In plants, as in most eukaryotic cells, import of nuclear-encoded cytosolic tRNAs is an essential process for mitochondrial biogenesis. Despite its broad occurrence, the mechanisms governing RNA transport into mitochondria are far less understood than protein import. This article demonstrates by Northwestern and gel-shift experiments that the plant mitochondrial voltage-dependent anion channel (VDAC) protein interacts with tRNA in vitro. It shows also that this porin, known to play a key role in metabolite transport, is a major component of the channel involved in the tRNA translocation step through the plant mitochondrial outer membrane, as supported by inhibition of tRNA import into isolated mitochondria by VDAC antibodies and Ruthenium red. However VDAC is not a tRNA receptor on the outer membrane. Rather, two major components from the TOM (translocase of the outer mitochondrial membrane) complex, namely TOM20 and TOM40, are important for tRNA binding at the surface of mitochondria, suggesting that they are also involved in tRNA import. Finally, we show that proteins and tRNAs are translocated into plant mitochondria by different pathways. Together, these findings identify unexpected components of the tRNA import machinery and suggest that the plant tRNA import pathway has evolved by recruiting multifunctional proteins. mitochondrial porin ͉ cytosolic tRNAs ͉ import factor
Expression of higher plant mitochondrial (mt) genes is regulated at the transcriptional, posttranscriptional, and translational levels, but the vast majority of the mtDNA and RNA-binding proteins involved remain to be identified. Plant mt single-stranded nucleic acid-binding proteins were purified by affinity chromatography, and corresponding genes have been identified. A majority of these proteins belong to a family of RNA-binding proteins characterized by the presence of an N-terminal RNA-recognition motif (RRM) sequence. They diverge in their C-terminal sequences, suggesting that they can be involved in different plant mt regulation processes. Mitochondrial localization of the proteins was confirmed both in vitro and in vivo and by immunolocalization. Binding experiments showed that several proteins have a preference for poly(U)-rich sequences. This mt protein family contains the ubiquitous RRM motif and has no known mt counterpart in non-plant species. Phylogenetic and functional analysis suggest a common ancestor with RNA-binding glycine-rich proteins (GRP), a family of developmentally regulated proteins of unknown function. As with several plant, cyanobacteria, and animal proteins that have similar structures, the expression of one of the Arabidopsis thaliana mt RNA-binding protein genes is induced by low temperatures.H igher plants require mitochondrial (mt) function for their survival, which depends on proper mtDNA maintenance and expression (1). At the structural level, the mtDNA of plants is relatively large, and in most species it is constantly reorganized by recombination between repeated sequences (2). Although large portions of mtDNA have been moved around during evolution, the plant mt genome evolves very slowly through nucleotide substitution; plant mt gene sequences have remained remarkably constant, suggesting the existence of very efficient DNA repair systems. On the other hand, the expression of plant mt genes is also complexly regulated, both at the transcriptional, posttranscriptional, and translational levels. For proper maturation of its transcripts, mitochondria puts to play complex processes of intron splicing, 5Ј and 3Ј RNA trimming, extensive RNA editing by C to U conversions, and regulation of transcript stability by secondary structures and polyadenylation (3, 4). Despite the importance of these mt processes in plant development, little is known about the factors involved, but at the core of the protein complexes must be DNA-binding proteins involved in mtDNA replication, recombination, repair and transcription, and RNA-binding proteins involved in posttranscriptional RNA maturation and translation. As a first step in the dissection of these complexes, we undertook to purify and identify nucleic acid-binding proteins from plant mitochondria. Most of the proteins identified are plant-specific, and many belong to a previously undescribed family of mt RNA-binding proteins (mRBP) characterized by the presence of an N-terminal RNA recognition motif (RRM). This family of plant mRBPs is ph...
Some of the mitochondrial tRNAs of higher plants are nuclearly encoded and imported into mitochondria. The import of tRNAs encoded in the nucleus has been shown to be essential for proper protein translation within mitochondria of a variety of organisms. Here, we report the development of an in vitro assay for import of nuclearly encoded tRNAs into plant mitochondria. This in vitro system utilizes isolated mitochondria from Solanum tuberosum and synthetic tRNAs transcribed from cloned nuclear tRNA genes. Although incubation of radioactively labeled in vitro-transcribed tRNA Ala , tRNA Phe , and tRNA Met-e with isolated potato mitochondria resulted in importation, as measured by nuclease protection, the amount of tRNA transcripts protected at saturation was at least five times higher for tRNA Ala than for the two other tRNAs. This difference in in vitro saturation levels of import is consistent with the in vivo localization of these tRNAs, since cytosolic tRNA Ala is naturally imported into potato mitochondria whereas tRNA Phe and tRNA Met-e are not. Characterization of in vitro tRNA import requirements indicates that mitochondrial tRNA import proceeds in the absence of any added cytosolic protein fraction, involves at least one protein component on the surface of mitochondria, and requires ATP-dependent step(s) and a membrane potential.
SummaryIn higher plants, one-third to one-half of the mitochondrial tRNAs are encoded in the nucleus and are imported into mitochondria. This process appears to be highly speci®c for some tRNAs, but the factors that interact with tRNAs before and/or during import, as well as the signals present on the tRNAs, still need to be identi®ed. The rare experiments performed so far suggest that, besides the probable implication of aminoacyl-tRNA synthetases, at least one additional import factor and/or structural features shared by imported tRNAs must be involved in plant mitochondrial tRNA import. To look for determinants that direct tRNA import into higher plant mitochondria, we have transformed BY2 tobacco cells with Arabidopsis thaliana cytosolic tRNA Val (AAC) carrying various mutations. The nucleotide replacements introduced in this naturally imported tRNA correspond to the anticodon and/or D-domain of the non-imported cytosolic tRNA Met±e . Unlike the wild-type tRNA Val (AAC), a mutant tRNA Val carrying a methionine CAU anticodon that switches the aminoacylation of this tRNA from valine to methionine is not present in the mitochondrial fraction. Furthermore, mutant tRNAs Val carrying the D-domain of the tRNA Met±e , although still ef®ciently recognized by the valyl-tRNA synthetase, are not imported any more into mitochondria. These data demonstrate that in plants, besides identity elements required for the recognition by the cognate aminoacyl-tRNA synthetase, tRNA molecules contain other determinants that are essential for mitochondrial import selectivity. Indeed, this suggests that the tRNA import mechanism occurring in plant mitochondria may be different from what has been described so far in yeast or in protozoa.
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