André Dietrich

Plant mitochondria actively import DNA via the permeability transition pore complex

Plant mitochondria are remarkable with respect to their content in foreign, alien and plasmid‐like DNA, raising the question of the transfer of this information into the organelles. We demonstrate the existence of an active, transmembrane potential‐dependent mechanism of DNA uptake into plant mitochondria. The process is restricted to double‐strand DNA, but has no obvious sequence specificity. It is most efficient with linear fragments up to a few kilobase pairs. When containing appropriate information, imported sequences are transcribed within the organelles. The uptake likely involves the voltage‐dependent anion channel and the adenine nucleotide translocator, i.e. the core components of the mitochondrial permeability transition pore complex in animal cells, but it does not rely on known mitochondrial membrane permeabilization processes. We conclude that DNA import into plant mitochondria might represent a physiological phenomenon with some functional relevance.

In vivo import of a normal or mutagenized heterologous transfer RNA into the mitochondria of transgenic plants: towards novel ways of influencing mitochondrial gene expression?

Evidence that nuclear‐encoded RNAs are present inside mitochondria has been reported from a wide variety of organisms, and is presumed to be due to import of specific cytosolic RNAs. In plants, the first examples were the mitochondrial leucine transfer RNAs of bean. In all cases, the evidence is circumstantial, based on hybridization of the mitochondrial RNAs to nuclear and not mitochondrial DNA. Here we show that transgenic potato plants carrying a leucine tRNA gene from bean nuclear DNA contain RNA transcribed from the introduced gene both in the cytosol and inside mitochondria, providing proof that the mitochondrial leucine tRNA is derived from a nuclear gene and imported into the mitochondria. The same bean gene carrying a 4 bp insertion in the anticodon loop was also expressed in transgenic potato plants and the transcript found to be present inside mitochondria, suggesting that this natural RNA import system could eventually be used to introduce foreign RNA sequences into mitochondria.

DNA repair in organelles: Pathways, organization, regulation, relevance in disease and aging.

Both endogenous processes and exogenous physical and chemical sources generate deoxyribonucleic acid (DNA) damage in the nucleus and organelles of living cells. To prevent deleterious effects, damage is balanced by repair pathways. DNA repair was first documented for the nuclear compartment but evidence was subsequently extended to the organelles. Mitochondria and chloroplasts possess their own repair processes. These share a number of factors with the nucleus but also rely on original mechanisms. Base excision repair remains the best characterized. Repair is organized with the other DNA metabolism pathways in the organelle membrane-associated nucleoids. DNA repair in mitochondria is a regulated, stress-responsive process. Organelle genomes do not encode DNA repair enzymes and translocation of nuclear-encoded repair proteins from the cytosol seems to be a major control mechanism. Finally, changes in the fidelity and efficiency of mitochondrial DNA repair are likely to be involved in DNA damage accumulation, disease and aging. The present review successively addresses these different issues.

Mitochondrial Targeting and Membrane Anchoring of a Viral Replicase in Plant and Yeast Cells

ABSTRACT Replication of the Carnation Italian ringspot virus genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon. We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria. Using infection, in vivo expression of green fluorescent protein fusions in plant and yeast cells, and in vitro mitochondrial integration assays, we demonstrate here that both the 36-kDa protein and the complete replicase are targeted to mitochondria and anchor to the outer membrane with the N terminus and C terminus on the cytosolic side. Analysis of deletion mutants indicated that the anchor sequence is likely to correspond approximately to amino acids 84 to 196, containing two transmembrane domains. No evidence for a matrix-targeting presequence was found, and the data suggest that membrane insertion of the viral proteins is mediated by an import receptor-independent signal-anchor mechanism relying on the two transmembrane segments and multiple recognition signals present in the N-terminal part of ORF1.

The yeast aminoacyl-tRNA synthetases. Methodology for their complete or partial purification and comparison of their relative activities under various extraction conditions.

Several fractionation steps are described which can be applied to the partial purification of the 20 aminoacyl-tRNA synthetases from commercial bakers yeast. Comparative experiments performed in the presence or absence of protease inhibitors revealed that some enzymes prepared in the presence of the inhibitor exhibit much higher specific activities than the proteins extracted in the absence of the inhibitor. The methodology reported can be used for the simultaneous preparation of several pure aminoacyl-tRNA synthetases. As examples, the large scale purification of phenylalanyl-and valyl-tRNA synthetases are described.

In vitro import of a nuclearly encoded tRNA into mitochondria of Solanum tuberosum.

ABSTRACT 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 tRNAAla, tRNAPhe, and tRNAMet-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 tRNAAla 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 tRNAAla is naturally imported into potato mitochondria whereas tRNAPhe and tRNAMet-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.

The plant mitochondrial genome: Dynamics and maintenance

Plant mitochondria have a complex and peculiar genetic system. They have the largest genomes, as compared to organelles from other eukaryotic organisms. These can expand tremendously in some species, reaching the megabase range. Nevertheless, whichever the size, the gene content remains modest and restricted to a few polypeptides required for the biogenesis of the oxidative phosphorylation chain complexes, ribosomal proteins, transfer RNAs and ribosomal RNAs. The presence of autonomous plasmids of essentially unknown function further enhances the level of complexity. The physical organization of the plant mitochondrial DNA includes a set of sub-genomic forms resulting from homologous recombination between repeats, with a mixture of linear, circular and branched structures. This material is compacted into membrane-bound nucleoids, which are the inheritance units but also the centers of genome maintenance and expression. Recombination appears to be an essential characteristic of plant mitochondrial genetic processes, both in shaping and maintaining the genome. Under nuclear surveillance, recombination is also the basis for the generation of new mitotypes and is involved in the evolution of the mitochondrial DNA. In line with, or as a consequence of its complex physical organization, replication of the plant mitochondrial DNA is likely to occur through multiple mechanisms, potentially involving recombination processes. We give here a synthetic view of these aspects.

A single editing event is a prerequisite for efficient processing of potato mitochondrial phenylalanine tRNA.

In bean, potato, and Oenothera plants, the C encoded at position 4 (C4) in the mitochondrial tRNA Phe GAA gene is converted into a U in the mature tRNA. This nucleotide change corrects a mismatched C4-A69 base pair which appears when the gene sequence is folded into the cloverleaf structure. C-to-U conversions constitute the most common editing events occurring in plant mitochondrial mRNAs. While most of these conversions introduce changes in the amino acids specified by the mRNA and appear to be essential for the synthesis of functional proteins in plant mitochondria, the putative role of mitochondrial tRNA editing has not yet been defined. Since the edited form of the tRNA has the correct secondary and tertiary structures compared with the nonedited form, the two main processes which might be affected by a nucleotide conversion are aminoacylation and maturation. To test these possibilities, we determined the aminoacylation properties of unedited and edited potato mitochondrial tRNAPhe in vitro transcripts, as well as the processing efficiency of in vitro-synthesized potato mitochondrial tRNAPhe precursors. Reverse transcription-PCR amplification of natural precursors followed by cDNA sequencing was also used to investigate the influence of editing on processing. Our results show that C-to-U conversion at position 4 in the potato mitochondrial tRNA Phe GAA is not required for aminoacylation with phenylalanine but is likely to he essential for efficient processing of this tRNA.

Large scale purification and structural properties of yeast aspartyl-tRNA synthetase

A large scale purification procedure of bakers yeast aspartyl-tRNA synthetase is described which yields more than 200 mg pure protein starting from 30 Kg of wet commercial cells. The synthetase is an alpha 2 dimer of Mr = 125,000 +/- 5,000 which can be crystallized (J. Mol. Biol. 138, 1980, 129-135). The enzyme has an elongated shape with a Stokes radius of 50 A and a frictional ratio of 1.5. The synthetase has a tendency to aggregate but methods are described where this effect is overcome.

Plant mitochondria possess a short-patch base excision DNA repair pathway.

Despite constant threat of oxidative damage, sequence drift in mitochondrial and chloroplast DNA usually remains very low in plant species, indicating efficient defense and repair. Whereas the antioxidative defense in the different subcellular compartments is known, the information on DNA repair in plant organelles is still scarce. Focusing on the occurrence of uracil in the DNA, the present work demonstrates that plant mitochondria possess a base excision repair (BER) pathway. In vitro and in organello incision assays of double-stranded oligodeoxyribonucleotides showed that mitochondria isolated from plant cells contain DNA glycosylase activity specific for uracil cleavage. A major proportion of the uracil–DNA glycosylase (UDG) was associated with the membranes, in agreement with the current hypothesis that the DNA is replicated, proofread and repaired in inner membrane-bound nucleoids. Full repair, from uracil excision to thymidine insertion and religation, was obtained in organello following import of a uracil-containing DNA fragment into isolated plant mitochondria. Repair occurred through single nucleotide insertion, which points to short-patch BER. In vivo targeting and in vitro import of GFP fusions showed that the putative UDG encoded by the At3g18 630 locus might be the first enzyme of this mitochondrial pathway in Arabidopsis thaliana.