Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Australia
Correspondence: E-mail: t.lithgow{at}unimelb.edu.au.
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Abstract |
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Key Words: endosymbiont mitochondria targeting sequence protein import
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Introduction |
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In modern eukaryotes the solution to this problem, protein targeting into mitochondria, is an elaborate and now well-understood process. Each of the several hundred proteins carry discrete targeting sequences to be selected for import into the organelle and are recognized for import by a complex protein import machinery (Neupert 1997; Voos et al. 1999). Two highly improbable events, creation of mitochondrial targeting sequences and evolution of a protein import machinery in the outer membrane of the endosymbiont were required to initiate the conversion of the -proteobacter endosymbiont to mitochondria.
Some components of the import machinery we see now in mitochondria are derived from the protein secretion apparatus of bacteria. Peptidases and elements of the translocation complexes in the mitochondrial inner membrane were probably already present in the endosymbiont, having homologs in present-day bacteria (Hermann 2003). However, the creation and evolution of the translocase in the outer mitochondrial membrane (TOM complex) remains something we understand almost nothing about. It seems likely that the genes encoding subunits of the TOM complex, like a large number of other mitochondrial proteins, were derived from the host cell's genome (Karlberg et al. 2000). The TOM complex consists of receptors that bind mitochondrial targeting sequences (Tom20 and Tom22 [Endo and Kohda 2002]), a translocation channel (Tom40) and attendant subunits (Tom6 and Tom7), and three or more other subunits that seem more species specific (Neupert 1997; Schatz 1997; Voos et al. 1999; Gabriel, Buchanan, and Lithgow 2001; Pfanner and Chacinska 2002). (The proteins are named according to their size in kilodaltons; for example, Tom20 is the 20-kDa subunit of the TOM complex [Pfanner et al. 1996]). The need for a codependent evolution of the TOM complex and the targeting sequences for each of the imported protein substrates is a major weakness in the current model explaining the evolution of mitochondria.
Comparative genomics makes a strong argument for the later stages of the evolution of mitochondria (Brennicke et al. 1993; Kurland and Andersson 2000; Gray et al. 2001): (1) escape of the DNA (or an RNA copy) of the gene from the endosymbiont, (2) transfer of the gene fragment to the nucleus and integration in the genome, (3) adaptive rearrangements to allow gene expression, (4) exon shuffling, and other means to create a mitochondrial targeting sequence, (5) transcription, then translation, of the mitochondrial protein in the cytosol, and (6) gene inactivation of the redundant copy(s) of the gene in the mitochondria (Kurland and Andersson 2000; Gray et al. 2001). However, in its current form, this model assumes the preexistence of a TOM complex. Before the TOM complex, no pressure existed to select for the sequences on a protein substrate that could promote targeting back to the early endosymbiont.
Here we show, using two independent predictive strategies, that many proteins present in extant prokaryotes carry rudimentary features for mitochondrial targeting. Application of two predictors, Mitoprot II and Predotar, and computer simulations of feasible scenarios suggest that up to 5% of proteins encoded by the extant proteobacterium Escherichia coli have features that would serve as mitochondrial targeting information, with basic and amphipathic extensions at their amino-termini and "meso-hydrophobic" character throughout the proteins. One of the bacterial proteins, YhaR, serves as an instructional case in point. It is a member of a large family of highly conserved proteins found in at least eight diverse phyla of present-day bacteria. Multiple sequence alignments of the bacterial versions of these proteins show ragged amino-termini, with YhaR from E. coli having one of the longer amino-terminal extensions. Ectopic expression of bacterial YhaR results in targeting of the protein to yeast mitochondria, suggesting that in some cases, during the course of evolution, this preadaptation meant little or no mutagenesis of upstream regions in bacterial genes to render the proteins they encode competent for import into "protomitochondria." The presence of this preexisting "targeting" information would have provided selective pressure to initiate evolution of the TOM complex.
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Materials and Methods |
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Prediction of Mitochondrial Targeting Peptides
MitProtII was obtained from ftp://ftp.ens.fr/pub/molbio, and Predotar was obtained from http://www.inra.fr/predotar/. The precompiled Solaris executable of MitoProtII was used in calculations. For the processing of multiple sequences and analysis of results, a set of wrapper programs was developed in Perl.
Computer Simulations of Mitochondrial Predictors
For computer simulations, we assumed two predictors with the characteristics of Predotar (i.e., sensitivity = 0.86 and specificity = 0.50 [Emanuelsson and von Heijne 2001]) whose errors are uncorrelated. This is expected to be a reasonable assumption because Mitoprot II and Predotar use an entirely different approach for the prediction of mitochondrial targeting features. Simulations were carried out on genomes containing a total of 4,096 proteins, with the number of mitochondrial proteins ranging from 2% to 34%.
Given the set of assumed mitochondrial proteins, the predictions of two predictors were simulated, and the intersection of positive predictions was calculated. Given the percentage of mitochondrial proteins, the calculation was repeated 20 times, and for each run, the ratio of false negatives to false positives was evaluated. From these data, the mean and standard deviation of the ratio of false negatives to false positives was calculated. The mean, thus, represents our best estimate of this ratio, and the standard deviation gives an estimate of the variability between repeated simulations. Optimization experiments suggest the data are insensitive to small changes in the neighborhood of chosen properties of predictors.
Yeast Strains and Mitochondrial Isolation
A URA3 gene cassette was used to disrupt the coding sequence in the HMF1 gene after the codon corresponding to Gly34. The hmf1::URA3 fragment was transformed into W303a/ yeast cells to generate the strain YRG27 (Mata/
, leu2/leu2, his3/his3, ade2/ade2, ura3/ura3, trp1/trp1, can1/can1, and HMF1/hmf1::URA3). YRG27 cells were sporulated and tetrads dissected to obtain haploid null mutants (Mata, leu2, his3, ade2, ura3, trp1, can1, and hmf1::URA3). Yeast mitochondria were prepared as described in Gabriel, Egan, and Lithgow (2003). Where indicated, mitochondria were exposed to osmotic shock conditions (10mM HEPES pH 7.4) or to 0.1% Triton X-100.
Antibody Production
The open reading frame encoding Mmf1 was amplified by PCR using purified yeast genomic DNA as a template and cloned into pQE30 (QIAGEN) behind a 6x Histidine coding sequence. Hexahistidine-tagged Mmf1 was expressed in Escherichia coli strain M15 (Qiagen) by induction with 0.5 mM IPTG. Denatured protein was purified using Ni-NTA resin (Qiagen) according to manufacturer's directions and used to immunize rabbits for the production of antibodies.
Mitochondrial Protein Import Assays
In vitro translation of 35S-labeled YhaR in rabbit reticulocyte lysates and import into isolated mitochondria was as previously described (Gabriel, Egan, and Lithgow 2003).
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Results |
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Pmf1 is now known to be mitochondrial in S. pombe, and epitope-tagged Mmf1 has been located in mitochondria of S. cerevisiae (Oxelmark et al. 2000; Kim, Yoshikawa, and Shirahige 2001; Marchini et al. 2002). An antibody raised to purified, recombinant protein found Mmf1 exclusively in the mitochondria (data not shown) and further localized Mmf1 within the mitochondrial matrix (fig. 2), demonstrating Mmf1 is not simply attached to the mitochondrial surface but has been imported across the mitochondrial membranes. The shorter protein from S. cerevisiae, Hmf1, is found only in the cytosol (data not shown). To verify that the amino-terminal extension of Mmf1 is a cleavable mitochondrial targeting sequence, a preparation of matrix proteins was isolated from yeast mitochondria, separated by Tris-tricine SDS-PAGE (Schägger and von Jagow 1991) and blotted to PVDF membrane for amino-terminal sequencing. Mmf1 is processed between amino acids Gly17 and Ile18, truncating the mature protein close to the point conforming to the size of the shorter family members (see figure 1).
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The two programs rely on different methods for the prediction of mitochondrial targeting peptides, and, in the first approximation, one could assume that errors in the predictions made by MitoProtII and Predotar are uncorrelated. We investigated this scenario by computer simulations to determine how reliable a combined prediction of mitochondrial targeting sequences might be. The simulations assumed two predictors with properties similar to that of Predotar (i.e., with sensitivity of 0.86 and specificity of 0.50 [Emanuelsson and von Heijne 2001]). Given the number of "true" mitochondrial proteins, the data from the two predictors were simulated, and the number of proteins predicted to be mitochondrial by both (henceforth referred as the "combined prediction") were analyzed. We were specifically interested in the ratio of false negatives to false positives in the combined prediction. When this ratio is greater than 1, the predictor underestimates the total number mitochondrial proteins; when this ratio is less than 1 the predictor overestimates the number of mitochondrial proteins (one can calculate this ratio to be 0.16 when Predotar alone is used).
The properties of the combined prediction depend on the fraction of "true" mitochondrial proteins. Thus, we simulated genomes where the proportion of mitochondrial proteins has been set at values in the range 2% to 34% (see Materials and Methods). Computer simulations show that a combined prediction drastically reduces the number of false positives relative to each predictor, especially when the fraction of mitochondrial proteins is small. At the same time, the combined prediction roughly halves the number of total positives and also doubles the number of false negatives, independently of the fraction of mitochondrial proteins. The ratio of false negatives to false positives in the combined prediction is shown in figure 4B. For genomes with less than approximately 25% of mitochondrial proteins and the assumed predictors, the combined prediction always underestimates the number of true mitochondrial proteins.
Of the proteins encoded in the E. coli genome, 210 sequences (4.9%) have both a MitoProt probability greater than 0.7 and a Predotar score greater than 0.7 (fig. 4A). Based on the computer simulations, we suggest that this number represents an underestimate of the actual fraction of E. coli proteins predisposed for targeting to mitochondria. When checking through the identities of these proteins many, like YhaR, have functions conserved in mitochondria today, including targeting sequence processing, phospholipid biosynthesis, membrane transporters (for metal ions and small molecules), and ribosomal proteins. Furthermore, like YhaR, the ribosomal proteins L18 and S20 and the phosphoserine decarboxylases, show raggedness in their amino-terminal sequences in multiple sequence alignments. We conclude that at least 5% of proteins from E. coli are predisposed for targeting to mitochondria.
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Discussion |
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Plastids, the other organelle derived from endosymbiosis, have a protein translocation machinery built of components that are also found in cyanobacteria. These similarities suggest some form of primitive translocation machinery preexisted in the early cyanobacter-type endosymbiont (Bolter et al. 1998; Reumann and Keegstra 1999). Although the mitochondrial TOM complex does not have obvious homologs in proteobacter species, the central component of the translocation channel is a membrane-embedded ß-barrel protein called Tom40. Membrane-embedded ß-barrels are found only in the outer membrane of bacteria and in outer membranes of mitochondria and plastids. It is widely accepted that Tom40 evolved from a bacterial ß-barrel porin (Mannella, Neuwald, and Lawrence 1996; Bains and Lithgow 1999; Gabriel, Buchanan, and Lithgow. 2001; Hermann 2003), perhaps one that could fortuitously bind proteins displaying the preadaptive characteristics found in YhaR.
Once a simple protein translocation system was established for proteins such as YhaR, the selective pressure would be established for the genetic rearrangement of exon sequences to produce basic, amphipathic peptides to those proteins of the endosymbiont that did not already carry such characteristics. Previous work (Baker and Schatz 1987; Lemire et al. 1989) has shown a significant proportion of DNA fragments generated from random sequences can function as a mitochondrial targeting sequence if cloned into a cassette upstream from the mitochondrial protein CoxIV (engineered to have lost its own mitochondrial targeting sequence). Exon duplication and exon shuffling, as often required to propagate this process, is readily seen in the case of the relatively recent transfer of the rps11 gene from the mitochondrial genome of the plant Oryza sativa. Two copies of rps11 are now present in the nuclear genome of the plant: rps11a, transcribed to incorporate an exon duplicated from the sequence encoding the mitochondrial presequence of the ATPase ß-subunit and rps11b, transcribed with a presequence-encoding exon from the CoxIV gene (Kadowaki et al. 1996).
We suggest that the first steps in the evolution of protein import into protomitochondria involved a very primitive set-up: a ß-barrel protein in the outer membrane of the internalized bacteria and a few substrates predisposed for targeting to mitochondria (fig. 5). Once these few substrates could be acquired from the cytosol of the earliest eukaryotic cells, a scenario would have been set to allow for the inactivation and loss of the first copies of genes from within the bacterial endosymbiont (Kurland and Andersson 2000). With this as impetus, the evolution of receptor subunits for the TOM complex would have been selected for codependently as the number of substrate proteins for import increased and the diversity of their targeting sequences expanded.
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Acknowledgements |
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Footnotes |
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