MOLECULAR AND CELLULAR BIOLOGY, Oct. 2007, p. 6876–6888
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 27, No. 19
Pentatricopeptide Repeat Proteins in Trypanosoma brucei Function in
Mascha Pusnik,1Ian Small,2Laurie K. Read,3Thomas Fabbro,1and Andre ´ Schneider1*
Department of Biology/Cell and Developmental Biology, University of Fribourg, CH-1700 Fribourg, Switzerland1; ARC Centre of
Excellence in Plant Energy Biology, University of Western Australia, Crawley, Perth 6009, WA, Australia2; and
Department Microbiology and Immunology, SUNY Buffalo School of Medicine, Buffalo, New York 142143
Received 23 April 2007/Returned for modification 9 May 2007/Accepted 13 July 2007
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.
Sequencing of the Arabidopsis genome led to the discovery
of a very large protein family that is defined by degenerate
35-amino-acid pentatricopeptide repeat (PPR) motifs. PPR
proteins contain 2 to 26 PPR motifs that usually are arranged
as tandem arrays (45). The genome of Arabidopsis encodes 450
distinct PPR proteins. Experimental and bioinformatic analy-
ses have shown that most of these are targeted to organelles
(approximately 75% to mitochondria and 25% to plastids)
(22). Inactivation of plant PPR proteins frequently causes em-
bryonic lethality (10, 22), showing that despite the large size of
the PPR protein family, there is little functional redundancy.
Studies of individual PPR proteins indicate that they function
at essentially all levels of organellar gene expression, including
transcription, RNA processing and editing, and RNA stability
and translation (1, 31, 41). The simplest explanation for these
diverse functions is that PPR proteins are sequence-specific
RNA binding proteins capable of recruiting effector enzymes
to defined sites on organellar RNAs (22). This idea is sup-
ported by structural models of the PPR motif and by the fact
that a number of PPR proteins are able to bind RNAs in vitro.
However, evidence of binding specificity for physiological sub-
strates has been shown in only a few cases (27, 30, 34).
One of the most intriguing aspects of PPR proteins is their
phylogenetic distribution. They seem to be absent from the bac-
terial domain. No PPR proteins have been identified in the ge-
nomes of Escherichia coli, Rickettsia prowazekii, and Synechocystis
(22), the latter two being the closest extant relatives of mitochon-
dria and plastids, respectively. However, with a few exceptions,
extraordinary discrepancy between their numbers in plant and
nonplant organisms. Whereas plants have several hundred PPR
proteins, only five and six putative PPR proteins are encoded by
the yeast and human genomes, respectively (22).
A number of nonplant PPR proteins have been studied as
well. PET309, the first PPR protein to be investigated, is a
yeast protein essential for expression of the mitochondrial cox1
gene (23). A similar PPR protein in humans also is required for
correct expression of COX1, and mutations in this PPR gene
are linked to genetic myopathies (29). The picture emerging
from these and other studies (reviewed in reference 1) is that,
as in plants, PPR proteins are involved in the expression of
specific mitochondrial RNAs. However, their mode of action
remains very poorly understood.
A recent study identified 23 distinct putative PPR proteins in
the genome of the parasitic protozoan Trypanosoma brucei
(28). Our own analysis of the T. brucei genome using different
bioinformatic methods detected 28 distinct PPR proteins (Ta-
ble 1; also see Fig. S1 in the supplemental material). These
numbers are much higher than those for any other nonplant
organism. The mitochondrial RNA metabolism of T. brucei is
known to have many unique features. The most prominent
ones in the context of this work are RNA editing and aberrant
short rRNAs. Most mitochondrial mRNAs in T. brucei require
extensive RNA editing by multiple uridine insertions and/or
deletions. However, unlike the case in plant organelles, where
the specificity of editing is likely determined by PPR proteins
(20, 41), in trypanosomatids the specificity of RNA editing is
mediated by short RNA transcripts, termed guide RNAs. The
12S large subunit (LSU) and 9S small subunit (SSU) rRNAs of
trypanosomatid mitochondria are among the smallest found in
nature (11, 12); as a consequence, the mitochondrial ribosomes
* Corresponding author. Mailing address: Department of Biology/
Cell and Developmental Biology, University of Fribourg, Chemin du
Muse ´e 10, CH-1700 Fribourg, Switzerland. Phone: (41)263008877.
Fax: (41)263009741. E-mail: email@example.com.
† Supplemental material for this article may be found at http://mcb
?Published ahead of print on 23 July 2007.
(mitoribosomes) of T. brucei are expected to have a higher
protein content than other ribosomes (24, 25).
approximately an order of magnitude compared to those of other
eukaryotes makes trypanosomes an excellent system to study the
function of nonplant PPR proteins. Here we present a compara-
tive analysis of eight putative PPR proteins of T. brucei.
MATERIALS AND METHODS
Bioinformatic analysis. Predicted T. brucei protein sequences were obtained
from the version 4 release of the genome annotation (3). The T. brucei proteome
was screened for PPR proteins using TPRpred (18). Proteins with a TPRpred
probability score of over 25% were retained. PPR motifs are characteristically
found in tandem arrays, and therefore all proteins not identified as containing
tandem arrays of motifs were investigated further using BLAST searches against
the total nonredundant protein database at NCBI. Twelve proteins lacking ob-
vious matches to multiple known PPR proteins across a region of over 70 amino
acids (indicating at least two motifs in tandem) were eliminated from the study.
The resulting set of 28 T. brucei proteins was used to recover putative Leishmania
major orthologues from GeneDB (16). Putative PPR motifs in the L. major
proteins then were detected as described above. The complete set of 53 putative
trypanosomatid PPR proteins was aligned with ClustalW.
Epitope tagging. To localize the eight PPR proteins, we produced transgenic
cell lines expressing protein variants containing a carboxy-terminal Ty-1 tag (for
TbPPR2, TbPPR4, TbPPR6, TbPPR7, and TbPPR8) or a hemagglutinin (HA)
tag (for TbPPR1, TbPPR3, and TbPPR5), respectively. Both tags are routinely
used with T. brucei (2, 40). The Ty-1 tag is detected by the monoclonal BB2
antibody, and the HA tag is visualized by the monoclonal antibody HA11 (Co-
vance Research Products). Localization was analyzed either by immunofluores-
cence or by immunoblotting. Cell fractionation was done by digitonin extraction
as described previously (7). For the experiment shown in Fig. 7B, we used
TbMRPL21 (Tb927.7.4140), a mitochondrial ribosomal protein of the LSU,
containing three copies of the c-myc tag at its C terminus. The genes encoding
the tagged TbPPR1, TbPPR3, TbPPR5, and TbMRPL21 were integrated into
their own genomic regions to achieve levels of expression that were as close as
possible to natural levels (32, 40). The tagged TbPPR2, TbPPR4, TbPPR6,
TbPPR7, and TbPPR8 were cloned into a derivative of the plasmid pLew-100
(47), which allows tetracycline-inducible overexpression of the tagged proteins
(2). None of the cell lines expressing tagged PPR proteins exhibited a growth
phenotype in SDM-79 medium.
Production and analysis of RNAi cell lines. RNA interference (RNAi) was
performed using stem-loop constructs containing the puromycin resistance gene
as described previously (5). As inserts, we used the sequences of the following
open reading frames (nucleotide positions are indicated in parentheses): TbPPR1,
Tb927.2.3180 (639 to 1190); TbPPR2, Tb927.1.2990 (446 to 1003); TbPPR3,
Tb927.1.1160 (385 to 863); TbPPR4, Tb10.389.0260 (555 to 1123); TbPPR5,
(487 to 979); and TbPPR8, Tb11.01.6040 (567 to 1087). Transfection of T. brucei,
previously (26). All transgenic cell lines used in this study are based on T. brucei strain
29-13, which was grown in SDM-79 medium supplemented with 15% fetal calf serum
(FCS), 50 ?g/ml hygromycin, and 15 ?g/ml G-418. Growth of RNAi cells also was
analyzed in SDM-80 medium, a modification of SDM-79 that lacks glucose and that is
and in the absence of 1 ?g/ml of tetracycline.
Northern blots. Total RNA (5 ?g each) was separated on 1% (wt/vol) agarose
gels containing 0.2 M formaldehyde and was electrophoresed in 20 mM mor-
pholinepropanesulfonic acid-NaOH, pH 7.0, and 0.2 M formaldehyde. After
being transferred, the GeneScreen Plus membranes were UV cross-linked and
baked. As probes, we used random hexamer [?-32P]dCTP-labeled double-
stranded DNA fragments corresponding to the following regions (nucleotide
positions are indicated in parentheses) of the indicated mitochondrion-encoded
proteins: COX1 (18 to 1526), COX2 (113 to 620), CYTB (87 to 1032), unedited
RPS12 (2 to 221), and unedited A6 (182 to 400). Furthermore, DNA fragments
corresponding to the indicated region of two edited mRNAs, RPS12 (2 to 325)
and A6 (374 to 819), were used. Finally, detection of the mitochondrial rRNAs
was done by the following32P-kinase oligonucleotides: 9S rRNA, TTGGTTAA
ATCAGCACTTAAC, and 12S rRNA, CTTGTTAACCTGCTCGAACC.
For the Northern blot analysis shown in Fig. 4, RNA was isolated (i) 24 h
before the growth arrest, (ii) at the time the growth arrest became apparent, and
TABLE 1. Genomic analysis of PPR proteins of T. brucei
T. bruceiL. major
aScore obtained by the TPRpred tool for identifying proteins carrying repeated helical repeats (18).
bPredicted subcellular localization by Predotar (44) and Mitoprot (9). Cyto., cytosolic; Mito., mitochondrial.
cY indicates a PPR protein identified previously by Mingler et al. (28).
dY indicates a PPR protein identified in the proteomics study of L. tarentolae mitoribosomal particles by Maslov et al. (25).
VOL. 27, 2007PPR PROTEINS IN T. BRUCEI6877
(iii) 24 h after the growth arrest. In all cases, essentially identical results were
obtained. Thus, for the statistical analysis (see Fig. 4B), all data points within this
48-h time period were combined.
Subfractionation of mitochondria. Mitochondria were isolated using the hy-
potonic procedure and were resuspended in 20 mM Tris-HCl, pH 8.0, 0.25 M
sucrose, 2 mM EDTA (37). Next, 15 ?l (approximately 300 ?g proteins) of the
mitochondrial fraction was diluted with 135 ?l of 10 mM MgCl2and was sub-
jected to 10 successive cycles of flash freezing in liquid N2and thawing at 25°C.
The resulting suspension was centrifuged for 5 min at 10,000 ? g at 4°C, yielding
a supernatant (matrix fraction) and a pellet (membrane fraction) that were
resuspended in 150 ?l of 10 mM MgCl2. A volume of 12.5 (supernatant) and 100
?l (pellet) of each fraction was analyzed by immunoblotting and, after RNA
extraction, by Northern blotting, respectively.
Immunoprecipitation. Twenty-five microliters (approximately 500 ?g total
protein) of hypotonically isolated mitochondria was lysed at 4°C using 500 ?l of
lysis buffer (25 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM EDTA,0.5% Triton
X-100). The suspension was cleared by centrifugation (10 min at 10,000 ? g), and
the resulting supernatant, corresponding to the total fraction, was combined with
50 ?l of anti-HA affinity matrix (Roche Applied Science) and incubated on a
rotator for 3 h at 4°C. The beads were reisolated, yielding a supernatant corre-
sponding to the unbound fraction. The pellet was washed thrice using lysis buffer
containing 0.1% Triton X-100. Half of each of the washed pellets representing
the bound fraction were boiled in sodium dodecyl sulfate sample buffer to
analyze the bound proteins or were subjected to RNA extraction to analyze the
Miscellaneous notes. All RNA isolations were done by the acid guanidinium
method (8). ATP production assays using digitonin-purified mitochondria from
RNAi cell lines were done as described previously (5, 36). Assays were done at
the time when the growth arrest became apparent. Carbonate extraction of
mitochondrial membranes was done as described previously (35).
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, sen-
sitive 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 1).
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 1). 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 indi-
cated represents an underestimation, and the total number of
PPR proteins in the T. brucei proteome still may be an under-
estimate. The likely subcellular localization of the candidate
PPR proteins was predicted using Predotar (44) and Mitoprot
(9). Both programs strongly predicted a mitochondrial local-
ization for almost all of these proteins. The exceptions are
the clusters related to Tb927.6.4190, Tb927.8.6040, and
Tb11.01.7210 (Table 1). The predicted mitochondrial localiza-
tion is consistent with the current understanding of PPR pro-
tein function. The L. major genome (17) contains putative
orthologues for 25 of 28 T. brucei PPR proteins (discounting
one recently duplicated T. brucei gene). This is a greater pro-
portion than that for the genome as a whole; overall, only 77%
of T. brucei genes have clear orthologues in L. major (13).
Thus, PPR genes are particularly well conserved in trypanoso-
matids 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 ex-
perimentally, we prepared transgenic cell lines allowing expres-
sion 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. 1A).
The other four cell lines did not give a signal in immunofluo-
rescence and therefore were subjected to a more sensitive
FIG. 1. Localization
(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 anti-
serum 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 ? 107cell equivalents each of
total cellular (Tot.), crude cytosolic (Cyt.), and crude mitochondrial (Mit.)
extracts for the presence of the indicated tagged PPR proteins (top pan-
els). 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).
6878PUSNIK ET AL.MOL. CELL. BIOL.
biochemical analysis. Digitonin extractions were used to pre-
pare mitochondrial and cytosolic fractions that could be ana-
lyzed by immunoblotting (7). The results showed that the
tagged TbPPR3, TbPPR6, and TbPPR7 copurify with the mi-
tochondrial marker (Fig. 1B).
Tagged TbPPR8 is predominantly localized in the cytosol,
even though it is predicted to have a mitochondrial import
signal (Table 1). However, while most of TbPPR8 is cytosolic,
it cannot be excluded that a small fraction of it is imported into
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. 2,
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 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.
VOL. 27, 2007PPR PROTEINS IN T. BRUCEI6879
Growth of uninduced and induced RNAi cell lines was
tested in both the standard culture medium SDM-79 (6) (Fig.
2, 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. 2, right graphs, ?Glc). Three
distinct phenotypes were observed in SDM-79. Ablation of
TbPPR2, TbPPR3, TbPPR4, and TbPPR5 caused growth ar-
rest, 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
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 pyru-
vate) is used for mitochondrial substrate-level phosphorylation
(SUBPHOS) in the trypanosome-specific acetate:succinate co-
enzyme 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, 21). In glucose-free SDM-80, how-
ever, 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 effi-
The mitochondrial gene products of T. brucei include cyto-
chrome 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 pro-
teins are expected to be involved in posttranscriptional pro-
cesses 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 predic-
tion. 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 quan-
tify the different modes of ATP production in isolated mito-
chondria of T. brucei (5). 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 trypano-
some-specific acetate:succinate CoA transferase/succinyl-CoA
synthetase cycle (5, 33). To measure OXPHOS, mitochondria
are incubated with ADP and succinate. To measure SUBPHOS
in the citric acid cycle, ?-ketoglutarate is used as a substrate,
whereas measuring SUBPHOS in the acetate:succinate CoA
transferase/succinyl-CoA synthetase cycle requires the addi-
tion of pyruvate as well as the cosubstrate succinate (5). Atrac-
tyloside 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 knock-
down cell lines to perform either OXPHOS or SUBPHOS
using both methods.
Figure 3 shows that ablation of TbPPR1 to TbPPR7 selec-
tively knocks down OXPHOS (induced by succinate) but does
not interfere with either of the two forms of SUBPHOS (in-
duced by ?-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. 2), and it indicates that ablation of TbPPR6 only
slightly interferes with the level of OXPHOS activity required
for normal growth in SDM-79 medium.
Furthermore, we also tested TbPPR8 (Fig. 3B). It is re-
quired for growth in glucose-free SDM-80 medium, despite
being mainly or even only extramitochondrially localized (Fig.
1B). 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. 3B, 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
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 pheno-
type, 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 ana-
lyzed 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. 4). These correspond to two mRNA populations hav-
ing poly(A) tails of different lengths (approximately 20 nucle-
otides 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 circu-
lar 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
6880 PUSNIK ET AL.MOL. CELL. BIOL.
consistent with the absence of the long poly(A) tail popu-
lation in the knockdown cells (Fig. 5A). Shortening of the
poly(A) tail was seen for all mature mRNAs, at least at later
times of induction (in Fig. 4 this is best visible for COX2,
CYTB, and edited RPS12). This suggests that it is an indi-
rect 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. 5B). 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 mi-
tochondrial TrpRS shows a selective inhibition of OXPHOS
identical to that observed in the induced TbPPR1 to Tb-
PPR5 RNAi cell lines (Fig. 3). Figure 5 shows that no
qualitative differences of COX1, COX2, and CYTB mRNAs
were observed in cells ablated for cytosolic TrpRS. How-
ever, in cells ablated for the mitochondrial enzyme, only the
lower band was detected. Thus, ablation of the mitochon-
drial TrpRS had the same effect on mitochondrial polyade-
nylation as knockdown of six of the eight tested PPR pro-
teins. 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. 2)
FIG. 3. Ablation of TbPPR1 to TbPPR8 selectively abolishes OXPHOS. Succinate-, ?-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.
VOL. 27, 2007PPR PROTEINS IN T. BRUCEI6881
and with the only partial inhibition of OXPHOS observed in
the TbPPR8 cell lines (Fig. 3B).
To obtain accurate quantitative information on mitochon-
drial 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 4B 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 pheno-
types: (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 eight-
fold 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. 2 and 3). 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.
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.
6882 PUSNIK ET AL.MOL. CELL. BIOL.
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 6 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 pro-
The rRNA depletion is specific to the TbPPR2 to TbPPR7
RNAi cell lines. It does not occur in the TbPPR1 RNAi cell
line (Fig. 6B) and therefore is a general consequence of nei-
ther the growth arrest nor the inhibition of OXPHOS. Fur-
thermore, degradation of rRNAs also does not occur in cells
that are ablated for TbPPR8 (Fig. 4) or for mitochondrial
TrpRS (Fig. 5B), 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 af-
fected 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 or-
ganellar 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. 7A. Both 9S and 12S
rRNAs as well as a tagged ribosomal protein of the LSU (Fig.
7B) are quantitatively associated with the membrane fractions.
tRNAs and ?-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 con-
tain a single transmembrane segment, none of the trypanoso-
matidal 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 car-
bonate extraction, whereas most of the integral membrane
protein cytochrome oxidase subunit 4 remains in the pellet
(Fig. 7A, bottom panels). In summary, these results are con-
sistent with an association of the tested PPR proteins with
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. 7C).
Thus, these results are consistent with the idea that TbPPR2 to
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).
VOL. 27, 2007PPR PROTEINS IN T. BRUCEI6883
TbPPR5 and the mitochondrial rRNAs are part of the same
TbPPR5 is associated with 12S rRNA. Purification of mito-
ribosomes 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 techni-
cally very challenging (24, 25, 42). As an alternative, we per-
formed 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 ribo-
somal) complexes that hide the epitope from the antibody, a
significant fraction of the tagged TbPPR5 was recovered from
the bound fraction (Fig. 8B). 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 exper-
iment directly shows that TbPPR5 is directly or indirectly as-
sociated with the LSU of the mitoribosome.
FIG. 6. Kinetics of mitochondrial rRNA depletion. (A) Graph showing
the relative changes in the levels of mitochondrial rRNAs during ablation of
cytosolic TrpRS. For each cell line, the growth phenotype was monitored in
parallel. The gray bar indicates the time interval at which the growth pheno-
type became apparent. The interval encompasses 24 h before and 24 h after
at least twice, and the variation between the same time points in the two
experiments was less than 10%. (B) Kinetics of mitochondrial rRNA deple-
tion in the TbPPR1 RNAi cell line.
FIG. 7. PPR proteins required for rRNA accumulation are mem-
brane associated. (A) Mitochondria of cell lines expressing the indi-
cated 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.
6884PUSNIK ET AL.MOL. CELL. BIOL.
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 simulta-
neous inducible expression of tagged TbPPR2 or/and TbPPR6,
respectively (Fig. 9). 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 ap-
proximately 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.
Overexpression of PPR proteins slows down RNAi-induced
phenotypes. Unexpectedly, expression of the tagged PPR pro-
teins delayed and reduced the extent of depletion of the mi-
tochondrial 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. 9, middle panels).
Causing a delay of rRNA degradation appears to be a general
effect of PPR protein overexpression; it was seen in all com-
binations 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. 9B). 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. 2 and 3). Thus, it is unlikely that overexpres-
sion of a PPR protein allows it to bind to an RNA sequence
that normally is recognized by the ablated PPR protein. How-
ever, 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 comple-
mentation would require both correct protein-protein and cor-
rect RNA-protein interactions and therefore cannot be
achieved by overexpression of heterologous PPR proteins. In
summary, even though the underlying mechanism of how over-
expression of one set of PPR proteins delays the RNAi-in-
duced phenotypes of another set is unknown, the results un-
derscore that rRNA-affecting PPR proteins are intricately
connected to each other and to the mitochondrial rRNAs.
The genome of T. brucei encodes 28 distinct PPR proteins,
which is approximately five times more PPR proteins than
most other nonplant eukaryotes. Eight of these proteins
(TbPPR1 to TbPPR8) were chosen for experimental analysis.
Epitope tagging has shown that TbPPR1 to TbPPR7 are local-
ized in mitochondria (Fig. 1). Moreover, they all are essential
for OXPHOS and thus for growth in medium lacking glucose
(Fig. 2 and 3). Surprisingly, six out of the seven mitochondrial
PPR proteins (TbPPR2 to TbPPR7) are functionally linked to
A recent proteomics study of an abundant mitochondrial
ribonucleoprotein complex of Leishmania that contains the 9S
rRNA and many SSU ribosomal proteins but no 12S rRNA
identified three PPR proteins (25). Homologues of two of
them also were detected in our bioinformatic analysis of the T.
brucei genome but were not investigated experimentally (Table
FIG. 8. TbPPR5 is associated with 12S rRNA. Mitochondrial ex-
tract 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
tRNAIleusing Northern blotting. The percentages of the total samples
that were analyzed in the different lanes are indicated at the bottom.
VOL. 27, 2007PPR PROTEINS IN T. BRUCEI 6885
1). Furthermore, the complex also contained at least a small
amount of the leishmanial orthologue of TbPPR5 (25).
Thus, in T. brucei at least eight distinct PPR proteins are
functionally or structurally connected to mitoribosomes.
TbPPR1 to TbPPR8, which were selected for experimental
analysis, were chosen essentially randomly, the only condition
being that they contained a putative mitochondrial targeting
signal. This is true for 25 out of the 28 trypanosomal PPR
proteins (Table 1). Thus, finding that six (75%) out of eight
randomly selected PPR proteins were linked to the mitochon-
drial rRNAs, together with the two distinct 9S rRNA-associ-
ated PPR proteins identified in Leishmania (25), predicts that
many more of the remaining 21 trypanosomal PPR proteins
will target the mitochondrial 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 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.
6886PUSNIK ET AL.MOL. CELL. BIOL.
What role could TbPPR2 to TbPPR7 play in ribosome func-
tion? We think that TbPPR2 to TbPPR7 are either required
for ribosome biogenesis or bona fide components of the mito-
ribosomes. In the former case, they may be associated with the
rRNA-containing particles but not or only transiently with
mature ribosomes. In the latter case, they are expected to be
stably and permanently associated with the mature ribosomes.
It is not possible at present to distinguish between these two
scenarios due to the difficulties of purifying mitoribosomes of
trypanosomatids. Furthermore, two recent studies have shown
that there are at least six stable rRNA-containing particles that
can be isolated from trypanosomatid mitochondria (24, 25).
The 9S rRNA was recovered in four distinct ribonucleoprotein
complexes that had sedimentation constants of 30, 45, 50, and
65S. A similar situation was seen for the 12S rRNAs, which
were found in 40, 50, and 65S particles. The 50S particle
contained both rRNA and SSU and LSU ribosomal proteins
and thus probably corresponds to the fully assembled ribo-
some. The 40S complex contained 12S rRNA and LSU ribo-
somal proteins and therefore probably represents the LSU of
the ribosome. The role of all other particles is unclear.
TbPPR5 is stably associated with the 12S rRNA (Fig. 8) and
thus is a good candidate for a bona fide component of the LSU
of the ribosome. The facts that the rRNA-affecting trypanoso-
mal PPR proteins are membrane bound and are coregulated
with the rRNAs argue for a stable association with the rRNAs.
Moreover, the fact that ablation of all rRNA-affecting PPR
proteins, except for TbPPR4, first and most extensively affects
the 12S rRNA suggests that they are associated with the 12S
rather than the 9S rRNA. However, since we extrapolate that
the function of a large fraction of the mitochondrial PPR
proteins of T. brucei is connected to ribosomes, it is reasonable
to assume that some are actual ribosomal proteins, whereas
others might be required only for the biogenesis of the ribo-
somes and thus at steady state might not be associated with
Mitoribosomes are of the bacterial type, and with the excep-
tion of plants, there is an evolutionary trend to reduce the
length of their rRNAs (38). Thus, whereas the 16S and 23S
rRNAs in E. coli are 1,542 and 2,904 nucleotides in length,
respectively, the rRNAs in human mitochondria have been
reduced to a length of 953 and 1,555 nucleotides. In trypano-
somatids (e.g., T. brucei), this reduction is taken much further,
and the 9S and 12S rRNA are only 611 and 1,150 nucleotides
long (11, 12, 14, 43). This makes them the shortest rRNAs
known to date. However, even in the mitochondrial rRNAs of
trypanosomatids, most domains of bacterial rRNAs have been
retained, though some stems and loops have been drastically
reduced or completely eliminated. There is evidence that the
length reduction of the rRNAs in mitoribosomes of mammals
is compensated for by having more numerous and larger ribo-
somal proteins (39). Since the mitochondrial rRNAs in
trypanosomatids are even shorter, it is tempting to speculate
that one of the main functions of PPR proteins in trypanoso-
matids is to functionally and structurally compensate for the
lacking parts of the truncated rRNAs.
In summary, our results are in agreement with the postu-
lated role of PPR proteins as sequence-specific RNA binding
proteins and suggest that the global function of the PPR pro-
tein family-mediating organellar gene expression is conserved
within eukaryotes. On the other hand, the fact that, in T.
brucei, a large fraction of the trypanosomal PPR proteins act
on rRNAs was unexpected. However, association of PPR pro-
teins with organellar ribosomes is not restricted to trypanoso-
matids. Data obtained from maize have shown that a null
mutant for a plastid PPR protein is devoid of plastid ribosomes
(46). Moreover, single PPR proteins associated with the SSU
of the mitoribosome recently have been described for yeast
(15) and mammals (19). Thus, while it is most prominent in T.
brucei, a role in rRNA maintenance and thus in ribosome
function is a conserved function of PPR proteins in all species.
This leads to the interesting situation in which eukaryote-
specific PPR proteins have become essential components of
bacterial-type organellar ribosomes.
We thank G. Cross, J. Lukes, D. Speijer, and E. Ullu for cell lines,
plasmids, and antisera.
This work was supported by grant 3100A0-109311 of the Swiss Na-
tional Foundation (A.S.), NIH RO3 AI063237 (L.K.R.), and Austra-
lian Research Council grant CE0561495 (I.S.).
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