In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects

Background: Adenine nucleotide/phosphate carriers (APCs) from mammals and yeast are commonly known to adapt the mitochondrial adenine nucleotide pool in accordance to cellular demands. They catalyze adenine nucleotide - particularly ATP-Mg - and phosphate exchange and their activity is regulated by calcium. Our current knowledge about corresponding proteins from plants is comparably limited. Recently, the three putative APCs from Arabidopsis thaliana were shown to restore the specific growth phenotype of APC yeast loss-of-function mutants and to interact with calcium via their N-terminal EF-hand motifs in vitro. In this study, we performed biochemical characterization of all three APC isoforms from A. thaliana to gain further insights into their functional properties. Results: Recombinant plant APCs were functionally reconstituted into liposomes and their biochemical characteristics were determined by transport measurements using radiolabeled substrates. All three plant APCs were capable of ATP, ADP and phosphate exchange, however, high preference for ATP-Mg, as shown for orthologous carriers, was not detectable. By contrast, the obtained data suggest that in the liposomal system the plant APCs rather favor ATP-Ca as substrate. Moreover, investigation of a representative mutant APC protein revealed that the observed calcium effects on ATP transport did not primarily/essentially involve Ca2+-binding to the EF-hand motifs in the N-terminal domain of the carrier. Conclusion: Biochemical characteristics suggest that plant APCs can mediate net transport of adenine nucleotides and hence, like their pendants from animals and yeast, might be involved in the alteration of the mitochondrial adenine nucleotide pool. Although, ATP-Ca was identified as an apparent import substrate of plant APCs in vitro it is arguable whether ATP-Ca formation and thus the corresponding transport can take place in vivo

Pyrimidine salvage: Physiological functions and interaction with chloroplast viogenesis

International audienceThe synthesis of pyrimidine nucleotides, an essential process in every organism, is accomplished by de novo synthesis or by salvaging pyrimdines from e.g. nucleic acid turnover. Here, we identify two Arabidopsis (Arabidopsis thaliana) uridine/cytidine kinases, UCK1 and UCK2, which are located in the cytosol and are responsible for the majority of pyrimidine salvage activity in vivo. In addition, the chloroplast has an active uracil salvage pathway. Uracil phosphoribosyltransferase (UPP) catalyzes the initial step in this pathway and is required for the establishment of photosynthesis, as revealed by analysis of upp mutants. The upp knockout mutants are unable to grow photoautotrophically, and knockdown mutants exhibit a variegated phenotype, with leaves that have chlorotic pale areas. Moreover, the upp mutants did not show altered expression of chloroplast-encoded genes, but transcript accumulation of the LIGHT HARVESTING COMPLEX B nuclear genes LHCB1.2 and LHCB2.3 was markedly reduced. An active UPP homolog from Escherichia colt failed to complement the upp mutant phenotype when targeted to the chloroplast, suggesting that the catalytic function of UPP is not the important factor for the chloroplast phenotype. Indeed, the expression of catalytically inactive Arabidopsis UPP, generated by introduction of point mutations, did complement the upp chloroplast phenotype. These results suggest that UPP has a vital function in chloroplast biogenesis unrelated to its catalytic activity and driven by a moonlighting function

Nucleobase and nucleoside transport and integration into plant metabolism

Nucleotide metabolism is an essential process in all living organisms. Besides newly synthesized nucleotides, the recycling (salvage) of partially degraded nucleotides i.e. nucleosides and nucleobases serves to keep the homeostasis of the nucleotide pool. Both types of metabolites are substrates of at least six families of transport proteins in Arabidopsis thaliana (Arabidopsis) with a total of 49 members. In the last years several members of such transport proteins have been analyzed allowing to present a more detailed picture of nucleoside and nucleobase transport and the physiological function of these processes. Besides functioning in nucleotide metabolism it turned out that individual members of the before named transporters exhibit the capacity to transport a wide range of different substrates including vitamins and phytohormones. The aim of this review is to summarize the current knowledge on nucleobase and nucleoside transport processes in plants and integrate this into nucleotide metabolism in general. Thereby, we will focus on those proteins which have been characterized at the biochemical level

Additional file 4: Figure S4. of In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects

Heterologous expression and ATP transport analysis of N- terminally truncated AtAPC2. (A) SDS-PAGE of 5μg and (B) Western-blot and immunodetection of 0.5 μg of the inclusion bodies fraction from E. coli cells expressing the N-terminally truncated (lanes 1). To enable detection of the molecular mass reduction due to loss of the N-terminal extension the full-length protein was included in this analysis (lanes 2). The Western-blot was immuno-decorated with a monoclonal anti poly His IgG (Sigma, Taufkirchen, Germany). M, prestained molecular weight marker (Thermo Fisher Scientific). (C) Time dependent import of 50 μM [α32P]-ATP via N- terminally truncated AtAPC2 into ATP loaded (black rhombs), Pi loaded (gray circles) and non-loaded (non-filled rhombs) liposomes. (PDF 156 kb

Additional file 2: Figure S2. of In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects

Time dependent ADP transport via AtAPC1-3. Transport of 50 ÎźM [Îą32P]-ADP into Pi (A, C, E) and into ADP (B, D, F) loaded proteoliposomes with reconstituted AtAPC1 (A, B), AtAPC2 (C, D) and AtAPC3 (E, F). Non-loaded liposomes (non-filled rhombs; negative control) showed only marginal accumulation of radioactivity when compared to proteoliposomes loaded with Pi or ADP (black rhombs). Data represent mean values of three independent replicates, standard errors are given. (PDF 82 kb

Additional file 6: Figure S6. of In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects

Effects of rising MgCl2 concentrations on [45Ca] transport via the N- terminally truncated AtAPC2. Transport of 20 ÎźM [45Ca] into Pi loaded (dark gray bars) and non- loaded (light gray bars) proteoliposomes was allowed for 10 min (given as nmol mg protein-1 h- 1). The transport medium was supplemented with 100 ÎźM non-labeled ATP and the indicated MgCl2 concentrations. Data represent mean values of three independent replicates. Standard errors are indicated. (PDF 102 kb

Additional file 7: Figure S7. of In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects

Alignment of APC proteins from different organisms. Amino acid sequence alignment of APCs from A. thaliana (AtAPC1-3 [GenBank:At5g61810; At5g51050; At5g07320]), S. cerevisiae (Sal1p [GenBank: YNL083w]) and human (HsSCaMC1-3 [GenBank:SLC25A24; SLC25A25; SLC25A23] using ClustalW2 ( http://www.ebi.ac.uk ). To allow easy detection of the N-terminal extension mitochondrial AAC2 from S. cerevisiae (ScPET9 [GenBank:YBL030C]) was included as a representative MCF protein. Shading of conserved amino acid residues was performed with Boxshade at the Swiss EMBnet server ( http://www.ch.embnet.org/index.html ). Residues of the N-terminal domains of AtAPC1-3 proposed to be involved in Ca2 +-interaction are highlighted by different colors. Residues predicted by Scanprosite ( http://prosite.expasy.org/scanprosite ) are marked in green and by molecular Ca2+ docking analyses with AutoDock vina (see also Additional file 8: Figure S8) are marked in orange. Ca2 +-interacting residues predicted by Scanprosite and molecular docking studies are marked in yellow. EF-hands I and III (orange boxes) exhibit lower support for Ca2 +-interaction (Scanprosite) than EF-hands II and IV (green boxes). (PDF 476 kb