC family members, the T. brucei proteins display highest similarity to CLC-6 and CLC-7. TbVCL1, but not TbVCL2 and TbVCL3 is able to complement growth of a CLC-deficient Saccharomyces cerevisiae mutant. All TbVCL-HA fusion proteins localize intracellulary in procyclic form trypanosomes. TbVCL1 localizes close to the Golgi apparatus and TbVCL2 and TbVCL3 to the endoplasmic reticulum. Upon expression in Xenopus oocytes, all three proteins induce similar outward rectifying chloride ion currents. Currents are sensitive to low concentrations of DIDS, insensitive to the pH in the range 5.4 to 8.4 and larger in nitrate than in chloride medium.

Comparative Proteomic Analysis of Lysine Acetylation in Trypanosomes.

Protein acetylation is a post-translational modification regulating diverse cellular processes. By using proteomic approaches, we identified N-terminal and ε-lysine acetylated proteins in Trypanosoma cruzi and Trypanosoma brucei, which are protozoan parasites that cause significant human and animal diseases. We detected 288 lysine acetylation sites in 210 proteins of procyclic form, an insect stage of T.brucei, and 380 acetylation sites in 285 proteins in the form of the parasite that replicates in mammalian bloodstream. In T.cruzi insect proliferative form we found 389 ε-lysine-acetylated sites in 235 proteins. Notably, we found distinct acetylation profiles according to the developmental stage and species, with only 44 common proteins between T.brucei stages and 18 in common between the two species. While K-ac proteins from T.cruzi are enriched in enzymes involved in oxidation/reduction balance, required for the parasite survival in the host, in T.brucei, most K-ac proteins are enriched in metabolic processes, essential for its adaptation in its hosts. We also identified in both parasites a quite variable N-terminal acetylation sites. Our results suggest that protein acetylation is involved in differential regulation of multiple cellular processes in Trypanosomes, contributing to our understanding of the essential mechanisms for parasite infection and survival.

Mitogen-Activated Protein Kinase Kinase 5 Regulates Proliferation and Biosynthetic Processes in Procyclic Forms of Trypanosoma brucei.

The pathogenic protozoan T. brucei alternates into distinct developmental stages in the mammalian and insect hosts. The mitogen-activated protein kinase (MAPK) signaling pathways transduce extracellular stimuli into a range of cellular responses, which ultimately lead to the adaptation to the external environment. Here, we combined a loss of function approach with stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry (MS) to investigate the role of the mitogen-activated protein kinase kinase 5 (MKK5) in T. brucei. The silencing of MKK5 significantly decreased the proliferation of procyclic forms of T. brucei. To shed light on the molecular alterations associated with this phenotype, we measured the total proteome and phosphoproteome of cells silenced for MKK5. In the total proteome, we observed a general decrease in proteins related to ribosome and translation as well as down-regulation of several components of the fatty acids biosynthesis pathway. In addition, we observed alterations in the protein levels and phosphorylation of key metabolic enzymes, which point toward a suppression of the oxidative metabolism. Taken together, our findings show that the silencing of MKK5 alters cell growth, energy metabolism, protein and fatty acids biosynthesis in procyclic T. brucei.

Involvement of SNARE protein Ykt6 in glycosome biogenesis in Trypanosoma brucei

The kinetoplastid parasites Trypanosoma and Leishmania are etiologic agents of diseases like African sleeping sickness, Chagas and leishmaniasis that inflict many tropical and subtropical parts of the world. These parasites are distinctive in that they compartmentalize most of the usually cytosolic enzymes of the glycolytic pathway within a peroxisome-like organelle called the glycosome. Functional glycosomes are essential in both the procyclic and bloodstream forms of trypanosomatid parasites, and mislocalization of glycosomal enzymes to the cytosol is fatal for the parasite. The life cycle of these parasites is intimately linked to their efficient protein and vesicular trafficking machinery that helps them in immune evasion, host-pathogen interaction and organelle biogenesis and integrity. Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins play important roles in vesicular trafficking and mediate a wide range of protein–protein interactions in eukaryotes. We show here that the SNARE protein Ykt6 is necessary for glycosome biogenesis and function in Trypanosoma brucei. RNAi-mediated depletion of Ykt6 in both the procyclic and bloodstream forms of T. brucei leads to mislocalization of glycosomal matrix proteins to the cytosol, pronounced reduction in glycosome number, and cell death. GFP-tagged Ykt6 appears as punctate structures in the T. brucei cell and colocalizes in part to glycosomes. Our results constitute the first demonstration of a role for SNARE proteins in the biogenesis of peroxisomal organelles.

Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues.

Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model.

Insights into the role of endonuclease V in RNA metabolism in Trypanosoma brucei

Inosine may arise in DNA as a result of oxidative deamination of adenine or misincorporation of deoxyinosine triphosphate during replication. On the other hand, the occurrence of inosine in RNA is considered a normal and essential modification induced by specific adenosine deaminases acting on mRNA and tRNA. In prokaryotes, endonuclease V (EndoV) can recognize and cleave inosine-containing DNA. In contrast, mammalian EndoVs preferentially cleave inosine-containing RNA, suggesting a role in RNA metabolism for the eukaryotic members of this protein family. We have performed a biochemical characterization of EndoV from the protozoan parasite Trypanosoma brucei. In vitro, TbEndoV efficiently processes single-stranded RNA oligonucleotides with inosine, including A to I-edited tRNA-like substrates but exhibits weak activity over DNA, except when a ribonucleotide is placed 3′ to the inosine. Immunolocalization studies performed in procyclic forms indicate that TbEndoV is mainly cytosolic yet upon nutritional stress it redistributes and accumulates in stress granules colocalizing with the DEAD-box helicase TbDhh1. RNAi-mediated depletion of TbEndoV results in moderate growth defects in procyclic cells while the two EndoV alleles could be readily knocked out in bloodstream forms. Taken together, these observations suggest an important role of TbEndoV in RNA metabolism in procyclic forms of the parasite.

The essential functions of KREPB4 are developmentally distinct and required for endonuclease association with editosomes.

Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei, and several transcripts are differentially edited in bloodstream (BF) and procyclic form (PF) cells correlating with changes in mitochondrial function. Editing is catalyzed by three ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III KREN1, N2, and N3 endonucleases with distinct cleavage specificities. KREPB4 is a common editosome protein that has a degenerate RNase III domain lacking conserved catalytic residues, in addition to zinc-finger and Pumilio/fem-3 mRNA binding factor (PUF) motifs. Here we show that KREPB4 is essential for BF and PF growth, in vivo RNA editing, and editosome integrity, but that loss of KREPB4 has differential effects on editosome components and complexes between BF and PF cells. We used targeted mutagenesis to investigate the functions of the conserved PUF and RNase III domains in both life-cycle stages and show that the PUF motif is not essential for function in BF or PF. In contrast, specific mutations in the RNase III domain severely inhibit BF and PF growth and editing, and disrupt ∼20S editosomes, while others indicate that the RNase III domain is noncatalytic. We further show that KREPB4, specifically the noncatalytic RNase III domain, is required for the association of KREN1, N2, and N3 with PF editosomes. These results, combined with previous studies, support a model in which KREPB4 acts as a pseudoenzyme to form the noncatalytic half of an RNase III heterodimer with the editing endonucleases.

Expression of the RNA-binding protein RBP10 promotes the bloodstream-form differentiation state in Trypanosoma brucei.

In nearly all eukaryotes, cellular differentiation is governed by changes in transcription, and stabilized by chromatin and DNA modification. Gene expression control in the pathogen Trypanosoma brucei, in contrast, relies almost exclusively on post-transcriptional mechanisms, so RNA binding proteins must assume the burden that is usually borne by transcription factors. T. brucei multiply in the blood of mammals as bloodstream forms, and in the midgut of Tsetse flies as procyclic forms. We show here that a single RNA-binding protein, RBP10, promotes the bloodstream-form trypanosome differentiation state. Depletion of RBP10 from bloodstream-form trypanosomes gives cells that can grow only as procyclic forms; conversely, expression of RBP10 in procyclic forms converts them to bloodstream forms. RBP10 binds to procyclic-specific mRNAs containing an UAUUUUUU motif, targeting them for translation repression and destruction. Products of RBP10 target mRNAs include not only the major procyclic surface protein and enzymes of energy metabolism, but also protein kinases and stage-specific RNA-binding proteins: this suggests that alterations in RBP10 trigger a regulatory cascade.

The proteome and transcriptome of the infectious metacyclic form of Trypanosoma brucei define quiescent cells primed for mammalian invasion.

The infectious metacyclic forms of Trypanosoma brucei result from a complex development in the tsetse fly vector. When they infect mammals, they cause African sleeping sickness in humans. Due to scarcity of biological material and difficulties of the tsetse fly as an experimental system, very limited information is available concerning the gene expression profile of metacyclic forms. We used an in vitro system based on expressing the RNA binding protein 6 to obtain infectious metacyclics and determined their protein and mRNA repertoires by mass-spectrometry (MS) based proteomics and mRNA sequencing (RNA-Seq) in comparison to non-infectious procyclic trypanosomes. We showed that metacyclics are quiescent cells, and propose this influences the choice of a monocistronic variant surface glycoprotein expression site. Metacyclics have a largely bloodstream-form type transcriptome, and thus are programmed to translate a bloodstream-form type proteome upon entry into the mammalian host and resumption of cell division. Genes encoding cell surface components showed the largest changes between procyclics and metacyclics, observed at both the transcript and protein levels. Genes encoding metabolic enzymes exhibited expression in metacyclics with features of both procyclic and bloodstream forms, suggesting that this intermediate-type metabolism is dictated by the availability of nutrients in the tsetse fly vector.

The Trypanosoma brucei dihydroxyacetonephosphate acyltransferase TbDAT is dispensable for normal growth but important for synthesis of ether glycerophospholipids.

Glycerophospholipids are the most abundant constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans and nagana in cattle. They are essential cellular components that fulfill various important functions beyond their structural role in biological membranes such as in signal transduction, regulation of membrane trafficking or control of cell cycle progression. Our previous studies have established that the glycerol-3-phosphate acyltransferase TbGAT is dispensable for growth, viability, and ester lipid biosynthesis suggesting the existence of another initial acyltransferase(s). This work presents the characterization of the alternative, dihydroxyacetonephosphate acyltransferase TbDAT, which acylates primarily dihydroxyacetonephosphate and prefers palmitoyl-CoA as an acyl-CoA donor. TbDAT restores the viability of a yeast double null mutant that lacks glycerol-3-phosphate and dihydroxyacetonephosphate acyltransferase activities. A conditional null mutant of TbDAT in T. brucei procyclic form was created and characterized. TbDAT was important for survival during stationary phase and synthesis of ether lipids. In contrast, TbDAT was dispensable for normal growth. Our results show that in T. brucei procyclic forms i) TbDAT but not TbGAT is the physiologically relevant initial acyltransferase and ii) ether lipid precursors are primarily made by TbDAT.

Conversion of procyclic-form Trypanosoma brucei to the bloodstream form by transient expression of RBP10

Bloodstream-form Trypanosoma brucei can lose the ability to differentiate to the procyclic form during prolonged in vitro culture. This can pose a problem during complicated genetic manipulation experiments, especially when the differentiation phenotype is under investigation. Ideally, to preserve differentiation competence, parasites should be cycled after every genetic manipulation step. Conversion of bloodstream-form Trypanosoma brucei to the procyclic form in vitro is routine, but conversion of procyclic forms to bloodstream forms has hitherto only been achieved in transgenic parasites with tetracycline-inducible expression of proteins with RNA-binding domains − either RBP6 or RBP10. This method, however, requires use of a selectable marker which might be needed for other purposes, and restricts options for tetracycline-inducible expression or repression of other genes. A simple method for inter-conversion that does not require permanent genetic manipulation would therefore be useful. Induced expression of RBP10 in procyclic forms gives faster differentiation than expression of RBP6, with a switch towards bloodstream forms within 48h. We here show that bloodstream forms can be obtained by transient transfection of procyclic forms with a circular plasmid designed for expression of RBP10 from an rRNA promoter. This method enables routine cycling of T. brucei without permanent genetic manipulation.

The ubiquitin-conjugating enzyme CDC34 is essential for cytokinesis in contrast to putative subunits of a SCF complex in Trypanosoma brucei.

The ubiquitin-proteasome system is a post-translational regulatory pathway for controlling protein stability and activity that underlies many fundamental cellular processes, including cell cycle progression. Target proteins are tagged with ubiquitin molecules through the action of an enzymatic cascade composed of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. One of the E3 ligases known to be responsible for the ubiquitination of cell cycle regulators in eukaryotes is the SKP1-CUL1-F-box complex (SCFC). In this work, we identified and studied the function of homologue proteins of the SCFC in the life cycle of Trypanosoma brucei, the causal agent of the African sleeping sickness. Depletion of trypanosomal SCFC components TbRBX1, TbSKP1, and TbCDC34 by RNAi resulted in decreased growth rate and contrasting cell cycle abnormalities for both procyclic (PCF) and bloodstream (BSF) forms. Depletion of TbRBX1 in PCF cells interfered with kinetoplast replication, whilst depletion of TbSKP1 arrested PCF and BSF cells in the G1/S transition. Silencing of TbCDC34 in BSF cells resulted in a block in cytokinesis and caused rapid clearance of parasites from infected mice. We also show that TbCDC34 is able to conjugate ubiquitin in vitro and in vivo, and that its activity is necessary for T. brucei infection progression in mice. This study reveals that different components of a putative SCFC have contrasting phenotypes once depleted from the cells, and that TbCDC34 is essential for trypanosome replication, making it a potential target for therapeutic intervention.

The role of the zinc finger protein ZC3H32 in bloodstream-form Trypanosoma brucei

Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, with regulation of mRNA processing, translation and degradation by RNA-binding proteins. ZC3H32 is a cytosolic mRNA-binding protein with three non-canonical CCCH zinc finger domains. It is much more abundant in bloodstream-form Trypanosoma brucei than in procyclic forms. Tethering of ZC3H32 to a reporter mRNA suppressed translation and resulted in mRNA degradation, and deletion analysis suggested that this activity was present in both the N- and C-terminal domains, but not the central zinc finger-containing domain. Tandem affinity purification, however, revealed no interaction partners that might account for this activity. RNASeq analyses did not yield any evidence for sequence-specific binding or regulation of specific mRNAs. The presence of ZC3H32 homologues in monogenetic and free-living Euglenids also argues against a role in developmental regulation, although its function may have diverged in evolution. T. brucei ZC3H32 might be implicated in basal mRNA metabolism, with this role perhaps being taken over by another protein in procyclic forms.

Dynamin-like proteins in Trypanosoma brucei: A division of labour between two paralogs?

Dynamins and dynamin-like proteins (DLPs) belong to a family of large GTPases involved in membrane remodelling events. These include both fusion and fission processes with different dynamin proteins often having a specialised function within the same organism. Trypanosoma brucei is thought to have only one multifunctional DLP (TbDLP). While this was initially reported to function in mitochondrial division only, an additional role in endocytosis and cytokinesis was later also proposed. Since there are two copies of TbDLP present in the trypanosome genome, we investigated potential functional differences between these two paralogs by re-expressing either protein in a TbDLP RNAi background. These paralogs, called TbDLP1 and TbDLP2, are almost identical bar a few amino acid substitutions. Our results, based on cell lines carrying tagged and RNAi-resistant versions of each protein, show that overexpression of TbDLP1 alone is able to rescue the observed endocytosis and growth defects in the mammalian bloodstream form (BSF) of the parasite. While TbDLP2 shows no rescue in our experiments in BSF, this might also be due to lower expression levels of the protein in this life stage. In contrast, both TbDLP proteins apparently play more complementary roles in the insect procyclic form (PCF) since neither TbDLP1 nor TbDLP2 alone can fully restore wildtype growth and morphology in TbDLP-depleted parasites.

PH regulation in glycosomes of procyclic form Trypanosoma brucei

Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form Trypanosoma brucei When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30-60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na+ ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na+/H+ exchangers are required for glycosomal pH regulation.

An efficient cumate-inducible system for procyclic and bloodstream form Trypanosoma brucei

In Trypanosoma brucei, the tetracycline-inducible system enables tightly-regulated, highly-efficient expression of recombinant proteins or double-stranded RNA in both procyclic and bloodstream form cells, providing useful molecular genetic tools to study gene functions. An alternative, vanillic acid-inducible system is recently described for procyclic T. brucei, providing ∼18-fold increase in GFP reporter expression upon induction (Sunter JD. Mol. Biochem. Parasitol. 2016, 207:45–48). Here we describe a cumate-inducible system that allows efficient, tunable gene expression showing >300-fold increase in GFP expression upon induction. The cumate-inducible system can be used alone or together with the tetracycline-inducible system, in both procyclic and bloodstream form T. brucei. Efficient cumate-inducible expression is also achieved in T. brucei-infected mice.

Glycosome biogenesis in trypanosomes and the de novo dilemma.

Trypanosomatid parasites, including Trypanosoma and Leishmania, are the causative agents of lethal diseases threatening millions of people around the world. These organisms compartmentalize glycolysis in essential, specialized peroxisomes called glycosomes. Peroxisome proliferation can occur through growth and division of existing organelles and de novo biogenesis from the endoplasmic reticulum. The level that each pathway contributes is debated. Current evidence supports the concerted contribution of both mechanisms in an equilibrium that can vary depending on environmental conditions and metabolic requirements of the cell. Homologs of a number of peroxins, the proteins involved in peroxisome biogenesis and matrix protein import, have been identified in T. brucei. Based on these findings, it is widely accepted that glycosomes proliferate through growth and division of existing organelles; however, to our knowledge, a de novo mechanism of biogenesis has not been directly demonstrated. Here, we review recent findings that provide support for the existence of an endoplasmic reticulum (ER)-derived de novo pathway of glycosome biogenesis in T. brucei. Two studies recently identified PEX13.1, a peroxin involved in matrix protein import, in the ER of procyclic form T. brucei. In other eukaryotes, peroxins including PEX13 have been found in the ER of cells undergoing de novo biogenesis of peroxisomes. In addition, PEX16 and PEX19 have been characterized in T. brucei, both of which are important for de novo biogenesis in other eukaryotes. Because glycosomes are rapidly remodeled via autophagy during life cycle differentiation, de novo biogenesis could provide a method of restoring glycosome populations following turnover. Together, the findings we summarize provide support for the hypothesis that glycosome proliferation occurs through growth and division of pre-existing organelles and de novo biogenesis of new organelles from the ER and that the level each mechanism contributes is influenced by glucose availability.

Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite.

The mitochondrial (mt) FoF1-ATP synthase of the digenetic parasite, Trypanosoma brucei, generates ATP during the insect procyclic form (PF), but becomes a perpetual consumer of ATP in the mammalian bloodstream form (BF), which lacks a canonical respiratory chain. This unconventional dependence on FoF1-ATPase is required to maintain the essential mt membrane potential (Δψm). Normally, ATP hydrolysis by this rotary molecular motor is restricted to when eukaryotic cells experience sporadic hypoxic conditions, during which this compulsory function quickly depletes the cellular ATP pool. To protect against this cellular treason, the highly conserved inhibitory factor 1 (IF1) binds the enzyme in a manner that solely inhibits the hydrolytic activity. Intriguingly, we were able to identify the IF1 homolog in T. brucei (TbIF1), but determined that its expression in the mitochondrion is tightly regulated throughout the life cycle as it is only detected in PF cells. TbIF1 appears to primarily function as an emergency brake in PF cells, where it prevented the restoration of the Δψm by FoF1-ATPase when respiration was chemically inhibited. In vitro, TbIF1 overexpression specifically inhibits the hydrolytic activity but not the synthetic capability of the FoF1-ATP synthase in PF mitochondria. Furthermore, low μM amounts of recombinant TbIF1 achieve the same inhibition of total mt ATPase activity as the FoF1-ATPase specific inhibitors, azide and oligomycin. Therefore, even minimal ectopic expression of TbIF1 in BF cells proved lethal as the indispensable Δψm collapsed due to inhibited FoF1-ATPase. In summary, we provide evidence that T. brucei harbors a natural and potent unidirectional inhibitor of the vital FoF1-ATPase activity that can be exploited for future structure-based drug design.

Characterization of RBP9 and RBP10, two developmentally regulated RNA-binding proteins in Trypanosoma brucei.

The fate of an mRNA is determined by its interaction with proteins and small RNAs within dynamic complexes called ribonucleoprotein complexes (mRNPs). In Trypanosoma brucei and related kinetoplastids, responses to internal and external signals are mainly mediated by post-transcriptional processes. Here, we used proximity-dependent biotin identification (BioID) combined with RNA-seq to investigate the changes resulting from ectopic expression of RBP10 and RBP9, two developmentally regulated RNA-binding proteins (RBPs). Both RBPs have reduced expression in insect procyclic forms (PCFs) compared with bloodstream forms (BSFs). Upon overexpression in PCFs, both proteins were recruited to cytoplasmic foci, co-localizing with the processing body marker SCD6. Further, both RBPs altered the transcriptome from a PCF- to a BSF-like pattern. Notably, upon expression of BirA*-RBP9 and BirA*-RBP10, BioID yielded more than 200 high confidence protein interactors (more than 10-fold enriched); 45 (RBP9) and 31 (RBP10) were directly related to mRNA metabolism. This study validates the use of BioID for investigating mRNP components but also illustrates the complexity of mRNP function.