Novel functions of the mitochondrial nucleoside diphosphate kinase in plants

Nucleoside diphosphate kinases (NDPKs) are ubiquitous enzymes found in almost all species. Their main function is to transfer a γ-phosphate from ATP to a cognate nucleoside diphosphate, thereby balancing the nucleoside pool. Early studies showed that the ablation of NDPK genes had different effects depending on species and isoform, indicating a multi-functionality of the NDPK enzymes. Indeed, additional functions, including nuclease and protein kinase activities, have been demonstrated and are continuously being discovered. Attention was especially paid to these additional effects when it was shown that a human NDPK (nm23) is involved in the progression of cancer. In plants there are three NDPK isoforms. NDPK1 is located in the cytosol, NDPK2 in the chloroplast stroma and NDPK3 in the mitochondrial inner membrane and intermembrane space (IMS). The plant NDPKs have been implicated in stress and hormone response, as well as in light signalling. This work concerns the plant NDPK genes and proteins, with a specific focus on the mitochondrial NDPK3. Phylogenetic analyses revealed that the evolution of separate plant NDPK isoforms is evolutionary old. In Arabidopsis thaliana the NDPK3 gene has been duplicated relatively recently, resulting in two genes, NDPK3a and NDPK3b. Quantitative RT-PCR showed that the expression of the NDPK genes differs between tissues, with NDPK1 being the overall most abundant transcript. The expression of the NDPK3a gene is relatively high in all tissues, especially in rapidly dividing cells in the flower bud. In addition, this gene appears to respond to the cell energy level, as shown by the more than 2-fold induction after sucrose and glucose applications. Interestingly, a specific expression of the NDPK3b gene was seen in ovules and tapetum cells of stage 12 flower buds, suggesting a specific function of this protein in these tissues despite a very low overall expression. By GFP-fusions and immunoblotting we demonstrated that the pea NDPK3 is dually targeted to chloroplasts and mitochondria. In addition, we found that recombinant NDPK3 is able to interact with the mitochondrial Adenylate kinase 1 (AK1). Activity measurements showed an inhibition of AK1 activity in parallel to an augmented NDPK3 activity when both reecombinant enzymes were present in the same assay. We were also able to show that the recombinant NDPK3 protein can cleave DNA and RNA molecules, an ability that was independent of the capacity to form a hexameric structure of the protein, and which was inhibited by ATP. This is the first time a nuclease has been identified in the plant IMS