Pentatricopeptide repeat proteins in Trypanosoma brucei function in mitochondrial ribosomes

Abstract

The pentatricopeptide repeat (PPR), a degenerate 35-amino-acid motif, defines a novel eukaryotic protein family. Plants have 400 to 500 distinct PPR proteins, whereas other eukaryotes generally have fewer than 5. The few PPR proteins that have been studied have roles in organellar gene expression, probably via direct interaction with RNA. Here we show that the parasitic protozoan Trypanosoma brucei encodes 28 distinct PPR proteins, an extraordinarily high number for a nonplant organism. A comparative analysis shows that seven out of eight selected PPR proteins are mitochondrially localized and essential for oxidative phosphorylation. Six of these are required for the stabilization of mitochondrial rRNAs and, like ribosomes, are associated with the mitochondrial membranes. Furthermore, one of the PPR proteins copurifies with the large subunit rRNA. Finally, ablation of all of the PPR proteins that were tested induces degradation of the other PPR proteins, indicating that they function in concert. Our results show that a significant number of trypanosomal PPR proteins are individually essential for the maintenance and/or biogenesis of mitochondrial rRNAs.

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 a polyclonal antiserum directed against a subunit of the mitochondrial ATPase (ATPase). A merged picture of the antitag antibody and the ATPase staining (Merge) as well as the Nomarski picture (Nom.) is shown in the bottom two panels. (B) Immunoblot analysis of 0.3 × 10⁷ cell equivalents each of total cellular (Tot.), crude cytosolic (Cyt.), and crude mitochondrial (Mit.) extracts for the presence of the indicated tagged PPR proteins (top panels). Only the relevant regions of the blots are shown. Comparison to molecular size markers showed that the sizes of the tagged proteins were consistent with the predictions (data not shown). Elongation factor 1a (EF-1a) served as a cytosolic marker (middle panel), and KDH served as a mitochondrial marker (bottom panel).

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 culture medium SDM-79 (left graphs) and in culture medium SDM-80 that lacks glucose (−Glc) (right graphs). The crosses indicate that further incubation led to the death of the whole population. Insets depict Northern blots of the corresponding TbPPR mRNAs. The RNA from induced cells was isolated at the time of the growth arrest (arrows). The rRNAs in the lower panel serve as loading controls.

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 to TbPPR8 RNAi cell lines. The substrates tested and the additions of antimycin (Antim.) and atractyloside (Atract.) are indicated at the top. ATP production in mitochondria isolated from uninduced cells tested without antimycin or atractyloside is set to 100%. The bars represent means expressed as percentages. Standard errors and the number of independent replicates (n) are indicated.

FIG. 4.

Lack of six out of eight PPR proteins affects mitochondrial rRNAs. (A) Total RNA from the indicated uninduced (−Tet) and induced (+Tet) RNAi cell lines was analyzed for the levels of COX1, COX2, CYTB, preedited (PE) A6 and edited (E) A6, preedited and edited RPS12 RNAs, and 9S and 12S rRNAs using Northern hybridization. To normalize for loading differences, each filter was reprobed for the mRNA of the cytosolic TrpRSs (lower part of each panel). (B) Graph showing the means of log₂-transformed normalized levels of each RNA species relative to that in uninduced cells [log₂(RNA_+Tet/RNA_−Tet)] obtained from three to six independent RNAi experiments. The 95% confidence intervals (means ± 2 standard errors) are indicated.

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 H and oligo(dT) are indicated. (B) Total RNA isolated from uninduced and induced cytosolic (Cyto.) or mitochondrial (Mito.) TrpRS RNAi cell lines was analyzed for COX1, COX2, and CYTB mRNAs (and for 9S and 12S rRNA in the case of the mitochondrial TrpRS) using Northern hybridization. As a loading control, filters from the cytosolic TrpRS RNAi cell line were reprobed for the mRNA of mitochondrial TrpRS and vice versa (lower part of each panel).

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 normalized by reprobing the same blots with cytosolic TrpRS. For each cell line, the growth phenotype was monitored in parallel. The gray bar indicates the time interval at which the growth phenotype became apparent. The interval encompasses 24 h before and 24 h after the first appearance of the growth phenotype. Each experiment was repeated at least twice, and the variation between the same time points in the two experiments was less than 10%. (B) Kinetics of mitochondrial rRNA depletion in the TbPPR1 RNAi cell line.

FIG. 7.

PPR proteins required for rRNA accumulation are membrane associated. (A) Mitochondria of cell lines expressing the indicated tagged PPR proteins were fractionated into membrane (Mem.) and matrix (Mat.) fractions. The top two panels show the ethidium bromide staining of RNA isolated from the two fractions; only the regions corresponding to the mitochondrial rRNAs and tRNAs are shown. The middle two panels show immunoblot analyses for the tagged PPR proteins and the mitochondrial marker KDH. The bottom two panels show immunoblot analyses for the tagged PPR proteins and the integral membrane protein COX4 of the pellet (P) and supernatant (S) fractions from carbonate-extracted mitochondrial membranes. The images in panels B and C are similar to those in panel A, but cell lines expressing a tagged mitochondrial LSU protein (TbMRPL21) or tagged TbPPR1 were analyzed. The membrane and matrix fractions that are compared correspond to equal cell equivalents.

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 for the presence of HA-tagged TbPPR5 or KDH (top panel) using immunoblots and were analyzed for mitochondrial rRNAs and tRNA^Ile using Northern blotting. The percentages of the total samples that were analyzed in the different lanes are indicated at the bottom.

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 mitochondrial rRNA depletion as well as for depletion of tagged TbPPR2. The top panels show immunoblot analyses for the tagged TbPPR2 and KDH as a loading control, respectively. The middle panels show Northern blot analyses of the RNAi-targeted TbPPR4 mRNA and the corresponding ethidium bromide stain of the cytosolic rRNAs as a loading control, respectively. The bottom panels show Northern blot analyses of the epitope-tagged TbPPR2 encoding mRNA and the corresponding ethidium bromide stain of the cytosolic rRNAs as a loading control, respectively. The lower and upper arrows indicate the position of the wild-type TbPPR2 mRNA and the mRNA-encoding epitope-tagged TbPPR2, respectively. The upper graph shows the relative changes in the levels of mitochondrial 9S rRNA and of tagged TbPPR2 during RNAi-induced ablation of TbPPR4. The graph at the bottom shows the same for the 12S rRNA. In both graphs the rRNA depletion kinetics in the parent TbPPR4 RNAi cell line that does not express the tagged PPR protein are indicated in light gray. These curves are identical to the ones shown in Fig. 6. The middle column is similar to the first column, but the analysis was done for a TbPPR4 RNAi cell line expressing tagged TbPPR6. The right column is similar to the other two columns, but the analysis was done for a TbPPR5 RNAi cell line expressing tagged TbPPR6. The images in panel B are similar to those in panel A, but analysis was done for a TbPPR4 RNAi cell line expressing both tagged TbPPR2 and tagged TbPPR6, respectively. All curves were determined twice in two independent experiments, yielding very similar results.