A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl-tRNA synthetase in Arabidopsis thaliana.

One-fifth of the tRNAs used in plant mitochondrial translation is coded for by chloroplast-derived tRNA genes. To understand how aminoacyl-tRNA synthetases have adapted to the presence of these tRNAs in mitochondria, we have cloned an Arabidopsis thaliana cDNA coding for a methionyl-tRNA synthetase. This enzyme was chosen because chloroplast-like elongator tRNAMet genes have been described in several plant species, including A. thaliana. We demonstrate here that the isolated cDNA codes for both the chloroplastic and the mitochondrial methionyl-tRNA synthetase (MetRS). The protein is transported into isolated chloroplasts and mitochondria and is processed to its mature form in both organelles. Transient expression assays using the green fluorescent protein demonstrated that the N-terminal region of the MetRS is sufficient to address the protein to both chloroplasts and mitochondria. Moreover, characterization of MetRS activities from mitochondria and chloroplasts of pea showed that only one MetRS activity exists in each organelle and that both are indistinguishable by their behavior on ion exchange and hydrophobic chromatographies. The high degree of sequence similarity between A. thaliana and Synechocystis MetRS strongly suggests that the A. thaliana MetRS gene described here is of chloroplast origin.

[1]  W. Webb,et al.  The green fluorescent protein as a marker to visualize plant mitochondria in vivo. , 1997, The Plant journal : for cell and molecular biology.

[2]  J. Weil,et al.  Aminoacylation of Phaseolus vulgaris cytoplasmic, chloroplastic and mitochondrial tRNAsMet and of Escherichia coli tRNAsMet by homologous and heterologous enzymes. , 1975, Biochimica et biophysica acta.

[3]  Alison G. Smith,et al.  A Single Precursor Protein for Ferrochelatase-I fromArabidopsis Is Imported in Vitro into Both Chloroplasts and Mitochondria* , 1997, The Journal of Biological Chemistry.

[4]  Frans,et al.  Genes Galore: A Summary of Methods for Accessing Results from Large-Scale Partial Sequencing of Anonymous Arabidopsis cDNA Clones , 1994, Plant physiology.

[5]  J. Mathur,et al.  Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[6]  I. Small,et al.  The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases. , 1996, The Plant cell.

[7]  K. Keegstra,et al.  Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes. , 1989, The Journal of biological chemistry.

[8]  W. Webb,et al.  Exchange of protein molecules through connections between higher plant plastids. , 1997, Science.

[9]  R. Julien,et al.  Chloroplastic methionyl-tRNA synthetase from wheat. , 1981, Biochemical and biophysical research communications.

[10]  A. Steinmetz,et al.  Aminoacylation of tRNA-Leu species from Escherichia coli and from the cytoplasm, chloroplasts and mitochondria of Phaseolus vulgaris by homologous and heterologous enzymes. , 1975, Biochimica et Biophysica Acta.

[11]  Protein Import into Plant Mitochondria , 1994 .

[12]  L. Marechal-Drouard,et al.  Import of several tRNAs from the cytoplasm into the mitochondria in bean Phaseolus vulgaris. , 1988, Nucleic acids research.

[13]  A. Brennicke,et al.  The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides , 1997, Nature Genetics.

[14]  P. Joyce,et al.  Chloroplast-like transfer RNA genes expressed in wheat mitochondria. , 1989, Nucleic acids research.

[15]  D. Youvan,et al.  Structure and fluorescence mechanism of GFP , 1996, Nature Biotechnology.

[16]  L. Marechal-Drouard,et al.  TRANSFER RNAs AND TRANSFER RNA GENES IN PLANTS , 1993 .

[17]  P. Mullineaux,et al.  Simultaneous targeting of pea glutathione reductase and of a bacterial fusion protein to chloroplasts and mitochondria in transgenic tobacco. , 1995, The Plant journal : for cell and molecular biology.

[18]  Pierre Guillemaut,et al.  Transfer RNAs of potato (Solanum tuberosum) mitochondria have different genetic origins , 1990, Nucleic Acids Res..

[19]  L. Marechal-Drouard,et al.  Transfer RNA import into plant mitochondria. , 1995, Methods in enzymology.

[20]  L. Marechal-Drouard,et al.  A single base change prevents import of cytosolic tRNA(Ala) into mitochondria in transgenic plants. , 1996, The Plant journal : for cell and molecular biology.

[21]  S. Blanquet,et al.  Molecular cloning and primary structure of the Escherichia coli methionyl-tRNA synthetase gene , 1984, Journal of bacteriology.

[22]  A. Steinmetz,et al.  Isolation and characterization of chloroplast and cytoplasmic transfer RNAs , 1986 .