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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.


Related in: MedlinePlus

Ca2+-impact on ATP transport via AtAPC1-3. Effect of rising Ca2+-concentrations (0–500 μM) on transport mediated by recombinant AtAPC1 (a, b), AtAPC2 (c, d) and AtAPC3 (e, f). Transport of 50 μM [α32P]-ATP was conducted in absence (black rhombs) and presence of supplemental MgCl2 (gray rhombs). Transport was allowed for 5 min and is given in nmol mg protein−1 h−1. Ca2+-dependent stimulation of ATP/Pi hetero-exchanges (a, c, e) and ATP/ATP homo-exchanges (b, d, f). Non-loaded liposomes (non-filled rhombs; negative control) showed only marginal accumulation of ATP and the corresponding rates were unaffected by MgCl2 addition. Data represent mean values of three independent replicates, standard errors are given
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Fig2: Ca2+-impact on ATP transport via AtAPC1-3. Effect of rising Ca2+-concentrations (0–500 μM) on transport mediated by recombinant AtAPC1 (a, b), AtAPC2 (c, d) and AtAPC3 (e, f). Transport of 50 μM [α32P]-ATP was conducted in absence (black rhombs) and presence of supplemental MgCl2 (gray rhombs). Transport was allowed for 5 min and is given in nmol mg protein−1 h−1. Ca2+-dependent stimulation of ATP/Pi hetero-exchanges (a, c, e) and ATP/ATP homo-exchanges (b, d, f). Non-loaded liposomes (non-filled rhombs; negative control) showed only marginal accumulation of ATP and the corresponding rates were unaffected by MgCl2 addition. Data represent mean values of three independent replicates, standard errors are given

Mentions: Comparison of Ca2+ effects on ATP and ATP-Mg transport indeed revealed interesting results that support this assumption. Mg2+ addition causes marginal (AtAPC2) to moderate (AtAPC1 and 3) increase in ATP transport when no extra Ca2+ is present (Figs. 1 and 2). With rising Ca2+ concentration the positive impact of Mg2+ becomes abolished and even reverted into a negative one (Fig. 2). More precisely, with higher Ca2+ concentrations (>10 μM AtAPC2; > 50 μM AtAPC1 and 3) the rates of ATP transport in absence of Mg2+ exceed the rates of the corresponding exchange in presence of Mg2+. Accordingly, in presence of Mg2+ higher concentrations of Ca2+ are apparently required to achieve ATP-transport saturation.Fig. 2


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)

Ca2+-impact on ATP transport via AtAPC1-3. Effect of rising Ca2+-concentrations (0–500 μM) on transport mediated by recombinant AtAPC1 (a, b), AtAPC2 (c, d) and AtAPC3 (e, f). Transport of 50 μM [α32P]-ATP was conducted in absence (black rhombs) and presence of supplemental MgCl2 (gray rhombs). Transport was allowed for 5 min and is given in nmol mg protein−1 h−1. Ca2+-dependent stimulation of ATP/Pi hetero-exchanges (a, c, e) and ATP/ATP homo-exchanges (b, d, f). Non-loaded liposomes (non-filled rhombs; negative control) showed only marginal accumulation of ATP and the corresponding rates were unaffected by MgCl2 addition. Data represent mean values of three independent replicates, standard errors are given
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Ca2+-impact on ATP transport via AtAPC1-3. Effect of rising Ca2+-concentrations (0–500 μM) on transport mediated by recombinant AtAPC1 (a, b), AtAPC2 (c, d) and AtAPC3 (e, f). Transport of 50 μM [α32P]-ATP was conducted in absence (black rhombs) and presence of supplemental MgCl2 (gray rhombs). Transport was allowed for 5 min and is given in nmol mg protein−1 h−1. Ca2+-dependent stimulation of ATP/Pi hetero-exchanges (a, c, e) and ATP/ATP homo-exchanges (b, d, f). Non-loaded liposomes (non-filled rhombs; negative control) showed only marginal accumulation of ATP and the corresponding rates were unaffected by MgCl2 addition. Data represent mean values of three independent replicates, standard errors are given
Mentions: Comparison of Ca2+ effects on ATP and ATP-Mg transport indeed revealed interesting results that support this assumption. Mg2+ addition causes marginal (AtAPC2) to moderate (AtAPC1 and 3) increase in ATP transport when no extra Ca2+ is present (Figs. 1 and 2). With rising Ca2+ concentration the positive impact of Mg2+ becomes abolished and even reverted into a negative one (Fig. 2). More precisely, with higher Ca2+ concentrations (>10 μM AtAPC2; > 50 μM AtAPC1 and 3) the rates of ATP transport in absence of Mg2+ exceed the rates of the corresponding exchange in presence of Mg2+. Accordingly, in presence of Mg2+ higher concentrations of Ca2+ are apparently required to achieve ATP-transport saturation.Fig. 2

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.


Related in: MedlinePlus