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In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca(2+)-effects.

Lorenz A, Lorenz M, Vothknecht UC, Niopek-Witz S, Neuhaus HE, Haferkamp I - BMC Plant Biol. (2015)

Bottom Line: Moreover, investigation of a representative mutant APC protein revealed that the observed calcium effects on ATP transport did not primarily/essentially involve Ca(2+)-binding to the EF-hand motifs in the N-terminal domain of the carrier.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.

View Article: PubMed Central - PubMed

Affiliation: Cellular Physiology/Membrane Transport, University of Kaiserslautern, 67653, Kaiserslautern, Germany. anlorenz@rhrk.uni-kl.de.

ABSTRACT

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 Ca(2+)-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.

No MeSH data available.


Structural alignment of the N-terminal domains of AtAPC1-3 and human SCaMC1. Three-dimensional homology models of AtAPC1 (residues 34–189, green), AtAPC2 (residues 38–194, yellow) and AtAPC3 (residues 35–189, orange). N-terminal domains were built using HHPred server and Modeller based on the crystal structure of the Ca2+-binding N-terminal domain of human SCaMC1 (blue; PDB ID: 4N5X) in complex with four calcium ions (gray spheres). The sequence alignment followed by a structural superimposition of the models was carried out using PyMOL (version 1.3)
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Fig7: Structural alignment of the N-terminal domains of AtAPC1-3 and human SCaMC1. Three-dimensional homology models of AtAPC1 (residues 34–189, green), AtAPC2 (residues 38–194, yellow) and AtAPC3 (residues 35–189, orange). N-terminal domains were built using HHPred server and Modeller based on the crystal structure of the Ca2+-binding N-terminal domain of human SCaMC1 (blue; PDB ID: 4N5X) in complex with four calcium ions (gray spheres). The sequence alignment followed by a structural superimposition of the models was carried out using PyMOL (version 1.3)

Mentions: Although Ca2+-dependent activity regulation of human and yeast APCs has been well known for a long time, first insights into the mechanistic principle were only gained recently. Sophisticated interaction studies with human SCaMC1 suggest that in absence of Ca2+ the quite flexible N-terminal domain caps the transmembrane part whereas Ca2+-binding turns the N-terminal domain into a more rigid state which leads to its dissociation and opening of the translocation pore [21, 22]. Superimposition of the corresponding regions in a structural alignment visualizes a high degree of conservation among the N-terminal domains of plant APCs and human SCaMC1 (Fig. 7). These structural similarities as well as computer based docking analyses (Additional file 8: Figure S8) suggest that the N-terminal domains of the plant APCs also interact with four Ca2+ ions. Moreover, amino acid sequence similarity to Sal1p and human SCaMC isoforms suggest that plant APCs are likewise regulated by Ca2+ (Additional file 7: Figure S7, Fig. 7).Fig. 7


In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca(2+)-effects.

Lorenz A, Lorenz M, Vothknecht UC, Niopek-Witz S, Neuhaus HE, Haferkamp I - BMC Plant Biol. (2015)

Structural alignment of the N-terminal domains of AtAPC1-3 and human SCaMC1. Three-dimensional homology models of AtAPC1 (residues 34–189, green), AtAPC2 (residues 38–194, yellow) and AtAPC3 (residues 35–189, orange). N-terminal domains were built using HHPred server and Modeller based on the crystal structure of the Ca2+-binding N-terminal domain of human SCaMC1 (blue; PDB ID: 4N5X) in complex with four calcium ions (gray spheres). The sequence alignment followed by a structural superimposition of the models was carried out using PyMOL (version 1.3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4595200&req=5

Fig7: Structural alignment of the N-terminal domains of AtAPC1-3 and human SCaMC1. Three-dimensional homology models of AtAPC1 (residues 34–189, green), AtAPC2 (residues 38–194, yellow) and AtAPC3 (residues 35–189, orange). N-terminal domains were built using HHPred server and Modeller based on the crystal structure of the Ca2+-binding N-terminal domain of human SCaMC1 (blue; PDB ID: 4N5X) in complex with four calcium ions (gray spheres). The sequence alignment followed by a structural superimposition of the models was carried out using PyMOL (version 1.3)
Mentions: Although Ca2+-dependent activity regulation of human and yeast APCs has been well known for a long time, first insights into the mechanistic principle were only gained recently. Sophisticated interaction studies with human SCaMC1 suggest that in absence of Ca2+ the quite flexible N-terminal domain caps the transmembrane part whereas Ca2+-binding turns the N-terminal domain into a more rigid state which leads to its dissociation and opening of the translocation pore [21, 22]. Superimposition of the corresponding regions in a structural alignment visualizes a high degree of conservation among the N-terminal domains of plant APCs and human SCaMC1 (Fig. 7). These structural similarities as well as computer based docking analyses (Additional file 8: Figure S8) suggest that the N-terminal domains of the plant APCs also interact with four Ca2+ ions. Moreover, amino acid sequence similarity to Sal1p and human SCaMC isoforms suggest that plant APCs are likewise regulated by Ca2+ (Additional file 7: Figure S7, Fig. 7).Fig. 7

Bottom Line: Moreover, investigation of a representative mutant APC protein revealed that the observed calcium effects on ATP transport did not primarily/essentially involve Ca(2+)-binding to the EF-hand motifs in the N-terminal domain of the carrier.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.

View Article: PubMed Central - PubMed

Affiliation: Cellular Physiology/Membrane Transport, University of Kaiserslautern, 67653, Kaiserslautern, Germany. anlorenz@rhrk.uni-kl.de.

ABSTRACT

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 Ca(2+)-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.

No MeSH data available.