TY - JOUR
AU1 - Tsaousis, Anastasios D.
AU2 - Gaston, Daniel
AU3 - Stechmann, Alexandra
AU4 - Walker, Peter B.
AU5 - Lithgow, Trevor
AU6 - Roger, Andrew J.
AB - Abstract 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. 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 α-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 translocated to the intermembrane space. As part of its role as a preprotein receptor, Tom70 functions as a co-chaperone 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). 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 μl 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 μl; 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 μl of ice-cold nuclease buffer (20 mM pH 8.8 Tris–HCl and 2 mM CaCl2) and 10 μl 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 μl of 3% sodium dodecyl sulfate/10% β-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 μl 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 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. Immunolocalization 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. 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 constructed S. cerevisiae—S. cerevisiae hybrid tom70 genes with these added codons as controls. 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-μl aliquots were serially diluted 5-fold 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. Bioinformatic Tools 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. 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. 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 + 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 (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 α-helical content according to HNN (67.2% for Blastocystis and 55.11% for S. cerevisiae) (Guermeur 1997), while neither has significant β-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, 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 α-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 α-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. FIG. 1. View largeDownload slide 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 C-terminal region (TPR9–11) in red. Non-TPR regions of Tom70 are shown in black including the dimer interface α-helices. FIG. 1. View largeDownload slide 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 C-terminal region (TPR9–11) in red. Non-TPR regions of Tom70 are shown in black including the dimer interface α-helices. 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 α-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 α-helical character. This may be an artifact of the model, an error in secondary structure prediction, or indicate a true break of this α-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 α-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 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 signal in immunofluorescence microscopy (supplementary fig. S2, Supplementary Material online). FIG. 2. View largeDownload slide 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. FIG. 2. View largeDownload slide 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. FIG. 3. View largeDownload slide 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 μm. FIG. 3. View largeDownload slide 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 μm. Complementation of Tom70 Knockouts in S. cerevisiae 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. FIG. 4. View largeDownload slide Yeast complementation growth assays. Serial dilution growth assays (5-μl aliquots were serially diluted 5-fold; from left to the right) of wild-type and Δtom70/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 α-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 red-colored 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. FIG. 4. View largeDownload slide Yeast complementation growth assays. Serial dilution growth assays (5-μl aliquots were serially diluted 5-fold; from left to the right) of wild-type and Δtom70/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 α-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 red-colored 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. The four S. cerevisiae–Blastocystis hybrid constructs (with the S. cerevisiae–S. cerevisiae construct as a positive control) were transfected into S. cerevisiae Δtom70/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 Δtom70/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 Δtom70/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 (ScTM-1-8/Bh9-11), which consists of only the C-terminal region of Blastocystis Tom70, showed a strong growth defect at 37 °C on nonfermentable media (YPEG) (fig. 4d), with growth equivalent to that of the Δtom70/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 homologue 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). 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, 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 monophyletic, when these topologies were compared with the AU test (P = 0.168) (Shimodaira 2002), SH test (P = 0.064), and ELW test (P = 0.057) (Strimmer and Rambaut 2002). FIG. 5. View largeDownload slide The Tom70 phylogeny. The tree shown was estimated from 344 aligned amino acids (44 taxa) using the Whelan and Goldman (WAG) + G + F model with RAxML. Support values are shown next to branches as maximum likelihood (ML) bootstrap support (WAG + G + F model, RAxML)/posterior probability (WAG + G + 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. FIG. 5. View largeDownload slide The Tom70 phylogeny. The tree shown was estimated from 344 aligned amino acids (44 taxa) using the Whelan and Goldman (WAG) + G + F model with RAxML. Support values are shown next to branches as maximum likelihood (ML) bootstrap support (WAG + G + F model, RAxML)/posterior probability (WAG + G + 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 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. 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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TI - A Functional Tom70 in the Human Parasite Blastocystis sp.: Implications for the Evolution of the Mitochondrial Import Apparatus
JF - Molecular Biology and Evolution
DO - 10.1093/molbev/msq252
DA - 2010-09-22
UR - https://www.deepdyve.com/lp/oxford-university-press/a-functional-tom70-in-the-human-parasite-blastocystis-sp-implications-8UD0lMI8jx
SP - 781
EP - 791
VL - 28
IS - 1
DP - DeepDyve
ER -