The T. brucei genome encodes 28 putative PPR proteins.
To identify PPR proteins in trypanosomatids, we screened the predicted T. brucei
proteome with TPRpred (18
), a new, sensitive tool for identifying proteins carrying repeated helical repeats such as PPR and TPR motifs. In this manner, we could increase the already unexpectedly large number of putative PPR proteins known in this group of organisms (Table ). Mingler et al. (28
) reported 23 PPR proteins in T. brucei
, whereas our analysis identifies 28, of which 10 were unique to our study (Table ). Conversely, five of the proteins identified in the previous analysis did not pass the criteria we used to define PPR proteins. Figure S1 in the supplemental material shows the distance tree obtained by aligning the putative PPR proteins of T. brucei
and L. major
together with schematic views of each protein, including the positions of the identified PPR motifs. The number of formally detected PPR motifs in these proteins ranges from 1 to 20, but in virtually all cases the sequence alignments and secondary structure predictions (data not shown) strongly suggest that structurally similar motifs adjoin, or are interspersed with, the motifs shown in Fig. S1 in the supplemental material. Thus, the number of motifs indicated represents an underestimation, and the total number of PPR proteins in the T. brucei
proteome still may be an underestimate. The likely subcellular localization of the candidate PPR proteins was predicted using Predotar (44
) and Mitoprot (9
). Both programs strongly predicted a mitochondrial localization for almost all of these proteins. The exceptions are the clusters related to Tb927.6.4190, Tb927.8.6040, and Tb11.01.7210 (Table ). The predicted mitochondrial localization is consistent with the current understanding of PPR protein function. The L. major
) contains putative orthologues for 25 of 28 T. brucei
PPR proteins (discounting one recently duplicated T. brucei
gene). This is a greater proportion than that for the genome as a whole; overall, only 77% of T. brucei
genes have clear orthologues in L. major
). Thus, PPR genes are particularly well conserved in trypanosomatids and are likely to have conserved functions. To begin to delineate what these functions might be, we selected eight putative PPR proteins of T. brucei
, termed TbPPR1 to TbPPR8, for further analysis.
Seven out of eight selected PPR proteins are mitochondrial.
All selected PPR proteins are predicted to have mitochondrial targeting signals. In order to determine their localization experimentally, we prepared transgenic cell lines allowing expression of the eight PPR proteins carrying either the Ty-1 peptide or the HA tag at their carboxy termini. Immunofluorescence analysis using antitag antibodies showed a colocalization of tagged TbPPR1, TbPPR2, TbPPR4, and TbPPR5 with the mitochondrial marker (Fig. ).
FIG. 1. Localization of epitope-tagged TbPPR1 to TbPPR8. (A) Double immunofluorescence analysis of T. brucei cell lines expressing the Ty-1 tag or the HA tag at their carboxy termini. The cells were double stained with monoclonal antitag antibodies (Tag) and (more ...)
The other four cell lines did not give a signal in immunofluorescence and therefore were subjected to a more sensitive biochemical analysis. Digitonin extractions were used to prepare mitochondrial and cytosolic fractions that could be analyzed by immunoblotting (7
). The results showed that the tagged TbPPR3, TbPPR6, and TbPPR7 copurify with the mitochondrial marker (Fig. ).
Tagged TbPPR8 is predominantly localized in the cytosol, even though it is predicted to have a mitochondrial import signal (Table ). However, while most of TbPPR8 is cytosolic, it cannot be excluded that a small fraction of it is imported into the mitochondrion.
In summary, except for TbPPR8, all selected PPR proteins are exclusively localized in mitochondria.
All tested PPR proteins are required for normal growth.
To study the function of the selected PPR proteins, we established stable transgenic cell lines that allow inducible RNAi-mediated ablation of each of the eight proteins. For each cell line, the efficiency of RNAi was verified by Northern analysis (Fig. , insets). Furthermore, even though PPR proteins belong to the same family, their nucleotide sequences show little similarity. Off-target effects of the RNAi can therefore be excluded.
FIG. 2. TbPPR1 to TbPPR8 are required for growth and survival in glucose-free culture medium. Shown are representative growth curves of uninduced (− Tet) and induced (+ Tet) representative clonal T. brucei TbPPR1 to TbPPR8 RNAi cell lines in standard (more ...)
Growth of uninduced and induced RNAi cell lines was tested in both the standard culture medium SDM-79 (6
) (Fig. , left graphs), which contains proline and glucose as the major energy sources, and in SDM-80, a modified version of SDM-79 that lacks glucose (21
) (Fig. , right graphs, −Glc). Three distinct phenotypes were observed in SDM-79. Ablation of TbPPR2, TbPPR3, TbPPR4, and TbPPR5 caused growth arrest, whereas induced TbPPR1, TbPPR6, and TbPPR7 RNAi cell lines kept growing, although at a much lower rate. Finally, growth of cells downregulated for TbPPR8 was not or was only marginally affected.
Interestingly, however, in SDM-80 all induced RNAi cell lines showed the same phenotype: they stopped growing, and a few days later they started to die. Thus, all eight tested PPR proteins are essential for survival in SDM-80.
In procyclic T. brucei
, glucose (after conversion into pyruvate) is used for mitochondrial substrate-level phosphorylation (SUBPHOS) in the trypanosome-specific acetate:succinate coenzyme A (CoA) transferase/succinyl-CoA synthetase cycle (33
). When grown in SDM-79, where glucose is available, the energy needs of T. brucei
can be fulfilled by substrate-level phosphorylation alone (5
). In glucose-free SDM-80, however, the sole energy source is proline that can be utilized only by oxidative phosporylation (OXPHOS) (21
). Growth in SDM-80 therefore selects for cells capable of performing efficient OXPHOS.
The mitochondrial gene products of T. brucei include cytochrome oxidase subunits (COX1 to COX3) and cytochrome b (CYTB), subunit 6 of the ATPase (A6), a ribosomal protein (RPS12), and the SSU and LSU rRNAs (9S and 12S rRNA). These gene products either function directly in OXPHOS or are components of the mitochondrial translation machinery, the function of which is to produce components of the OXPHOS complexes. Based on results with plants, PPR proteins are expected to be involved in posttranscriptional processes required for mitochondrial gene expression. Should this also be the case for T. brucei, we predict that the lack of PPR proteins ultimately will affect OXPHOS. The fact that RNAi-mediated ablation of all tested trypanosomal PPR proteins causes cell death in glucose-free SDM-80 medium but much milder phenotypes in SDM-79 medium supports this prediction. In conclusion, the results show that all eight PPR proteins analyzed perform nonredundant functions that ultimately are required for OXPHOS.
All tested PPR proteins are required for efficient OXPHOS.
We have recently established an assay that allows us to quantify the different modes of ATP production in isolated mitochondria of T. brucei
). This assay enables us to confirm whether the lack of growth of induced RNAi cell lines on glucose-free medium is indeed due to deficient OXPHOS. Besides OXPHOS, T. brucei
mitochondria can produce ATP either by SUBPHOS in the citric acid cycle or in the trypanosome-specific acetate:succinate CoA transferase/succinyl-CoA synthetase cycle (5
). To measure OXPHOS, mitochondria are incubated with ADP and succinate. To measure SUBPHOS in the citric acid cycle, α-ketoglutarate is used as a substrate, whereas measuring SUBPHOS in the acetate:succinate CoA transferase/succinyl-CoA synthetase cycle requires the addition of pyruvate as well as the cosubstrate succinate (5
). Atractyloside treatment prevents mitochondrial import of the added ADP and thus will inhibit all forms of mitochondrial ATP production. OXPHOS, in contrast to either form of SUBPHOS, is antimycin sensitive.
We tested the ability of mitochondria from all RNAi knockdown cell lines to perform either OXPHOS or SUBPHOS using both methods.
Figure shows that ablation of TbPPR1 to TbPPR7 selectively knocks down OXPHOS (induced by succinate) but does not interfere with either of the two forms of SUBPHOS (induced by α-ketoglutarate or pyruvate). Whereas OXPHOS was completely inhibited in TbPPR1 to TbPPR5 and TbPPR7 RNAi cell lines, it was only approximately 50% reduced in TbPPR6 RNAi cells. This is consistent with the relatively weak growth phenotype that ablation of TbPPR6 causes on SDM-79 medium (Fig. ), and it indicates that ablation of TbPPR6 only slightly interferes with the level of OXPHOS activity required for normal growth in SDM-79 medium.
FIG. 3. Ablation of TbPPR1 to TbPPR8 selectively abolishes OXPHOS. Succinate-, α-ketoglutarate-, and pyruvate-induced mitochondrial ATP production in crude mitochondrial fractions from uninduced (−) and induced cells (+) is shown for TbPPR1 (more ...)
Furthermore, we also tested TbPPR8 (Fig. ). It is required for growth in glucose-free SDM-80 medium, despite being mainly or even only extramitochondrially localized (Fig. ). SDM-80 medium selects for cells having maximal OXPHOS activity, suggesting that TbPPR8 function is at least indirectly connected to OXPHOS. This is supported by the ATP production assays presented in Fig. , which show that ablation of TbPPR8 causes a partial reduction of OXPHOS to approximately 60%. Thus, these results explain why the lack of TbPPR8 does not affect growth in SDM-79 medium that allows cells to grow even if their OXPHOS activity is suboptimal. It is possible that TbPPR8 stabilizes a cytosolic mRNA encoding a mitochondrial protein that is required for efficient OXPHOS. However, we did not further study TbPPR8, since the focus of the present work was on the mitochondrially localized PPR proteins.
Lack of six out of eight PPR proteins affects mitochondrial rRNAs.
Total RNA from uninduced and induced RNAi cell lines, isolated at the time of the first apparent growth phenotype, was analyzed by Northern hybridization to determine the steady-state levels of mitochondrially encoded RNAs. Blots were probed for COX1, COX2, and CYTB (for TbPPR1 to TbPPR8) as well as for RPS12 and A6 mRNAs (for TbPPR1 to TbPPR5). COX2 and CYTB transcripts are edited in a small domain only. The hybridization probes therefore detected both edited and unedited RNAs. RPS12 and A6 transcripts, on the other hand, are extensively edited. Thus, two probes were used: one that detects unedited and minimally edited RNAs, and another one that recognizes extensively and fully edited molecules. Finally, the levels of 9S and 12S rRNAs were analyzed in all eight RNAi cell lines.
As expected based on previous analyses, most mRNAs in the uninduced cell lines showed a doublet of closely spaced bands (4
) (Fig. ). These correspond to two mRNA populations having poly(A) tails of different lengths (approximately 20 nucleotides and 200 nucleotides). In six out of the eight induced cell lines, the two bands converged into a single, more intense band corresponding in size to the population having a short poly(A) tail. The presence of a short poly(A) tail on the COX1 mRNA in induced TbPPR2 cells was experimentally verified by circular reverse transcription-PCR (data not shown). Furthermore, comigration of oligo(dT)/Rnase H-treated CYTB mRNA in induced TbPPR2 with the corresponding untreated mRNA is consistent with the absence of the long poly(A) tail population in the knockdown cells (Fig. ). Shortening of the poly(A) tail was seen for all mature mRNAs, at least at later times of induction (in Fig. this is best visible for COX2, CYTB, and edited RPS12). This suggests that it is an indirect effect of PPR protein depletion caused by the reduced ATP levels due to the loss of OXPHOS. In order to test this possibility, we performed Northern analyses for COX1, COX2, and CYTB mRNAs in RNAi cell lines ablated for cytosolic and mitochondrial tryptophanyl-tRNA synthetases (TrpRS) (Fig. ). These two proteins are essential for the normal growth of procyclic T. brucei
, as was the case for TbPPR1 to TbPPR7 (7
). Furthermore, ablation of the mitochondrial TrpRS shows a selective inhibition of OXPHOS identical to that observed in the induced TbPPR1 to TbPPR5 RNAi cell lines (Fig. ). Figure shows that no qualitative differences of COX1, COX2, and CYTB mRNAs were observed in cells ablated for cytosolic TrpRS. However, in cells ablated for the mitochondrial enzyme, only the lower band was detected. Thus, ablation of the mitochondrial TrpRS had the same effect on mitochondrial polyadenylation as knockdown of six of the eight tested PPR proteins. Moreover, the lack of accumulation of the short poly(A) tails in the TbPPR6 and TbPPR8 knockdown cell lines is consistent with the observation that these two cell lines grow essentially normally on SDM-79 medium (Fig. ) and with the only partial inhibition of OXPHOS observed in the TbPPR8 cell lines (Fig. ).
FIG. 5. Short poly(A) tail is a consequence of the lack of OXPHOS. (A) Northern blot analysis of the poly(A) tail length of CYTB mRNAs in uninduced (−Tet) and induced (+Tet) TbPPR2 cells by oligo(dT)-induced RNase H digestion. Additions of RNase (more ...)
To obtain accurate quantitative information on mitochondrial RNA levels, we performed replicate Northern blots (n = 3 to 6) for each of the tested RNA species in all eight RNAi cell lines. The levels of the tested mRNAs were normalized using the mRNA of cytosolic TrpRS as an internal standard. Figure shows the means of the log2-transformed relative levels of each individually normalized RNA species of induced cells relative to the normalized one in uninduced cells.
This quantitative analysis revealed three distinct phenotypes: (i) induced TbPPR1 cell lines showed a specific 2.3-fold reduction of the COX1 mRNA level, (ii) ablation of the putative cytosolically localized TbPPR8 did not affect the level of any of the tested mitochondrial RNAs, and (iii) knockdown of TbPPR2 to TbPPR7 caused a three- to eightfold reduction of at least one mitochondrial rRNA. These results suggest that the mitochondrial rRNAs are the targets of six out of the seven tested mitochondrial PPR proteins. This is very different from the plant PPR proteins that are generally found to affect processing or expression of mRNAs. Furthermore, even though TbPPR2 to TbPPR7 all specifically affect rRNAs, there is no functional redundancy among these proteins, since all are individually essential for efficient OXPHOS (Fig. and ). It should be noted, though, that it is possible that the phenotypes of the RNAi cell lines might be more complex, since we did not test all mitochondrially encoded RNAs.
Kinetics of mitochondrial rRNA depletion.
In the next series of experiments, we analyzed the kinetics of 9S and 12S rRNA depletion in the TbPPR2 to TbPPR7 RNAi cell lines relative to each other and to the appearance of the growth phenotype. Figure shows that the loss of the mitochondrial rRNAs is very rapid. In all cell lines, the level of the two rRNAs already reached less than 50% for at least one rRNA species 24 h after induction of RNAi. This is long before the appearance of the growth phenotypes and suggests that rRNA depletion is a direct consequence of the ablation of the various PPR proteins.
FIG. 6. Kinetics of mitochondrial rRNA depletion. (A) Graph showing the relative changes in the levels of mitochondrial rRNAs during ablation of the indicated PPR proteins. The level of rRNAs in uninduced cells was set to 100%. All levels of rRNAs were (more ...)
The rRNA depletion is specific to the TbPPR2 to TbPPR7 RNAi cell lines. It does not occur in the TbPPR1 RNAi cell line (Fig. ) and therefore is a general consequence of neither the growth arrest nor the inhibition of OXPHOS. Furthermore, degradation of rRNAs also does not occur in cells that are ablated for TbPPR8 (Fig. ) or for mitochondrial TrpRS (Fig. ), an essential mitochondrial protein.
In all but the TbPPR4 RNAi cell line, degradation of the 12S rRNA is more extensive than degradation of the 9S rRNA. These results suggest that the selected PPR proteins, with the exception of TbPPR4, may act primarily on the 12S rRNA. However, it is also clear that eventually both rRNAs are affected in all cell lines, indicating that the levels of the two rRNAs are coregulated.
PPR proteins affecting rRNAs are membrane associated.
To investigate the physical connection of PPR proteins with organellar ribosomes, we purified mitochondria from cell lines expressing tagged TbPPR2 to TbPPR5 and fractionated them into membrane and matrix fractions. Mitoribosomes generally are associated with the mitochondrial inner membrane. This also applies for T. brucei, as seen in Fig. . Both 9S and 12S rRNAs as well as a tagged ribosomal protein of the LSU (Fig. ) are quantitatively associated with the membrane fractions. tRNAs and α-ketoglutarate dehydrogenase (KDH), however, mainly are recovered in the matrix fraction, as expected. Tagged TbPPR2 to TbPPR5 cofractionate with the membrane fraction and thus with the ribosomal markers.
Except for Tb927.7.1350 and Tb11.03.0440, as well as their orthologues LmjF26.0610 and LmjF25.0630, which likely contain a single transmembrane segment, none of the trypanosomatidal PPR proteins is predicted to be an integral membrane protein. In line with this, all tested membrane-associated PPR proteins are recovered in the supernatant fraction after carbonate extraction, whereas most of the integral membrane protein cytochrome oxidase subunit 4 remains in the pellet (Fig. , bottom panels). In summary, these results are consistent with an association of the tested PPR proteins with mitochondrial rRNAs.
Interestingly, however, tagged TbPPR1, whose function is not linked to mitochondrial rRNAs but to the COX1 mRNA, is at least partially recovered in the matrix fraction (Fig. ). Thus, these results are consistent with the idea that TbPPR2 to TbPPR5 and the mitochondrial rRNAs are part of the same macromolecular complexes.
TbPPR5 is associated with 12S rRNA.
Purification of mitoribosomes of trypanosomatids is complicated by the lack of a functional assay as well as by the presence of additional rRNA-containing particles, and therefore the purification is technically very challenging (24
). As an alternative, we performed immunoprecipitations from mitochondrial extracts that originate from cell lines expressing tagged TbPPR2, TbPPR3, and TbPPR5 using anti-HA tag antibodies. While we were not able to precipitate tagged TbPPR2 and TbPPR3, presumably because the proteins are in tight (perhaps ribosomal) complexes that hide the epitope from the antibody, a significant fraction of the tagged TbPPR5 was recovered from the bound fraction (Fig. ). Most interestingly, some of the 12S rRNA coprecipitated with the tagged protein, whereas this was not the case for the 9S rRNA or tRNAs. Thus, this experiment directly shows that TbPPR5 is directly or indirectly associated with the LSU of the mitoribosome.
FIG. 8. TbPPR5 is associated with 12S rRNA. Mitochondrial extract of a cell line expressing HA-tagged TbPPR5 was subjected to immunoprecipitation using anti-HA antibody. The total extract (Tot.), the unbound fraction (UB), and the bound fraction (B) were analyzed (more ...) rRNA-affecting PPR proteins and mitochondrial rRNAs are coregulated.
If the rRNA-affecting PPR proteins are in the same particle and/or are bound to the same RNAs, we might expect that ablation of one would lead to degradation of the other. To test this, we prepared RNAi cell lines allowing RNAi-induced ablation of TbPPR4 or TbPPR5 with simultaneous inducible expression of tagged TbPPR2 or/and TbPPR6, respectively (Fig. ). For all these cell lines we monitored the kinetics of rRNA depletion and the level of the tagged proteins during induction of RNAi. The results show that ablation of one set of PPR proteins leads to the degradation of tagged PPR proteins that are not targeted by RNAi. There are two main interpretations of these results: (i) the selected PPR proteins directly interact with each other and form a protein complex that is destabilized by ablation of one of its members, and (ii) the degradation of the tagged PPR proteins in the RNAi cell lines is caused indirectly by the depletion of the rRNAs. The observation that the degradation of tagged TbPPR2 in the TbPPR4 RNAi cell line is less extensive than the degradation of the rRNAs suggests that depletion of the tagged proteins might, in this case, mainly be triggered by the lack of protein binding partners rather than by the altered levels of the rRNAs. In the TbPPR4 RNAi cell line, however, both the rRNA and the tagged TbPPR6 are degraded to approximately the same extent, consistent with the idea that it is the ablation of the rRNA that causes the disappearance of the tagged protein. These two explanations, that degradation of the tagged PPR proteins is caused by either missing protein-protein or missing protein-RNA interactions, clearly are not mutually exclusive. Most importantly, both imply that the tested PPR proteins are functionally linked to rRNAs.
FIG. 9. Fate and effect of tagged PPR proteins in RNAi cell lines ablated for other PPR proteins. (A) For the left column, a cell line allowing inducible ablation of TbPPR4 with simultaneous inducible expression of tagged TbPPR2 was tested for the kinetics of (more ...) Overexpression of PPR proteins slows down RNAi-induced phenotypes.
Unexpectedly, expression of the tagged PPR proteins delayed and reduced the extent of depletion of the mitochondrial rRNAs compared to the amount of depletion in the parent RNAi cell lines. This is probably due to the fact that the tagged PPR proteins were overexpressed compared to the expression of endogenous protein (Fig. , middle panels). Causing a delay of rRNA degradation appears to be a general effect of PPR protein overexpression; it was seen in all combinations of tagged proteins and RNAi cell lines tested. In line with their presumed association with the 12S rRNA, the main effect of overexpressing tagged TbPPR2 and TbPPR6, when tested in TbPPR4 RNAi cell lines, was a slowing down of the degradation of the 12S rRNA. The 9S rRNA was affected only for 24 h. However, when tagged TbPPR6 was overexpressed in TbPPR5 RNAi cells, it slowed down the degradation of both 9S and 12S rRNAs. Moreover, in all cell lines, overexpression of tagged PPR proteins suppressed the growth phenotype caused by the RNAi by 24 h (data not shown). Finally, in a TbPPR4 RNAi cell line that simultaneously overexpresses both tagged TbPPR2 and TbPPR6, the complementation effect was cumulative to the one seen in the cell line expressing the tagged proteins individually (Fig. ). Thus, the degradation of the 12S rRNA essentially was abolished, and the growth arrest appeared 48 h later than that in the parent RNAi cell line.
PPR proteins are hypothesized to bind to specific RNA sequences. In agreement with this model, all mitochondrial PPR proteins we investigated were individually essential for OXPHOS (Fig. and ). Thus, it is unlikely that overexpression of a PPR protein allows it to bind to an RNA sequence that normally is recognized by the ablated PPR protein. However, there might be some redundancy in the putative protein-protein interactions. Thus, overexpression of a PPR protein might indeed allow it to stabilize a protein complex that lacks the ablated PPR protein, even though if it were not expressed at high levels it would not bind to it. However, full complementation would require both correct protein-protein and correct RNA-protein interactions and therefore cannot be achieved by overexpression of heterologous PPR proteins. In summary, even though the underlying mechanism of how overexpression of one set of PPR proteins delays the RNAi-induced phenotypes of another set is unknown, the results underscore that rRNA-affecting PPR proteins are intricately connected to each other and to the mitochondrial rRNAs.