A novel connection between nucleotide and carbohydrate metabolism in mitochondria: sugar regulation of the Arabidopsis nucleoside diphosphate kinase 3a gene.

Sugar metabolism is intricately connected with mitochondria through the conversion of sugars to ATP, and through the production of carbon skeletons that can be used in anabolic processes. Sugar molecules also take part in signalling cascades. In this study we investigated the impact of sucrose on the expression of the Arabidopsis thaliana Nucleoside Diphosphate Kinase gene family (NDPK, EC 2.7.4.6), focusing on NDPK3a, the product of which is located predominantly in mitochondria. Using quantitative PCR we show that the NDPK3a gene is subject to sucrose and glucose induction, while no other Arabidopsis NDPK gene are sucrose-inducible. The induction reaches a half-maximum after about 6 hours and is stable for at least 48 h. Sucrose and glucose inductions were found not to be affected by the presence of a hexokinase inhibitor, N-acetyl-glucosamine. Furthermore, turanose, a sucrose analogue that is not metabolised in plant cells, did not induce NDPK3a gene expression. An analysis of the NDPK3a gene revealed two WBOXHWISO1 boxes in the promoter region, elements that have previously been reported to be involved in sugar signalling in barley via the SUSIBA2 protein. SUSIBA2 belongs to the WRKY group of transcription factors. In this study we used two mutants containing T-DNA insertions in WRKY-genes, AtWrky4 and AtWrky34, to investigate the possible involvement of WRKY transcription factors in the sugar induction of NDPK3a.

The pea mitochondrial nucleoside diphosphate kinase cleaves DNA and RNA.

Here, we present the characterization of a plant NDPK exhibiting nuclease activity. This is the first identification of a nuclease localised in the intermembrane space of plant mitochondria. The recombinant pea NDPK3 protein cleaves not only supercoiled plasmid DNA, but also highly structured RNA molecules such as tRNAs or the 3'UTR of the atp9 mRNA suggesting that the NDPK3 nuclease activity has a structural requirement. ATP inhibits this nuclease activity, while ADP has no effect. Furthermore, studies on NDPK mutant proteins indicate that the nuclease- and the kinase-mechanisms are separate.

Plant mitochondria contain at least two i-AAA-like complexes.

The FtsH proteases, also called AAA proteases, are membrane-bound ATP-dependent metalloproteases. The Arabidopsis genome contains a total of 12 FtsH-like genes. Two of them, AtFtsH4 and AtFtsH11, encode proteins with a high similarity to Yme1p, a subunit of the i-AAA complex in yeast mitochondria. Phylogenetic analysis groups the AtFtsH4, AtFtsH11 and Yme1 proteins together, with AtFtsH4 being the most similar to Yme1. Using immunological method we demonstrate here that AtFtsH4 is an exclusively mitochondrial protein while AtFtsH11 is found in both chloroplasts and mitochondria. AtFtsH4 and AtFtsH11 proteases are integral parts of the inner mitochondrial membrane and expose their catalytic sites towards the intermembrane space, same as yeast i-AAA. Database searches revealed that orthologs of AtFtsH4 and AtFtsH11 are present in both monocotyledonous and dicotyledonous plants. The two plant i-AAA proteases differ significantly in their termini: the FtsH4 proteins have a characteristic alanine stretch at the C-terminal end while FtsH11s have long N-terminal extensions. Blue-native gel electrophoresis revealed that AtFtsH4 and AtFtsH11 form at least two complexes with apparent molecular masses of about 1500 kDa. This finding implies that plants, in contrast to fungi and metazoa, have more than one complex with a topology similar to that of yeast i-AAA.

Modified sucrose, starch, and ATP levels in two alloplasmic male-sterile lines of B. napus.

Alloplasmic lines of Brassica napus with rearranged Arabidopsis thaliana mitochondrial DNA are male sterile and vegetatively altered compared with B. napus cv. Hanna. The CMS lines contain pure nuclear and plastid genomes from B. napus. Cross-sections of leaves revealed elevated starch accumulation and a higher number of chloroplasts per cell area in CMS plants compared with B. napus. The increase in chloroplast density was found to be the result of the smaller mesophyll cells. Sucrose concentration in the leaves of the CMS lines was reduced both in green leaves as well as in leaves from 2 d-etiolated plants. Flower meristem, flower buds, and leaves from green and 2 d-etiolated plants were analysed for ATP and ADP contents. All CMS plant tissues, except for green leaves, possessed lower ATP levels than B. napus. The results indicate that the reduced availability of energy, i.e. ATP and sucrose in the CMS plants, limits plant growth. This is supported by the reduced levels of two D-type cyclin transcripts and the reduced capacity of the CMS plants to recover after etiolation.

Structure and mutational analysis of a plant mitochondrial nucleoside diphosphate kinase. Identification of residues involved in serine phosphorylation and oligomerization.

We report the first crystal structure of a plant (Pisum sativum L. cv Oregon sugarpod) mitochondrial nucleoside diphosphate kinase. Similar to other eukaryotic nucleoside diphosphate kinases, the plant enzyme is a hexamer; the six monomers in the asymmetric unit are arranged as trimers of dimers. Different functions of the kinase have been correlated with the oligomeric structure and the phosphorylation of Ser residues. We show that the occurrence of Ser autophosphorylation depends on enzymatic activity. The mutation of the strictly conserved Ser-119 to Ala reduced the Ser phosphorylation to about one-half of that observed in wild type with only a modest change of enzyme activity. We also show that mutating another strictly conserved Ser, Ser-69, to Ala reduces the enzyme activity to 6% and 14% of wild-type using dCDP and dTDP as acceptors, respectively. Changes in the oligomerization pattern of the S69A mutant were observed by cross-linking experiments. A reduction in trimer formation and a change in the dimer interaction could be detected with a concomitant increase of tetramers. We conclude that the S69 mutant is involved in the stabilization of the oligomeric state of this plant nucleoside diphosphate kinase.

Plant mitochondrial nucleoside diphosphate kinase is attached to the membrane through interaction with the adenine nucleotide translocator.

This study shows that the plant mitochondrial nucleoside diphosphate kinase (mNDPK) localizes to both the intermembrane space and to the mitochondrial inner membrane. We show that mNDPK is very firmly attached to the membrane. Co-immunoprecipitation experiments identified the adenine nucleotide translocator as an interaction partner. This is the first report showing a direct association between these two proteins, although previous studies have shown metabolic cooperation between them. Possible consequences for mitochondrial energy metabolism are discussed.

A higher plant mitochondrial homologue of the yeast m-AAA protease. Molecular cloning, localization, and putative function.

Mitochondrial AAA metalloproteases play a fundamental role in mitochondrial biogenesis and function. They have been identified in yeast and animals but not yet in plants. This work describes the isolation and sequence analysis of the full-length cDNA from the pea (Pisum sativum) with significant homology to the yeast matrix AAA (m-AAA) protease. The product of this clone was imported into isolated pea mitochondria where it was processed to its mature form (PsFtsH). We have shown that the central region of PsFtsH containing the chaperone domain is exposed to the matrix space. Furthermore, we have demonstrated that the pea protease can complement respiration deficiency in the yta10 and/or yta12 null yeast mutants, indicating that the plant protein can compensate for the loss of at least some of the important m-AAA functions in yeast. Based on biochemical experiments using isolated pea mitochondria, we propose that PsFtsH-like m-AAA is involved in the accumulation of the subunit 9 of the ATP synthase in the mitochondrial membrane.