MBE Advance Access originally published online on June 23, 2008
Molecular Biology and Evolution 2008 25(9):1791-1793; doi:10.1093/molbev/msn139
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Letter |
Presence of a Latent Mitochondrial Targeting Signal in Gene on Mitochondrial Genome
* Genetic Diversity Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan
Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan
E-mail: kadowaki{at}affrc.go.jp.
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Abstract |
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Organelles, such as mitochondria and chloroplasts, are derived from endosymbionts. Gene transfer events from organelles to the nucleus have occurred over evolutionary time. In the case that a transferred gene in the nucleus needs to go back to the original organelle, it must obtain targeting information for sorting its protein to that organelle. Here, we reveal that the genes for the ribosomal proteins L2 and S4 in the Arabidopsis thaliana mitochondrial (mt) genome contain information for protein targeting into the mitochondria. Similarly, the genes for the ribosomal proteins L2 and S19 in the Oryza sativa mt genome contain information for protein targeting into mitochondria. These results suggest that targeting information already existed in each gene in the plant mt genome before the transfer event to the nucleus occurred. We provide new insights into the timing of the appearance of targeting signals in evolution.
Key Words: targeting signal • gene transfer • mitochondria • ribosomal protein genes
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Organelles (mitochondria or chloroplasts) that are the descendant of endosymbionts still retain genes on own genome. However, most of the genes in the ancestral endosymbiont have either been translocated to the nuclear genome of the host cell or have been lost during evolution after the initial endosymbiotic event (Gray 1992
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In angiosperms, more genes (especially, ribosomal protein genes) are encoded in the mitochondrial (mt) genome than in vertebrates and fungi. Furthermore, the number of encoded genes in the mt genome varies among plant species. These clues suggest that gene transfer is still on going in angiosperms. Thus, the mt genome in angiosperms is a good tool for the study of gene transfer events from the mitochondria to the nucleus and provides a way of understanding the mechanism of gene transfer in angiosperms (Adams and Palmer 2003).
To complete gene transfer, a transferred gene must undergo several steps because of the differences in transcriptional and translational machinery between the mitochondria and nucleus (cytoplasm) (Brennicke et al. 1993). In one of these steps, the transferred gene must acquire a targeting signal to enable its protein to be sent to the mitochondria. To date, many genes transferred from the mitochondria to the nucleus in various species have been isolated, and several mechanisms for the acquisition of a targeting signal have been reported (see review, Adams and Palmer 2003). For example, a targeting signal duplication in a gene in the nucleus has been shown (Kadowaki et al. 1996), and another study has shown the use of one targeting signal by alternative splicing (Kubo et al. 1999). Generally, obvious N-terminal extension for the targeting signal occurs in transferred genes in comparison to genes that remain in the mt genome. In contrast, genes in which a distinguishable targeting signal could not be found have been also reported (e.g., the ribosomal protein S10 gene in several angiosperms [Adams et al. 2000; Kubo et al. 2000; Murcha et al. 2005] and the ribosomal protein L2 gene [rpl2] in Glycine max and Medicago truncatula [Adams et al. 2001]). A dual targeting signal to the mitochondria and chloroplasts has been discovered in the ribosomal protein S16 gene without an N-terminal extension (Ueda et al. 2008). Approximately 75% of the nuclear-encoded mt ribosomal proteins in Arabidopsis thaliana have N-terminal extensions (Bonen and Calixte 2006). The remaining ribosomal proteins may have an internal targeting signal without an N-terminal extension. Ribosomal proteins containing an internal targeting signal have also been found in Saccharomyces cerevisiae (Matsushita and Isono 1993). It is therefore possible that some genes encoded in the mt genome already have a targeting signal. Angiosperms that are still undergoing the process of gene transfer enable us to verify the above hypothesis.
The number of ribosomal protein genes on the A. thaliana and Oryza sativa mt genomes is 9 (Unseld et al. 1997) and 11 (Notsu et al. 2002), respectively. TargetP (Emanuelsson et al. 2000) and Predotar (Small et al. 2004) were used to predict whether ribosomal proteins of genes on the mt genome in these 2 plant species could be located to the mitochondria. Among them, the ribosomal protein S4 gene (rps4), ribosomal protein S19 gene (rps19), and rpl2 in O. sativa and the ribosomal protein S3 gene (rps3), rps4, and rpl2 in A. thaliana were predicted to be targeted to the mitochondria. The corresponding sequences of 100 amino acids from the N-termini of the above ribosomal proteins were used for localization analysis. For RPS19, the full-length protein (93 amino acids) was used. These sequences were fused upstream to the gene for green fluorescence protein (GFP) and subjected to overexpression by the cauliflower mosaic virus (CaMV) 35S promoter in a Nicotiana tabacum bright yellow-2 (BY-2) cell suspension. The results showed that the GFP-fused proteins for RPS19 and RPL2 in O. sativa and RPS4 and RPL2 in A. thaliana were located in the mitochondria (fig. 1), although rps3 in A. thaliana and rps4 in O. sativa were located in the cytoplasm or nuclei, not the mitochondria (fig. 1). These results suggest that mt rps4 and rpl2 in A. thaliana and mt rps19 and rpl2 in O. sativa were equipped with targeting information for their proteins to be located in the mitochondria, not requiring additional signals from nuclear genes. Therefore, it is possible that these genes became functional in the nucleus after gene translocation from the mitochondria by gaining a nuclear promoter and other regulatory elements in the nucleus without the acquisition of a targeting signal.
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As for rps4, A. thaliana RPS4 was targeted to the mitochondria, whereas O. sativa RPS4 was not. These results show that even the same genes in the mt genome of angiosperms do not always have the same latent targeting information (fig. 1). The deduced N-terminal 100 amino acids for A. thaliana and O. sativa mt RPS4 showed 68% identity. This amino acid sequence variation may explain differences in protein targeting.
Our research shows that targeting information has already been generated in some genes in the mt genome before they transfer to the nucleus. There are several studies showing that genes on the nuclear genome have an inner targeting signal for the mitochondria (Pfanner and Geissler 2001). Some of these genes might have obtained the targeting signal before the gene transfer event from the mitochondria to the nucleus; although it is likely that targeting signals were obtained after gene translocation from the organelle to the nuclear genome.
Why do some genes in the mt genome already contain targeting signals? Possible explanations could be a need for internal localization after translation on the mt ribosomes or a bias in nucleotide alteration in the mtDNAs.
After the engulfment of 
-proteobacteria that is an mt ancestor by primitive eukaryotic cells, most of -proteobacterial genes were either lost or transferred into the nuclear genome. Alternatively, genes of different organelle origin or novel genes in the nuclear genome substituted for mt-encoded genes, resulting in the structure of the present mt genome. Therefore, the component of the mitochondria has been changing, compared with that of its progenitor (references in Ueda et al. 2008). For non-mt genes to be newly located to the mitochondria, they must obtain an mt-targeting signal. Organelle genomes have been repeatedly integrated into the nucleus at a high rate (Timmis et al. 2004; Leister 2005), and many organelle genome sequences have been integrated into protein-coding exons in the nucleus (Noutsos et al. 2007). One of the possible sources of mt-targeting signal for them, the sequence derived from mt genes having targeting information might be used.
In this study, we have not examined whether the expressed mt-encoded genes in the nucleus are cleaved after they are imported into the mitochondria. We will need to analyze protein processing of them in the future.
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Methods |
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Total DNA and mtRNA were isolated as previously described by Ueda et al. (2006)
. For GFP fusion construction, each mt-encoded gene was amplified by the polymerase chain reaction (PCR) using total DNA as a template. In the case of mt rps3 in A. thaliana, a reverse transcribed–PCR product derived from mtRNA was used as a template. The primers used in this study were as follows and all restriction enzyme recognition sites within the primers are underlined: A. thaliana rps3 (5'-AAGTCACTCGAGATGGCACGAAAAGG-3') and (5'-TTCCCCATGGTCGTCCACCAC-3'); A. thaliana rps4 (5'-CAGGGTCGACATGTGGCTGCTTA-3') and (5'-GGATCCATGGGAAAAGGGATATATG-3'); A. thaliana rpl2 (5'-CGAGGTCGACAATGAGACCAGG-3') and (5'-ATGGCCATGGTGGTAGGTTCGAG-3'); O. sativa rps19 (5'-ATTGTCGACATGCCACGACGATCTATA-3') and (5'-GCTTACCATGGACTTTTTCCCCTTTC-3'); O. sativa rpl2, (5'-AGAGTCGACATGAGACAAAGCATAAAGG-3') and (5'-TGGCCATGGGGATCTTACGCGGCAG-3'); and O. sativa rps4 (5'-AAAGTCGACATGCCTGCATTAAGAT-3') and (5'-CAAACCATGGGACGAACCGGAATCAC-3'). All PCR products were inserted inframe with the gfp-coding sequence into an S65TGFP vector encoding the CaMV 35S promoter and 3' noparin synthase transcription terminator (Chiu et al. 1996). The nucleotide sequence of the resultant plasmid was confirmed by DNA sequencing. TargetP (Emanuelsson et al. 2000) and Predotar (Small et al. 2004) were used for the prediction of subcellular localization. Plasmid DNA (10 µg) was precipitated onto 1-µm spherical gold beads (Bio-Rad, Hercules, CA), and the beads were bombarded into suspension-cultured N. tabacum BY-2 cells using a PDS-1000 particle delivery system (Bio-Rad). After bombardment, the BY-2 cells were incubated in the dark at 24 °C for 6 h. GFP and MitoTracker Orange CMTMRos (Invitrogen, Carlsbad, CA) fluorescence were visualized with a confocal laser scanning microscope, as described previously (Arimura and Tsutsumi 2002; Ueda et al. 2006).
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Acknowledgements |
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We thank Dr Y. Niwa for providing the S65TGFP construct. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (grant 15208001) to N.T. and K.K. This research was also supported by a research fellowship of the Japan Society for the Promotion of Science for junior scientists to M.U. Institutions at which research was done: National Institute of Agrobiological Sciences and University of Tokyo.
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Footnotes |
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1 Present address: The ministry of Agriculture, Forestry and Fisheries of Japan, Chiyoda-ku, Tokyo, Japan.
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References |
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