Heat stress response in pea involves interaction of mitochondrial nucleoside diphosphate kinase with a novel 86-kilodalton protein.

In this work we have further characterized the first mitochondrial nucleoside diphosphate kinase (mtNDPK) isolated from plants. The mitochondrial isoform was found to be especially abundant in reproductive and young tissues. Expression of the pea (Pisum sativum L. cv Oregon sugarpod) mtNDPK was not affected by different stress conditions. However, the pea mtNDPK was found to interact with a novel 86-kD protein, which is de novo synthesized in pea leaves upon exposure to heat. Thus, we have evidence for the involvement of mtNDPK in mitochondrial heat response in pea in vivo. Studies on oligomerization revealed that mtNDPK was found in complexes of various sizes, corresponding to the sizes of e.g. hexamers, tetramers, and dimers, indicating flexibility in oligomerization. This flexibility, also found for other NDPK isoforms, has been correlated with the ability of this enzyme to interact with other proteins. We believe that the mtNDPK is involved in heat stress response in pea, possibly as a modulator of the 86-kD protein.

Cloning and characterisation of a pea mitochondrial NDPK.

Here we report the cloning of a cDNA encoding the first nucleoside diphosphate kinase (NDPK) isolated from plant mitochondria. Amplification of a 317 nt product was performed by PCR, using oligonucleotide primers based on partial amino acid sequences of the pea mitochondria NDPK and other NDPK isoforms. By screening of a pea cDNA library with this PCR product, a full length clone was obtained. Northern analysis revealed the presence of a 1.1 kb single transcript, with high expression in young leaves and reproductive tissues. The clone encodes a precursor protein of 232 amino acids (26 kDa), including an N-terminal extension of 80 amino acids (9 kDa). Analysis of the deduced amino acid sequence confirmed its identity with the sequences obtained from the purified mature pea mitochondrial NDPK. In vitro import experiments carried out in isolated pea mitochondria showed targeting and processing of the 27 kDa precursor into a 16.5 kDa mature form. Phylogenetic analysis of some vertebrate and plant isoforms of NDPK showed that the pea mtNDPK groups together with the NDPK3 isoform from A. thaliana and the chloroplastic NDPK III from spinach. We suggest that it is possible to design a novel classification of the different NDPK isoforms according to their subcellular localisation and origin.

Protein synthesis by isolated pea mitochondria is dependent on the activity of respiratory complex II.

In isolated pea (Pisum sativum L.) mitochondria incorporation of 35S-methionine into newly synthesised proteins was influenced by the presence of site-specific inhibitors of the respiratory electron-transport chain. These effects were not produced by changes in the rate of respiratory electron transport itself nor by changes in ATP concentration. Protein synthesis was inhibited by inhibitors of ubiquinone reduction but not by inhibitors of ubiquinol oxidation. By the use of additional inhibitors at specific sites of the respiratory chain, different oxidation-reduction states were obtained for the different complexes in the electron-transport chain. It was found that electron transport through succinate:ubiquinone oxidoreductase (respiratory complex II) was specifically required for protein synthesis, even when all the other conditions for protein synthesis were satisfied. We suggest that a subunit of complex II, or a component closely associated with complex II, is involved in a regulatory system that couples electron transport to protein synthesis.