Jean-Michel Grienenberger

Identification and characterization of a mitochondrial thioredoxin system in plants.

Plants possess two well described thioredoxin systems: a cytoplasmic system including several thioredoxins and an NADPH-dependent thioredoxin reductase and a specific chloroplastic system characterized by a ferredoxin-dependent thioredoxin reductase. On the basis of biochemical activities, plants also are supposed to have a mitochondrial thioredoxin system as described in yeast and mammals, but no gene encoding plant mitochondrial thioredoxin or thioredoxin reductase has been identified yet. We report the characterization of a plant thioredoxin system located in mitochondria. Arabidopsis thaliana genome sequencing has revealed numerous thioredoxin genes among which we have identified AtTRX-o1, a gene encoding a thioredoxin with a potential mitochondrial transit peptide. AtTRX-o1 and a second gene, AtTRX-o2, define, on the basis of the sequence and intron positions, a new thioredoxin type up to now specific to plants. We also have characterized AtNTRA, a gene encoding a protein highly similar to the previously described cytosolic NADPH-dependent thioredoxin reductase AtNTRB but with a putative presequence for import into mitochondria. Western blot analysis of A. thaliana subcellular and submitochondrial fractions and in vitro import experiments show that AtTRX-o1 and AtNTRA are targeted to the mitochondrial matrix through their cleavable N-terminal signal. The two proteins truncated to the estimated mature forms were produced in Escherichia coli; AtTRX-o1 efficiently reduces insulin in the presence of DTT and is reduced efficiently by AtNTRA and NADPH. Therefore, the thioredoxin and the NADPH-dependent thioredoxin reductase described here are proposed to constitute a functional plant mitochondrial thioredoxin system.

The genes coding for subunit 3 of NADH dehydrogenase and for ribosomal protein S12 are present in the wheat and maize mitochondrial genomes and are co-transcribed

SummaryA region of about 2 kb which is almost identical in the wheat and maize mitochondrial genomes has been sequenced. It contains a tRNASer gene, a pseudo-tRNA gene and two open reading frames coding for subunit 3 of the NADH dehydrogenase (118 amino acids) and for ribosomal protein S12 (125 amino acids). The two protein genes are separated by 47 bp and are co-transcribed in wheat and maize. Two transcripts of about 0.9 kb and 3.0 kb, cach coding for both proteins, have been characterized, but no monocistronic transcript was detected. Each gene is preceded by a putative ribosome binding site. The pseudo-tRNA gene is interrupted by two insertion sequences in wheat and by one in maize. The origin of the additional interrupting sequence found in the wheat pseudo-tRNA gene, which is also present elsewhere in the mitochondrial genomes, is discussed.

Editing of the wheat coxIII transcript: evidence for twelve C to U and one U to C conversions and for sequence similarities around editing sites

The complete cDNA sequence corresponding to the wheat coxIII gene transcript (coding for subunit 3 of cytochrome oxidase) has been determined by a method involving cDNA synthesis using specific oligonucleotides as primers followed by PCR amplification, cloning and sequencing of the amplification products. In 12 different clones, the same 13 nucleotide modifications have been found as compared to the genomic mitochondrial DNA sequence. Among these modifications, 12 are C----U conversions which change codons identities, thereby increasing the homology between the wheat COXIII protein and the corresponding protein of non-plant organisms. The 13th modification is a silent U----C conversion which seems to be an unfrequent editing eventin plant mitochondria. Homologies can be found between sequences surrounding editing sites in the coxIII transcript and in other wheat mitochondrial transcripts. The presence of such homology suggests that these sequences could base-pair with a common RNA molecule which might be involved in editing site recognition.

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

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.

Localization and organization of tRNA genes on the mitochondrial genomes of fertile and male sterile lines of maize

SummaryMaize mitochondrial (mt) tRNA genes were localized on the mt master circles of two fertile lines (WF9-N and B37-N) and of one cytoplasmic male sterile line (B37-cmsT) of maize. The three genomes contain 16 tRNA genes with 14 different anticodons which correspond to 13 amino acids. Out of these 16 tRNA genes, 6 show a high degree of homology with the corresponding chloroplast (cp) tRNA genes and were shown to originate from cp DNA insertions and to be expressed in the mitochondria. The organization of the mt tRNA genes in both fertile lines is similar. The same genes are found, in the same environment, as judged from the restriction maps, in fertile and male sterile lines that have the same nuclear background, but the relative organization of the mt tRNA genes on the master circle is completely different.

Location and nucleotide sequence of two tRNA genes and a tRNA pseudo-gene in the maize mitochondrial genome: evidence for the transcription of a chloroplast gene in mitochondria

SummaryWe report the nucleotide sequence of three tRNA genes from maize mitochondria. The genes are located in two BamHI fragments, 3.55 and 5.7 kb long, adjacent to the S2 sequence in the maize mitochondrial genome. On the 3.55 kb BamHI fragment, we have characterized a tRNACys (GCA) gene. A strong sequence homology of this tRNACys (GCA) gene with its chloroplast counterpart in wheat suggests that it may be part of a chloroplast DNA insertion into the mitochondrial genome. This gene has been found to be transcribed in the mitochondrion. Two tRNA genes are located on the 5.7 kb BamHI fragment, separated from each other by 250 bp. One is a mitochondrial tRNASer (GCU) gene. The other, a non-transcribed tRNAPhe-like gene, is interrupted by a 49 base-pair inserted DNA sequence in the variable loop and has a Leu (UAA) anticodon.

AtCCMA Interacts with AtCcmB to Form a Novel Mitochondrial ABC Transporter Involved in Cytochrome c Maturation in Arabidopsis

ABC transporters make a large and diverse family of proteins found in all phylae. AtCCMA is the nucleotide binding domain of a novel Arabidopsis mitochondrial ABC transporter. It is encoded in the nucleus and imported into mitochondria. Sub-organellar and topology studies find AtCCMA bound to the mitochondrial inner membrane, facing the matrix. AtCCMA exhibits an ATPase activity, and ATP/Mg2+ can facilitate its dissociation from membranes. Blue Native PAGE shows that it is part of a 480-kDa complex. Yeast two-hybrid assays reveal interactions between AtCCMA and domains of CcmB, the mitochondria-encoded transmembrane protein of a conserved ABC transporter. All these properties designate the protein as the ortholog in plant mitochondria of the bacterial CcmA required for cytochrome c maturation. The transporter that involves AtCCMA defines a new category of eukaryotic ABC proteins because its transmembrane and nucleotide binding domains are encoded by separate genomes.

Structure and transcription of the gene coding for subunit 3 of cytochrome oxidase in wheat mitochondria

SummaryThe wheat mitochondrial (mt) cox3 has been localized and sequenced. The gene exists as a single copy in the wheat mt master chromosome and is transcribed into a single 1.2 kb RNA, whose extremities have been mapped. Comparison of the wheat and Oenothera cox3 sequences gives ambiguous indications concerning the amino acid coded by the codon CGG. Upstream and downstream of the wheat cox3 gene, two short sequences of 43 bp and 69 bp respectively are present, which are almost identical to sequences present in the flanking regions of other plant mitochondrial genes. These common sequences seem to have played a role in the rearrangements which caused sequence divergence of the plant mt genomes during evolution. Furthermore, mapping of wheat and maize cox3 and cob transcripts suggests that some of these common sequences can play a role in the regulation of transcription or processing.

Structure of bean mitochondrial tRNAPhe and localization of the tRNAPhe gene on the mitochondrial genomes of maize and wheat

Bean mitochondrial tRNAPhe, purified by RPC‐5 chromatography and two‐dimensional gel electrophoresis, has been sequenced using in vitro post‐labeling techniques. It is the first plant mitochondrial tRNA sequenced. It shows 76% homology with bean chloroplast tRNAPhe and has many features characteristic of prokaryotic tRNAsPhe. It was used as a probe to localize the tRNAPhe gene on the mitochondrial genomes of maize and wheat.

RNA editing at a splicing site of NADH dehydrogenase subunit IV gene transcript in wheat mitochondria.

Comparison between the sequence of the gene coding for the wheat mitochondrial NADH dehydrogenase subunit IV (nad4) and the cDNA sequence obtained by reverse transcription, using total wheat mtRNA as template, has shown the presence of a uridine residue, not encoded by the genomic sequence, at the exon2‐exon3 junction of the spliced transcript. This U creates a non‐encoded CUG leucine codon which is essential for maintaining the reading frame, as shown by the conservation of the amino acid sequence of the NAD4 protein in various species. The addition of a U or the specific post‐transcriptional conversion of a C to a U could explain this phenomenon.