A Functional Tom70 in the Human Parasite Blastocystis sp.:
Implications for the Evolution of the Mitochondrial Import
Apparatus
Anastasios D. Tsaousis,*,1 Daniel Gaston,1 Alexandra Stechmann,1 Peter B. Walker,1 Trevor Lithgow,2
and Andrew J. Roger1
1
Department of Biochemistry and Molecular Biology, Centre for Comparative Genomics and Evolutionary Bioinformatics,
Dalhousie University, Halifax, Canada
2
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia
*Corresponding author: E-mail: [email protected].
Associate editor: Martin Embley
Core proteins of mitochondrial protein import are found in all mitochondria, suggesting a common origin of this import
machinery. Despite the presence of a universal core import mechanism, there are specific proteins found only in a few
groups of organisms. One of these proteins is the translocase of outer membrane 70 (Tom70), a protein that is essential for
the import of preproteins with internal targeting sequences into the mitochondrion. Until now, Tom70 has only been
found in animals and Fungi. We have identified a tom70 gene in the human parasitic anaerobic stramenopile Blastocystis
sp. that is neither an animal nor a fungus. Using a combination of bioinformatics, genetic complementation, and
immunofluorescence microscopy analyses, we demonstrate that this protein functions as a typical Tom70 in Blastocystis
mitochondrion-related organelles. Additionally, we identified putative tom70 genes in the genomes of other stramenopiles
and a haptophyte, that, in phylogenies, form a monophyletic group distinct from the animal and the fungal homologues.
The presence of Tom70 in these lineages significantly expands the evolutionary spectrum of eukaryotes that contain this
protein and suggests that it may have been part of the core mitochondrial protein import apparatus of the last common
ancestral eukaryote.
Key words: mitochondrial protein import, translocase of the outer membrane, Tom70, Blastocystis, yeast complementations,
phylogeny.
Introduction
The acquisition of mitochondria was a key event in the
origin of modern eukaryotic cells. Mitochondria originated
from a-proteobacterial endosymbionts, which subsequently lost many of their biosynthetic capabilities and
became integrated into the metabolism of their host
(Andersson et al. 1998). In the process, the endosymbionts
not only lost their autonomy, they also developed new
mechanisms for organelle biogenesis and metabolic exchange (Dyall et al. 2004). The ancestral mitochondrion experienced genome reduction through both gene loss and
the transfer of genes to the host nuclear genome, a process
known as endosymbiotic gene transfer (Timmis et al. 2004).
The origin of a mitochondrial protein import mechanism
facilitated this transfer of genes to the nucleus by allowing
their products to be correctly retargeted to the mitochondrial compartment. Evidence that has accumulated over
the past decade suggests that this mechanism is shared
by, and ancestral to, all mitochondrion-related organelles
(MROs) (mitosomes, hydrogenosomes, and anaerobic/
aerobic mitochondria) (Embley and Martin 2006). However, determining exactly which parts of the protein import
apparatus evolved prior to the divergence of all living
eukaryotes, and which parts evolved later in specific
eukaryote lineages remains an active area of investigation
(Dolezal et al. 2006).
The mitochondrial protein import apparatus consists of
five oligomeric complexes: the translocase of outer membrane (TOM) complex, the translocase of inner membrane
22 and 23 complexes, the sorting and assembling machinery, and the mitochondrial intermembrane space assembly.
The TOM complex is responsible for the recognition and
translocation of mitochondrial-targeted proteins through
the outer mitochondrial membrane. In the mitochondria
of animals and fungi (opisthokonts), the TOM complex
has at least two outer membrane surface receptors, Tom20
and Tom70 (Endo and Kohda 2002; Dolezal et al. 2006;
Chacinska et al. 2009). Tom20 is responsible for the recognition of canonical N-terminal-targeting sequences found
in mitochondrial preproteins, whereas Tom70 is responsible for binding more hydrophobic proteins that often have
internal (cryptic) targeting sequences. After the initial
recognition event, preproteins are directed to two components of the TOM complex: Tom40 and Tom22 (Neupert
and Herrmann 2007; Chacinska et al. 2009). Tom22p
appears to cooperate with Tom20 in binding of N-terminal
sequences and guides precursor proteins to the outer
membrane pore, Tom40. Tom40 forms the channel in
the outer membrane through which preproteins are
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Mol. Biol. Evol. 28(1):781–791. 2011 doi:10.1093/molbev/msq252
Advance Access publication September 22, 2010
781
Research article
Abstract
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Tsaousis et al. · doi:10.1093/molbev/msq252
translocated to the intermembrane space. As part of its
role as a preprotein receptor, Tom70 functions as a cochaperone that cooperates with cytosolic heat shock protein 70 (Hsp70) and Hsp90 in the delivery of preproteins to
mitochondria (Young et al. 2003). Tom70 is also of particular interest because, until now, it was believed to be present only in opisthokonts, suggesting an innovation in the
mitochondrial import apparatus after the split of animals
and fungi from other eukaryotic lineages (Chan et al. 2006).
All metazoan and fungal genomes examined to date
possess only one copy of tom70 gene, with the exception
of yeast species of the genus Saccharomyces that have
a functional tom70 paralog, tom71, derived from a relatively
recent gene duplication event (Kurtzman 2003; Chan et al.
2006). A common characteristic of all Tom70/71 proteins is
the presence of 11 tetratricopeptide repeat (TPR) motifs,
which form three functional regions: the ‘‘clamp’’ domain
(TPR1–3), suggested to be the domain responsible for binding the C-termini of the cytosolic chaperones; the ‘‘core’’ domain (TPR4–8) responsible for preprotein binding (Brix et al.
1999, 2000); and the C-terminal region (TPR9–11), diagnostic of the Tom70 family and required for efficient import
(Chan et al. 2006). The crystal structure of Tom70 was solved
as a dimer (Wu and Sha 2006), but recent structural data
suggest that this is unlikely to be the only functional form
of Tom70 (Mills et al. 2009). To better understand the function of this important protein, characterization of homologous proteins is required, especially for those that are more
divergent.
From an expressed sequence tag (EST) survey of the unicellular stramenopile Blastocystis sp. (Stechmann et al.
2008), we identified a partial sequence with similarities
to Tom70. The stramenopiles are a heterogeneous group
of eukaryotes that includes brown algae, diatoms, slime
nets, and oomycetes that are distantly related to opisthokonts. The possible presence of a Tom70-like protein encoded in the genome of a representative of this group raises
questions about the functionality, origin, and distribution
of Tom70 among eukaryotes. Here, we show by structural,
functional, and cell biological analysis that the Blastocystis
sp. genome does indeed encode a canonical Tom70 protein
that functions in its MROs. Furthermore, we identify
Tom70 homologues encoded in a number of related stramenopile genomes as well as in the more distantly related
haptophyte Emiliania huxleyi. These data suggest that
Tom70-based mitochondrial protein import occurs not only in animals and fungi but also in the eukaryote supergroup referred to as the chromalveolates. Our findings
are most straightforwardly interpreted as indicating that
Tom70 was part of the core protein import apparatus
of mitochondria in the last common ancestral eukaryote.
Materials and Methods
Cell Growth
Blastocystis sp. NandII was grown at 37 °C under anaerobic
conditions on whole beaten egg slants overlaid with Locke’s
solution as described in Zierdt et al. (1988).
782
DNA and RNA Extraction
Genomic DNA was extracted using the phenol:chloroform
protocol as described before (Sambrook et al. 2001). Total
RNA extraction was performed using TRIzol protocol as
described elsewhere (Stechmann et al. 2008). mRNA was
purified using the Oligotex Direct mRNA Kit (Qiagen).
The mRNA was used as a template for cDNA synthesis with
the GeneRacer Kit (Invitrogen).
Gene Walking and SiteFinding–Polymerase Chain
Reaction
Gene walking and SiteFinding–polymerase chain reaction
(PCR) protocols were performed on Blastocystis genomic
DNA as previously outlined in Katz et al. (2000) and Tan
et al. (2005), respectively. Both methods are good for characterizing genes where a portion of the gene is known, such
as an EST fragment. These two protocols employ the use of
random primers, which in combination with gene-specific
primers can be used to amplify segments of the gene and
upstream (or downstream) regions of the genome, and in
both cases, multiple primers are designed in the direction
of interest from the identified sequence.
Protein Extraction
A total of approximately 1 1010 cells were suspended in
600 ll of 0.15 M NaCl. Cells were disrupted using ultrasonication (30 s at 30-s intervals, six cycles) (Nasirudeen and
Tan 2004). Protease inhibitor cocktail (10 ll; Sigma) was
added to the sample, which was then centrifuged at
10,000 g for 10 min at 4 °C. After collecting the supernatant, 10 ll of ice-cold nuclease buffer (20 mM pH 8.8
Tris–HCl and 2 mM CaCl2) and 10 ll of protease inhibitor
cocktail were added. Thirty microliters of DNAase/RNAase
mix (MgCl2 50 mM, Tris–HCl 0.5 M pH 7.0) was added and
incubated on ice for 3 min after which 10 ll of 3% sodium
dodecyl sulfate/10% b-mercaptoethanol was added and
the mix was passed through a fine syringe. Samples were
stored at 20 °C in NuPAGE LDS sample buffer plus 10
sample reducing agent (Invitrogen). About 5–20 ll of the
supernatant (;10%) was analyzed using a polyacrylamide
minigel.
Antibody
Tom70 was purified from yeast mitochondria as described
previously (Hase et al. 1984), and the protein was injected
into rabbits. The antiserum was used in western blots as
described by Chan et al. (2006).
Western Blotting
The specificity of the antiserum was tested using western
blots. Extracts of Escherichia coli expressing Blastocystis
Tom70 or total protein extracts from Blastocystis sp. were
electrophoresed on a polyacrylamide gel and transferred to
a nitrocellulose membrane using a semidry transfer unit.
These membranes were blocked using 5% powdered
milk–tris buffered salts (TBS)-Tween solution for 30 min.
The blots were then rinsed in 0.5% milk–TBS-Tween solution for an additional 30 min. Titrations from 1:500 to
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1:10,000 of the antisera were made in solutions of 1% milk–
TBS-Tween. The membranes were incubated in these solutions overnight at 4 °C and then rinsed three times for 10
min in 1% milk–TBS-Tween. Membranes were then incubated for 1 h with a secondary antibody conjugated to peroxidase. After three rinses of 1 TBS for 10 min, the blots
were developed using the Amersham ECL detection kit.
The expressed recombinant protein of Blastocystis
Tom70 was differentiated from E. coli proteins by western
blot. Its identity as a fusion protein was verified by detection of the His-tag by incubation with a His-tag monoclonal
antibody (Abnova) using the manufacturer’s protocol. Labeling was detected using the Amersham ECL detection kit.
structed S. cerevisiae—S. cerevisiae hybrid tom70 genes
with these added codons as controls.
Immunolocalization
Bioinformatic Tools
Blastocystis cells were resuspended in 1 phosphate buffered saline (PBS) pH 7.4 and were transferred to pretreated
poly-L-lysine slides (Sigma). Slides were incubated at 4 °C
for 2 h and then washed for 5 min in 1 PBS. The cells on
the slides were fixed with 3.7% formaldehyde/0.5% acetic
acid for 15 min at 37 °C. Slides were washed for 5 min
in PBS/0.5% Tween-20 and then permeabilized with 0.1%
Triton X-100 for 5 min. Washes were performed three times
for 5 min in PBS/0.05% Triton X-100 for 5 min. Fixed cells
were incubated for 30 min with a blocking solution of 5%
skimmed milk powder in 1 PBS solution (w/v) and then
rinsed with 0.5% milk/PBS solution for 30 min. In some
cases, these two steps were substituted with an hour incubation with 10% horse serum in 1 PBS solution. The cells
were then incubated with a dilution of the antiserum in 1%
milk/PBS solution for 1 h at 25 °C or overnight at 4 °C.
Three different dilutions of each antiserum were tested
to determine optimal conditions. After three rinses in
1% milk/PBS, the slides were incubated with a fluorescent
dye–labeled (Alexa 488 green) goat secondary antibody at a
dilution of 1:200. For colocalization experiments, before fixation, cells were incubated for 20 min with 200 nm of
MitoTracker Red CMXros (Molecular Probes). Cover slips
were mounted with antifade mounting medium (Vectashield)
and observed under a laser scanning confocal microscope
(Zeiss LSM 510 Meta) using a 100 oil immersion lens.
For control experiments, the same conditions (including
antibody concentration and reaction volume) were used as
described previously. One milligram of Saccharomyces
cerevisiae total protein extracts was incubated with the
corresponding antiserum for 30 min prior to the overnight
incubation on the cells.
Transmembrane (TM) domains were predicted using the
TMHMM v.3 program (Krogh et al. 2001). TPR motifs were
predicted using the REP program (Andrade et al. 2000) and
HMMER3 (Eddy 1998, 2008). Other bioinformatic analyses
are discussed in detail in the following.
Preparation of Constructs
To prepare the constructs for yeast complementations, we
selected regions corresponding to the three domains based
on a structural alignment (supplementary fig. S1, Supplementary Material online), amplified these fragments adding appropriate 6 bp restriction endonuclease cutting sites
for digestion/ligation, and cloned the various pieces together into the S. cerevisiae expression vector. As the
restriction sites added two additional codons at the boundary region between the ligated fragments, we also con-
Complementation Studies
Yeast cells (strain MH272) were grown to mid-logarithmic
phase (optical density at 600 nm of 0.6) in selective minimal
medium (SD-URA) and diluted to an optical density at 600
nm of 0.2, and then, 5-ll aliquots were serially diluted 5fold and spotted onto YPAD (yeast extract 1%, peptone 2%,
adenine 0.1%, glucose 2%) and YPEG (yeast extract 1%,
peptone 2%, ethanol 3%, glycerol 3%) plates. Plates were
incubated at 25 °C, 30 °C, or 37 °C for 2–4 days until colonies were visible and then photographed.
Homology Modeling
Homology structures for putative Blastocystis Tom70 and
Blastocystis–Yeast Tom70 hybrids were generated from
sequence alignments using the Swiss-Model online suite
(Peitsch et al. 1995; Arnold et al. 2006; Kiefer et al.
2009). Returned homology models were then subjected
to energy minimization and equilibration using the NAMD
package (Phillips et al. 2005). The initial Blastocystis homology model was subjected to 10,000 steps of conjugate
gradient energy minimization followed by 1.5 ns of molecular dynamics and a further 10,000 steps of energy minimization. The procedure was identical for the hybrid
structures except using only 1 ns of molecular dynamics.
The Blastocystis homology model was evaluated with the
WHAT IF and ProSa web servers (Vriend 1990; Sippl
1993; Wiederstein and Sippl 2007) for quality and subjected
to further refinement using WHAT IF. Structural evaluation
(instead of valuation) valuation results were interpreted by
comparing the various predictions with the yeast Tom70
template structure (2GW1).
Tom70 Identification
The putative Tom70 of Blastocystis is very divergent in
comparison with the Tom70 sequences of animals and
fungi and was not identified as such using standard basic
local alignment search tool (BLAST) searching. Hidden
Markov model (HMM) profile–based searching using
HMMER (Eddy 1998) as described previously (Waller
et al. 2009) was used to confirm initial BLAST-based identifications of several divergent Tom70 sequences including
that of Blastocystis. Briefly, an HMM profile was constructed from a seed Tom70 alignment (Waller et al.
2009) using HMMER (Eddy 1998). The genomes of several
newly sequenced protists were then scanned with this profile using HMMER to identify putative tom70 genes. BLAST,
REP, and TMHMM (Krogh et al. 2001) were then used to
confirm the putative sequence as a Tom70.
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Phylogenetic Analyses
Multiple sequence alignments were created using
HMMER3 (Eddy 1998, 2008) based on a seed alignment
of Tom70 sequences (Waller et al. 2009). The multiple sequence alignment was iteratively refined using HMMER3 to
create new alignment-based profiles and realigning the sequences. This iterative refinement was carried out until
convergence or 100 iterations had passed. The completed
multiple sequence alignment was trimmed based on
HMMER3 column posterior probability scores, with all sites
having support values less than 8 being discarded. Some
high-scoring columns on the edges of poorly aligned
regions were also discarded from the analysis. Several
extremely divergent and partial sequences (microsporidia
and Aureococcus) were not included in the multiple alignment or phylogenetic analysis. Phylogenetic trees were estimated from alignments using RAxML 7.0.4 (Stamatakis
2006) using the Whelan and Goldman þ F model of amino
acid substitution and the gamma model of rate heterogeneity. Bayesian phylogenetic analysis was also carried out using
MrBayes (Huelsenbeck and Ronquist 2001). Topology testing
was performed using Consel (Shimodaira and Hasegawa 2001)
for the approximately unbiased (AU) test and RAxML 7.0.4
(Stamatakis 2006) for the shimodaira-hasegawa (SH) and
expected likelihood weights (ELW) tests.
Data Deposition
Data generated in this study are deposited at GenBank
under the following accession number: GU247896.
Results and Discussion
From a recently completed EST project of the NandII strain
of Blastocystis sp. (Stechmann et al. 2008), we identified
a partial sequence with similarity to Tom70. This sequence
codes for 282 amino acid residues and showed 25% sequence identity to TPR5–10 of Tom70 from S. cerevisiae
(Chan et al. 2006; Wu and Sha 2006). To amplify the whole
gene encoding this sequence, we used rapid amplification
of cDNA ends on Blastocystis cDNA and gene walking PCR
on Blastocystis genomic DNA (Katz et al. 2000). A putative
full-length tom70 gene sequence of 2,502 bp was obtained,
which corresponds to 833 amino acids, 52% longer than the
S. cerevisiae Tom70. Blastocystis Tom70 has 11 predicted
TPR motifs (see structure analysis in the following) and
a predicted TM domain extending from amino acid residues 7–26. The TM domain shows 42% sequence identity
to the corresponding region in the Tom70 of S. cerevisiae.
Curiously, the Blastocystis homologue contains an insertion between the TM domain and the first predicted TPR
motif that is approximately 300 amino acids in length; the
sequence has no in-frame stop codons and was present in
both the genomic and the cDNA sequences examined and
therefore is not an intron. Closer inspection revealed that
this segment consists of approximately 19–20 repeats of
slightly variable sequence and length, with a consensus sequence of PGKVEGDKXXXKXEF, as predicted by the
XSTREAM (Newman and Cooper 2007) and TRUST
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FIG. 1 A comparison of the Blastocystis Tom70 homology model and
the Saccharomyces cerevisiae Tom70 crystal structure. A comparison of the Blastocystis Tom70 homology model (A) and the S.
cerevisiae Tom70 crystal structure (PDB ID: 2GW1) (B) with the
three TPR regions highlighted. The clamp domain (TPR1–3) is
shown in blue, the core domain (TPR4–8) in green, and the Cterminal region (TPR9–11) in red. Non-TPR regions of Tom70 are
shown in black including the dimer interface a-helices.
(Szklarczyk and Heringa 2004) tandem repeat detection
tools. Similarity searches did not identify any significantly
similar sequences in other Blastocystis ESTs, nor in sequences in the nonredundant database. Secondary structure
prediction using a hierarchical neural network (HNN) on
the NPS@ server (Combet et al. 2000) indicates that this
repeat region is predominantly ‘‘coiled-coil/disordered.’’
This sequence repeat has not been found in any other
Tom70 homologue to date, including the other stramenopile
sequences discussed in the following.
Structural Interpretation
To investigate whether the Blastocystis protein is structurally similar to canonical Tom70 proteins, the Blastocystis
protein sequence was first analyzed with HNN to predict
its secondary structure content. Comparison of the
Blastocystis sequence with that of S. cerevisiae using
HNN shows that both have high levels of a-helical content
according to HNN (67.2% for Blastocystis and 55.11% for
S. cerevisiae) (Guermeur 1997), while neither has significant
b-sheet composition (6.94% and 6.81%, respectively). The
predictions for S. cerevisiae Tom70 are in broad agreement
with measurements by circular dichroism (Beddoe et al.
2004) and the observed crystal structure (Wu and Sha 2006).
Next, we aligned the Blastocystis protein sequence with
residues 39–617 from the crystal structure (Wu and Sha
2006) of the S. cerevisiae Tom70 (supplementary fig. S1,
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FIG. 2 Testing the specificity of anti–Saccharomyces cerevisiae Tom70 antisera on Blastocystis total protein extracts and Escherichia coli
expressing Blastocystis Tom70. (a) Identification of the Blastocystis recombinant protein using an antibody to the His-tag. The antibody
recognizes a band at an apparent relative molecular mass (Mr) of about 96 kDa, which is not present in E. coli protein extract controls. (b) A
western blot using heterologous antiserum to S. cerevisiae Tom70 against Blastocystis recombinant protein (the lane labeling is the same as in
a). The antiserum shows specific detection of Blastocystis recombinant Tom70 protein in both E. coli cells expressing the protein and the
purified Blastocystis Tom70 protein, but not in E. coli cells. The protein has an apparent Mr of about 96 kDa, similar to the band identified using
the His-tag antibody. (c) Analysis of the expression of Blastocystis sp. Tom70s in total protein extracts from the organism. Rabbit anti–
S. cerevisiae Tom70 antiserum (1:250 dilution) shows specific detection of Blastocystis Tom70 (see arrowhead), with an apparent Mr of about
93.2 kDa.
Supplementary Material online). This alignment was used to
generate a homology model of the tertiary structure of the
Blastocystis Tom70. Consistent with the HNN predictions,
and similar to S. cerevisiae Tom70 (Wu and Sha 2006)
(fig. 1b), the modeled structure of the Blastocystis Tom70
monomer consists of 24 a-helices. The majority of these helices form TPR motifs (TPR1–11) (fig. 1a), and all but one
correspond to the S. cerevisiae Tom70 crystal structure:
The second of the non-TPR a-helices that lies in this region
is predicted by a repeat detection method, REP (Andrade et al.
2000), to be a new TPR domain. This is in contrast to the
region that corresponds to S. cerevisiae TPR6, which was
not predicted by REP to be a TPR in the Blastocystis homologue. Although these results could be artifacts of the prediction algorithm, they are also considered minor changes in the
TPR topology of the two sequences, considering the distinct
evolutionary distance between the two species.
The predicted structural model of Blastocystis Tom70
(fig. 1a) was of good quality, as determined by ProSa
Web Server (Sippl 1993; Wiederstein and Sippl 2007). The
calculated Z-score of the S. cerevisiae Tom70 crystal structure
(2GW1; fig. 1b) was 8.54 and that of the homology model
was 7.34, both within the range of X-ray crystal structures of
a comparable size. After refinement, the predicted structure
of Blastocystis was structurally aligned to the S. cerevisiae template using FATCAT (Ye and Godzik 2004; Li et al. 2006) with
an average reported root mean square deviation value of
3.00 A° and deemed to be significantly similar with a reported
P value of 0.0 (raw score: 991.64, with 469 equivalent
positions).
The overall structure of the TPR-containing region of the
model is similar to that of the original S. cerevisiae structure
(fig. 1b); the WHAT IF server (Vriend 1990) indicates the
model is of high quality compared with the original
S. cerevisiae Tom70 template. There are a few problematic
areas of the predicted structure such as the a-helix between TPR3 and TPR4, which is not predicted by DSSP
(Kabsch and Sander 1983) or STRIDE (Frishman and Argos
1995; Heinig and Frishman 2004) to have significant
a-helical character. This may be an artifact of the model,
an error in secondary structure prediction, or indicate
a true break of this a-helix in the Blastocystis Tom70 structure compared with the original S. cerevisiae crystal structure
(Wu and Sha 2006). If the secondary structure prediction is
accurate, the lack of a helix in this region may indicate
a different mode of dimerization for the Blastocystis homologue as this a-helix is part of the dimerization interface in
S. cerevisiae Tom70. Consistent with this observation, residues within this interface are not well conserved within
Blastocystis compared with other Tom70 protein sequences
(supplementary fig. S1, Supplementary Material online) and,
in fact, display several hydrophobic to hydrophilic
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signal in immunofluorescence microscopy (supplementary
fig. S2, Supplementary Material online).
Complementation of Tom70 Knockouts in
S. cerevisiae
FIG. 3 Cellular localization of the Tom70 protein in Blastocystis. (a)
MitoTracker Red localizing to discrete structures corresponding to
MROs of Blastocystis. (b) Rabbit anti–Saccharomyces cerevisiae
Tom70 antiserum (1:100 dilution) detects discrete structures
corresponding to Blastocystis Tom70. (c) Colocalization of MitoTracker with anti–S. cerevisiae Tom70 antiserum in Blastocystis
MROs. (d) Differential interference contrast (DIC) images of the
cells used for immunofluorescence. Scale bar: 10 lm.
substitutions. However, the generally divergent nature of the
Blastocystis sequence makes it difficult to determine what, if
any, impact this may have on function.
Localization of Putative Tom70 within Blastocystis
sp. Cells
Using an antibody raised against S. cerevisiae Tom70 (Chan
et al. 2006), we demonstrated high specificity for a protein
of the expected size of Blastocystis Tom70 in total protein
extracts (93.2 kDa) (fig. 2c) and in E. coli cells heterologously
expressing the Blastocystis protein (fig. 2a and b). Staining
of Blastocystis cells with anti–S. cerevisiae Tom70 antiserum
showed a labeling distribution that was consistent with localizing to small compartments in the cytoplasm of Blastocystis and colocalized with the mitochondrion-specific
dye MitoTracker (fig. 3). As most subcellular organelles
in Blastocystis are located at the periphery of the cell
due to the presence of a large central vacuole, this colocalization pattern strongly suggests that the anti–S. cerevisiae
Tom70 antibody is labeling the Blastocystis Tom70 protein
on Blastocystis MROs. Similar localization distributions and
colocalizations with MitoTracker in Blastocystis have been
previously demonstrated in two studies using antibodies directed at the MRO-specific proteins anti-[FeFe]-hydrogenase
and anti-succinyl-CoA synthetase, respectively (Hamblin
et al. 2008; Stechmann et al. 2008). Addition of an excess
of S. cerevisiae total protein extract (1 mg) during the incubation of the primary antibody resulted on elimination of the
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Although sequence and predicted structural similarity to
the S. cerevisiae homologue and the clear localization pattern to MROs are suggestive of a canonical Tom70 function
for the Blastocystis homologue, we sought direct evidence
of its function by performing complementation studies using S. cerevisiae tom70/71 knockout strains (Chan et al.
2006). We focused attention on the different ‘‘domains’’
of Tom70 that are responsible for interaction with different
protein partners: 1) the ‘‘clamp’’ domain (TPR1–3) involved in binding cytosolic chaperonins, 2) the ‘‘core’’ domain (TPR4–8) involved in preprotein binding, and 3) the
C-terminal domain (TPR9–11) that most likely plays a role
in the structural flexibility of the protein (Mills et al. 2009).
Four constructs were made (fig. 4) where different segments of the gene encoding the N-terminal regions of
S. cerevisiae Tom70 were fused with the complementary
C-terminal encoding regions of the Blastocystis homologue.
Subsequently, we generated homology models of these
hybrids to check if, and how, the additional amino acids
and/or the Blastocystis domains affected the overall structure of the fusion protein (supplementary fig. S3, Supplementary Material online). With the exception of hybrid 3 in
both the S. cerevisiae–Blastocystis and the S. cerevisiae–S.
cerevisiae hybrid, the predicted structures conformed
closely to the S. cerevisiae crystal structure.
The four S. cerevisiae–Blastocystis hybrid constructs
(with the S. cerevisiae–S. cerevisiae construct as a positive
control) were transfected into S. cerevisiae Dtom70/tom71
cells (Chan et al. 2006; Waller et al. 2009). Of principle
interest is hybrid 1 that consists of the N-terminal TM domain of the S. cerevisiae Tom70 with the three functional
regions of the Blastocystis homologue (ScTM/Bh1-11).
Figure 4a demonstrates that hybrid 1 can functionally replace the S. cerevisiae Tom70 in the Dtom70/tom71 cells,
indicating that the Blastocystis Tom70 can function as
a protein import receptor. Hybrids 2 and 3 consist of
the clamp domain (TPR1–3) of S. cerevisiae Tom70 and
the core domain (TPR4–8) and C-terminal region
(TPR9–11) of the Blastocystis homologue. Hybrid 2
(ScTM-1-3/Bh4-11) differs from hybrid 3 (ScTM-1-3*/
Bh4-11) in whether the bridging segment between TPR3
and TPR4 derives from the Blastocystis (hybrid 2) or
S. cerevisiae (hybrid 3) homologue; this segment is disordered in the crystal structure of S. cerevisiae Tom70 (Chan
et al. 2006; Wu and Sha 2006). Both hybrid 2 and 3 demonstrated similar growth to the wild type in Dtom70/tom71
cells (fig. 4b and c) suggesting that both the core
domain that is responsible for preprotein binding and
the C-terminal region that is required for efficient import
are functional regardless of the nature of the clamp domain
and the bridging segment. Unexpectedly, hybrid 4 (ScTM1-8/Bh9-11), which consists of only the C-terminal region
of Blastocystis Tom70, showed a strong growth defect at
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FIG. 4 Yeast complementation growth assays. Serial dilution growth assays (5-ll aliquots were serially diluted 5-fold; from left to the right) of
wild-type and Dtom70/tom71 yeast cells transformed with plasmids expressing Blastocystis and Saccharomyces cerevisiae Tom70 hybrids.
Colored boxes and lines delimit structural features of Tom70. Narrow tall boxes indicate a-helices; wider boxes, TPR domains; and lines,
interdomain linker regions. Green indicates segments of the Blastocystis Tom70, whereas blue boxes indicate sequences derived from the
S. cerevisiae Tom70, or S. cerevisiae sequences that were incorporated in the corresponding plasmids shown in Chan et al. (2006). The redcolored block represents coding sequences for the TM domain of the S. cerevisiae Tom70. (a) Serial dilution growth assay with plasmid encoding all
of Blastocystis and S. cerevisiae TPRs. (b) Serial dilution growth assay with plasmid encoding the fusion protein composed from S. cerevisiae TPR1–3
with TPR4–11 of Blastocystis or S. cerevisiae. (c) Serial dilution growth assay with plasmid encoding S. cerevisiae TPR1–3 (including a segment that is
inherently disordered in the crystal structure) with TPR4–11 of Blastocystis or S. cerevisiae. (d) Serial dilution growth assay with plasmid encoding
S. cerevisiae TPR1–8 with TPR9–11 of Blastocystis or S. cerevisiae. Cells were incubated at 37 °C on YPEG plates for 2–4 days.
37 °C on nonfermentable media (YPEG) (fig. 4d), with
growth equivalent to that of the Dtom70/tom71 cells. Because the larger fragments of the Blastocystis Tom70 protein that included this region were apparently functional, it
is possible that hybrid 4 fails to complement because the
Blastocystis C-terminal segment (TPR9–11) does not function and/or fold properly in the absence of a neighboring
native Blastocystis core region (supplementary fig. S4, Supplementary Material online). Alternatively, the S. cerevisiae
core segment may require its native C-terminal domain to
properly fold and/or function. The latter explanation is
consistent with our observation that the Blastocystis ho-
mologue does not contain the residues at the C-terminal
domain responsible for the dimerization of the protein in
S. cerevisiae (supplementary fig. S1, Supplementary Material online). A final remote possibility is that Blastocystis
fragment (Bh9-11) interferes with the expression of the hybrid leading to an apparent lack of complementation. However, this is very unlikely because: 1) the hybrid constructs
containing larger sections of the Blastocystis gene including
this C-terminal segment clearly do not interfere with expression and 2) the deletion construct from which this hybrid was constructed has been previously shown to be
expressed at wild-type levels (Chan et al. 2006).
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Tsaousis et al. · doi:10.1093/molbev/msq252
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FIG. 5 The Tom70 phylogeny. The tree shown was estimated from 344 aligned amino acids (44 taxa) using the Whelan and Goldman (WAG) þ
G þ F model with RAxML. Support values are shown next to branches as maximum likelihood (ML) bootstrap support (WAG þ G þ F model,
RAxML)/posterior probability (WAG þ G þ F model, MrBayes)/posterior probability (C20 model, PHYLOBAYES). Branches that did not appear
in the majority-rule consensus tree of the posterior distribution of trees from the Bayesian analyses are indicated with a an asterisk (*). Only
bipartitions that received greater than 50% ML bootstrap support are labeled with support values. Stramenopiles and haptophyte are shown in
green, animals and unicellular opisthokonts in red, and fungi in fuchsia color.
The Phylogenetic Distribution of Tom70 among
Eukaryotes.
The discovery of a tom70 gene in a stramenopile such as
Blastocystis was unexpected because, until now, tom70
genes had only been found in the genomes of animals
and fungi and not in other model eukaryote systems such
as plants or ciliates. It was therefore thought to be a novel
innovation in the mitochondrial protein import apparatus
of opisthokonts (Chan et al. 2006). A potential explanation is that Blastocystis gained the tom70 gene from a recent horizontal gene transfer (HGT) from an opisthokont.
Alternatively, the presence of tom70 in Blastocystis could
reflect a more widespread distribution of these proteins
in related organisms. To investigate these alternatives
further, we mined the various newly available genome
and EST sequence databases using a phylogenetically diverse alignment profile based on the Blastocystis Tom70
sequence aligned with animal and fungal sequences. Using
this profile, we performed HMMER (Eddy 1998) searches
against a variety of available protistan, as well as some
recently completed invertebrate and basal metazoan,
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genomes (supplementary table S1, Supplementary Material online). We identified tom70 homologues in several
unicellular opisthokont protists including Monosiga brevicollis and Capsaspora owczarzaki. More surprisingly, we
also found putative tom70 genes in the genomes of a number of additional stramenopiles including species of Phaeodactylum, Thalassiosira, Phytophthora, and Aureococcus,
as well as the haptophyte E. huxleyi. Maximum likelihood
and Bayesian phylogenetic analyses of all available Tom70
proteins demonstrated that the stramenopile and haptophyte sequences branch together to the exclusion of
others, although only with weak bootstrap support and
posterior probability, respectively (fig. 5). These data suggest that the stramenopile and haptophyte sequences are
more closely related to each other than to the metazoan
and fungal Tom70s, including the yeast Tom71p sequences. Although Capsaspora and Monosiga Tom70 sequences group weakly as a sister clade to the stramenopile/
haptophyte clade, there is no significant difference between this optimal topology and the one where Metazoa
and metazoan-related protists are constrained to be
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Evolution of Mitochondrial Import · doi:10.1093/molbev/msq252
monophyletic, when these topologies were compared
with the AU test (P 5 0.168) (Shimodaira 2002), SH test
(P 5 0.064), and ELW test (P 5 0.057) (Strimmer and
Rambaut 2002).
The presence of Tom70 in stramenopiles, haptophytes,
and opisthokonts but in no other eukaryotes examined so
far is of particular interest because the former two lineages
are only distantly related to the latter in the current eukaryotic tree (Roger and Simpson 2009). Indeed, some evidence
suggests that the root of the eukaryote tree falls between
the so-called unikonts to which opisthokonts belong and
the bikonts that comprise lineages such as stramenopiles,
haptophytes, and a variety of other eukaryotes (Simpson
and Roger 2004; Richards and Cavalier-Smith 2005).
Although stramenopiles and haptophytes have been
suggested to group together as two anciently diverging
lineages within the chromalveolate supergroup, this
hypothesis remains hotly debated (Burki et al. 2009; Hampl
et al. 2009).
There are a number of possible scenarios to explain this
peculiar phylogenetic distribution of Tom70. The most
straightforward explanation is that Tom70 was part of
the core mitochondrial import apparatus in the common
ancestor of all living eukaryotes but was highly modified
to become unrecognizable, or lost completely, in eukaryotic
supergroups such as the Excavata and the Archaeplastida.
The fact that mitochondrial-targeted proteins that lack
N-terminal targeting peptides occur in disparate, apparently
Tom70-lacking, lineages including plants (Millar et al. 2005),
ciliates such as Tetrahymena (Smith et al. 2007), and anaerobic protists such as Trichomonas and Giardia (Dolezal et al.
2006) indicates that some system for cryptic targeting peptide detection must exist in these organisms. Localization of
mitochondrial proteins using heterologous expression systems (Dolezal et al. 2005; Burri et al. 2006) also suggests that
the mechanism of internal targeting sequence recognition is
conserved. Determining whether a divergent Tom70 protein
is present in these species as opposed to a completely distinct system for recognition of internal targeting signals will
require further functional and structural characterizations of
their TIM/TOM complexes.
A less likely explanation would posit the invention of
Tom70 in a common ancestor of either the opisthokonts
or the stramenopiles and haptophytes replacing an ancestral mechanism for recognizing internal cryptic targeting
sequences. Subsequently, an ancestor of either the stramenopile/haptophyte lineage or the opisthokont lineage
acquired the Tom70 protein by HGT from an early ancestor
of the other. If this scenario were correct, the Tom70 phylogeny we report could be interpreted as suggesting that
the transfer occurred from a unicellular opisthokont to
a common ancestor of the stramenopiles and haptophytes
within the ‘‘bikont’’ lineage. Better sampling of species in
this region of the tree could improve resolution to the
point where more definitive conclusions could be made.
In any case, these HGT scenarios all assume that a preexisting
system for recognizing internal cryptic targeting sequences
existed in the ancestral mitochondrion.
Conclusions
In conclusion, more than 50% of the proteins that are imported to mitochondria do not possess cleavable N-terminal
targeting peptides and instead rely on internal targeting
signals (Bolender et al. 2008; Gaston et al. 2009) that require
a receptor protein for their recognition. Here, we have
demonstrated that the receptor involved in this process
in opisthokonts, Tom70, is found in more eukaryotic lineages than was previously thought (Chan et al. 2006), suggesting it was likely present in the common ancestor of all
extant eukaryotes. Structural modeling, microscopy, and
functional investigations of the Blastocystis Tom70 indicate
that it performs the same function as in the opisthokonts,
serving as a protein import receptor in the outer mitochondrial membrane. The phylogenetic relationship between
Blastocystis Tom70 and the homologues we have identified
in other stramenopiles and haptophytes suggests that their
mitochondria also possess a Tom70-based protein import
mechanism. Genomic and transcriptomic data coupled
with functional studies of mitochondrial protein import
systems of more diverse unicellular bikont lineages are
needed to determine the prevalence of this system across
eukaryotic diversity as well as to further clarify its origin.
Supplementary Material
Supplementary figures S1–S4 and table S1 are available at
Molecular Biology and Evolution online (http://www
.mbe.oxfordjournals.org/).
Acknowledgments
This work was supported by a grant (MOP-62809) from the
Canadian Institutes of Health Research awarded to A.J.R.
A.D.T. was supported by the Tula Foundation and
a European Molecular Biology Organization postdoctoral
fellowship. A.J.R. was supported by the Integrated Microbial
Biodiversity program of the Canadian Institute for Advanced Research. T.L. is an Australian Research Council
Federation Fellow. We thank Jordan Pinder (Dalhousie
University) and Nickie Chan (CalTech) for their help on
the yeast complementation studies and Jacqueline de
Mestral (Dalhousie University) for her technical support.
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