Transfer of <i>rps10</i> from the mitochondrion to the nucleus in <i>Arabidopsis thaliana</i>: evidence for RNA‐mediated transfer and exon shuffling at the integration site

Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrialencoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first transmembrane region of the nuclear encoded protein compared with the organelle-encoded protein. Mitochondrial-encoded Cox2 could not be imported into mitochondria under the direction of the mitochondrial targeting sequence that readily supports the import of nuclear encoded Cox2. Removal of the first transmembrane region promotes import ability of the mitochondrial-encoded Cox2. Changing just two amino acids in the first transmembrane region of mitochondrial-encoded Cox2 to the corresponding amino acids in the nuclear encoded Cox2 also promotes import ability, whereas changing the same two amino acids in the nuclear encoded Cox2 to what they are in the mitochondrial-encoded copy prevents import. Therefore, changes in amino acids in the mature protein were necessary and sufficient for gene transfer to allow import under the direction of an appropriate signal to achieve the functional topology of Cox2.

Section: Discussionmentioning

confidence: 99%

Intracellular gene transfer: Reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochromecoxidase

Daley

Clifton

Whelan

2002

“…Precursor proteins were synthesized from the full-length cDNA clones by coupled transcription-translation in the presence of [ 35 S]methionine (TNT-coupled reticulocyte lysate system, Promega), and in vitro import assays were conducted as described (6). For in vivo intracellular localization, the gene coding sequences were cloned in the NcoI-BamHI sites of plasmid pCK-GFP3A (7), which allows the expression of protein-eGFP (enhanced green fluorescent protein) fusions under the control of a 35S promoter.…”

Section: Methodsmentioning

confidence: 99%

A family of RRM-type RNA-binding proteins specific to plant mitochondria

Vermel

Guermann

Delage

et al. 2002

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...

“…23), or by the protein being dispensable in certain plants. Six ribosomal protein genes (22)(23)(24)(25)(26)(27)(28) that are present in the mitochondrion of many flowering plants, along with the respiratory gene cox2 (18)(19)(20)(21), have been reported to have been transferred to the nucleus. Thus, the most likely explanation for loss of a gene from the mitochondrion is transfer to the nucleus.…”

Section: Ribosomal Protein Genes Are Lost Frequently Respiratory Genmentioning

confidence: 99%

Dynamic evolution of plant mitochondrial genomes: Mobile genes and introns and highly variable mutation rates

Palmer

Adams

Cho

et al. 2000

304

242

We summarize our recent studies showing that angiosperm mitochondrial (mt) genomes have experienced remarkably high rates of gene loss and concomitant transfer to the nucleus and of intron acquisition by horizontal transfer. Moreover, we find substantial lineage-specific variation in rates of these structural mutations and also point mutations. These findings mostly arise from a Southern blot survey of gene and intron distribution in 281 diverse angiosperms. These blots reveal numerous losses of mt ribosomal protein genes but, with one exception, only rare loss of respiratory genes. Some lineages of angiosperms have kept all of their mt ribosomal protein genes whereas others have lost most of them. These many losses appear to reflect remarkably high (and variable) rates of functional transfer of mt ribosomal protein genes to the nucleus in angiosperms. The recent transfer of cox2 to the nucleus in legumes provides both an example of interorganellar gene transfer in action and a starting point for discussion of the roles of mechanistic and selective forces in determining the distribution of genetic labor between organellar and nuclear genomes. Plant mt genomes also acquire sequences by horizontal transfer. A striking example of this is a homing group I intron in the mt cox1 gene. This extraordinarily invasive mobile element has probably been acquired over 1,000 times separately during angiosperm evolution via a recent wave of cross-species horizontal transfers. Finally, whereas all previously examined angiosperm mtDNAs have low rates of synonymous substitutions, mtDNAs of two distantly related angiosperms have highly accelerated substitution rates.