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In addition to this, the Fe-S proteome of this divergent protist remains vastly unexplored, thus providing an excellent opportunity to study these two biological questions. In this work, we demonstrate that the late-acting module of the CIA machinery is essential for the survival of this parasite in vitro , but not in vivo.

Only Tb CIA1 has been characterized to date [ 25 ]. As in humans, two genes encoding homologues of yeast Cia2 protein were found in T. Fixed parasites were probed with anti-V5 and anti-enolase antibodies Tb ENO [ 29 ] to detect the fusion proteins and the cytosolic marker, respectively. To further confirm this finding, the subcellular distribution of the CIA pathway components was analysed by a fractionation with digitonin. For this, we incubated the cells with a concentration of digitonin that liberates the cytosol, separating it from the mitochondrial fraction Fig 1B.

The pellet P denotes the insoluble fraction after solubilizing the mitochondrial fraction, which exhibits proteins that are membrane-bound, such as part of Tb mtHSP A parallel corroboration was performed by selective permeabilisation with digitonin. In this experiment, equal numbers of cells were incubated with increasing concentrations of the detergent, causing progressive cell membrane permeabilisation and sequential release of the cytosolic and organellar fractions.

Anti-V5 antibody green was used to detect the CIA proteins localized throughout the cell body. Enolase red was used as a cytosolic marker. The merge displays co-localization of enolase with the V5-tagged proteins. B Isolation of mitochondrial fraction with digitonin. PCF trypanosomes were incubated with 0. V5-tagged targets were visualized with anti-V5 monoclonal antibody. MtHSP70 and enolase were used as mitochondrial and cytosolic markers, respectively.

The graphs represent densitometric quantifications of the Western blots for each experiment. However, stringent pairwise knockdowns of the early-acting components of this pathway e. We sought to employ this phenomenon by silencing the expression of two CTC members simultaneously. Western blots shown under each growth curve were probed with anti-HA, anti-V5 or specific antibodies and were used to assess protein expression before and after RNAi induction D-F. Anti-tubulin or anti-enolase antibodies were used as loading controls. To assess the role of the CTC components on the pathogenicity of T.

As shown in S2 Fig , these infection experiments suggest that neither protein is essential in BSF, in agreement with the mild in vitro growth phenotypes described above, as well as with the initial observation of the double-knockdown cell lines in this life stage, in which at least two components of the pathway had to be ablated in order to obtain a clearer growth phenotype S1 Fig [ 25 ]. We next asked whether depleting the cells of individual CTC members would impact the activity of known Fe-S proteins. Aconitase Tb ACO , a Fe-S enzyme that catalyses the reversible isomerisation of citrate to isocitrate, is encoded by a single gene and has a dual subcellular localisation, being ca.

These features qualify it as a suitable surrogate for Fe-S cluster-dependent enzymatic activity in these two cellular compartments [ 27 ].

Several lines of evidence have linked the pathways for Fe-S cluster biogenesis to DNA repair processes in humans, yeast, and plants [ 13 , 14 , 34 — 37 ]. Surprisingly, even efficient depletion of the CTC members did not affect the ability of T.

When depleted of Tb CIA2B, both life stages were significantly more susceptible to DFO compared to those with normal levels of this protein Fig 3A and 3C , suggesting a decrease in the pool of available intracellular iron. Furthermore, ferene assays suggested that the content of iron bound to proteins in the cytosolic lysates of TbCIA2B knockdowns was lower than that found in uninduced cells, whereas protein-bound iron levels did not change in the organellar fractions Fig 3J.

The values shown in the inset of the curves are the mean DFO EC 50 s for induced or uninduced cultures. J PCF Tb CIA2B RNAi cells were grown for 4 days in the presence red or absence blue of tetracycline and the content of iron bound to proteins was measured in the cytosolic and organellar fractions of digitonin permeabilised parasites. Plasmids without insert or plasmids encoding endogenous yeast CIA genes were used as controls. Subsequently, these constructs were transformed into regulatable yeast Gal-CIA mutants, in which the expression of the cognate CIA gene is induced in the presence of galactose and repressed by the presence of glucose as described elsewhere [ 25 ].

These findings show that Tb CIA2B can partially take over the role of its yeast counterpart, suggesting that it performs an orthologous function. The result was reproduced at least three times with independent transformations. Individual interactions of the CTC proteins had only been mapped in detail for a few representatives of the eukaryotic supergroup Opisthokonta [ 44 ].

Moreover, the progress made in the field of Fe-S biology in the past decade suggests Fe-S proteins are diverse and abundant in a typical eukaryotic cell, but remained overlooked due to the difficulties related to their instability under aerobic conditions.

One of the most valuable tools that contributed to expanding the list of mammalian Fe-S proteins was the use of mass spectrometry MS and affinity purifications to detect potential Fe-S proteins interacting with the human CIA targeting complex [ 13 , 14 , 49 ]. Therefore, aiming to gain insight into the composition of the T.

In all cases, MS detected proteins co-purifying with the tagged baits. Eluates were resolved in Bis-Tris gels and sections were analysed by mass spectrometry for protein identification. H Schematic representation of the protein-protein interaction profile of the T. Next, in an attempt to identify potential target Fe-S proteins, we investigated other proteins co-eluting with members of the CTC.

To inquire if these proteins were known or could be predicted to contain Fe-S clusters, the amino acid sequences of the hits were retrieved from the TriTryp database [ 46 ] and analysed with MetalPredator [ 48 ], a tool to predict Fe-S clusters in polypeptide chains based on the presence of known Fe-S domains and metal-binding motifs. Equimolar amounts of purified recombinant proteins or glutathione S-transferase GST , used as a negative control, were coupled to glutathione Sepharose 4B beads, incubated with soluble extracts of PCF parasites expressing HA-tagged Tb FHc, and the interactions were assessed by Western blotting.

In order to verify possible protein-protein interactions of Tb CIA2B that could not be detected by other methods, this protein was expressed in E. This difference may reflect stage-specific requirement of the CIA pathway. Importantly, mutual interactions of the CTC members are the same in the BSF and PCF cells, although the sets of their targets differ from one another and require further analysis to determine their capability to bear an Fe-S cluster.

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We are currently pursuing the possibility that these proteins harbour an Fe-S cluster by heterologous expression and purification. Aiming to better understand the dynamics of the interactions amongst the CTC members, it was of interest to identify the site through which members of this complex interact. However, the amino acid sequence of Tb MMS19 is poorly conserved when compared to its human or yeast homologues [ 52 ], and no crystal structures of this protein have been elucidated so far. We hypothesised that the binding site for the CIA proteins would be different in trypanosomes, given the divergent amino acid composition and apparent different distribution of the repeats.

Growth curves of PCF T. E - G Parasites were grown for 2 or 4 days in the presence or absence of tetracycline.

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A Schematic representation of T. The N-terminal domain is depicted in blue and the C-terminal domain in red.

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Cell extracts of T. Labelled residues at the inset had the highest SVM-Hot spot scores. One possible explanation is that C1 adopted a fold that hinders the ability of Tb CIA2B to reach the interaction surface which could, in turn, be more accessible in C2. We also observed that the C3 and C4 fragments have an enhanced ability to bind Tb CIA2B in cell extracts in comparison to the purified recombinant protein, hinting at the presence of a factor that stabilises the complex.

The predicted structures were subsequently used to generate models of protein-protein interaction by in silico docking with ClusPro [ 59 ]. Given this reassuring overlap between the experimental data and in silico predictions, we aimed to refine this analysis by examining the best complex model with PredHS, a tool that integrates analysis of structural and energetic properties to identify regions at the contact surface, which are more likely to be crucial for protein-protein interactions i.

These residues are depicted in a scale of red in Fig 7E. This model fits satisfactorily our experimental data, which indicated that the C2 fragment Val -Lys binds tightly to r Tb CIA2B, while the C3 fragment Thr -Lys interacted very weakly with it, although this association was stabilised when the native protein was present in cell lysates.

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Collectively, these results indicate that Tb CIA2B binds directly and tightly to the C-terminal domain of Tb MMS19, but this interaction is likely to require additional factors to stabilise the complex. Since the subcellular localisation of the CIA components seems to depend upon the organism under study [ 34 , 62 — 66 ], we aimed to clarify the cellular compartment in which the late-acting module of the CIA machinery was present in trypanosomes. For this aim, a combination of immunofluorescence and immunoblot analyses of detergent-permeabilised cell extracts localised all four studied proteins to the cytosol, in agreement with data from mammalian cells [ 13 , 15 , 67 ].

However, Mms19 in Schizosaccharomyces pombe , and Cia1 in S. In the plant Arabidopsis thaliana , MMS19 is exclusively cytosolic, although other members of the CTC exist both in the nuclear and cytosolic compartments [ 66 ]. Moreover, Giardia intestinalis exhibits a dual localisation of Cia2, between the intermembrane space of the mitosome and the cytosol [ 45 ].

Interestingly, overexpressing the C-terminal domain of Tb MMS19 produced a dominant-negative phenotype with severe defects on the cell growth and concomitant up-regulation of the Tb CIA2B levels. One plausible explanation for this finding concerns the modes of interaction within the CTC, since the C-terminal domain of Tb MMS19 appears to be the docking site of the targeting complex, as recently described also for human cells [ 58 ].

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It is possible that high levels of this truncated protein can sequester Tb CIA2B, Tb CIA1, as well as client proteins into non-functional complexes, thus depleting the cell of at least two CTC members and mimicking the effect of a double knockdown. Though the MS data suggests non-redundant functions, such as the formation of different subcomplexes among various components of the CTC, the growth phenotype in the RNAi cell lines, as well as the capability of infection of BSF RNAi cell lines, hint at the possibility of function overlapping.

However, residual proteins escaping RNAi knockdown may be sufficient to maintain the functionality of the CIA machinery.

Conversely, the CIA proteins do not exert a direct impact on iron regulation in S. This is in line with previous studies, which demonstrated that the mitochondrial pool of this enzyme is matured by the ISC pathway, the mitochondrial machinery for Fe-S biogenesis [ 70 — 72 ], while the cytosolic fraction requires both the ISC and CIA machineries to obtain its cluster [ 25 , 73 ].

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The specificity of this effect was confirmed by incubating trypanosomes with deferoxamine pre-saturated with iron, which abolished its toxicity. This effect was consistent, although not as pronounced as in conditional null mutants of the cation channel mucolipin 1 that delivers iron to the cytosol of BSF flagellates [ 74 ]. Although IRP1-like mechanisms implicating the CIA machinery in iron sensing and regulation, such as those described in human cells [ 15 ], seem unlikely to exist in T.

Deferoxamine acts by scavenging the cellular labile iron pool LIP , thus preventing incorporation of this element into the newly synthesised apo-proteins [ 75 ]. The precise composition of LIP is uncertain, but free iron is seldom present in the intracellular milieu, given its capacity to generate reactive oxygen species via the Fenton reaction [ 26 , 76 ]. The source of iron for the assembly of Fe-S clusters in the cytosol remains unknown, although one line of thought speculates that the scaffold proteins for Fe-S cluster assembly can bind LIP directly [ 77 ].