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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.


Determination of Ca2+ transport via AtAPC2. Time dependent uptake of [45Ca] via full-length AtAPC2 (a) and via N-terminally truncated AtAPC2 (b) reconstituted into Pi (black rhombs) and non-loaded liposomes (gray squares). (c) Effects of rising MgCl2 concentrations on [45Ca] transport into Pi loaded (dark gray bars) and non-loaded (light gray bars) AtAPC2 proteoliposomes. Transport media contained 20 μM [45Ca] and were additionally supplemented with 100 μM non-labeled ATP and the indicated concentrations of MgCl2. For determination of the Mg2+-effects on Ca2+ transport via AtAPC2 uptake was allowed for 10 min (given as nmol mg protein−1 h−1). Data represent mean values of three independent replicates. Standard errors are indicated
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Fig6: Determination of Ca2+ transport via AtAPC2. Time dependent uptake of [45Ca] via full-length AtAPC2 (a) and via N-terminally truncated AtAPC2 (b) reconstituted into Pi (black rhombs) and non-loaded liposomes (gray squares). (c) Effects of rising MgCl2 concentrations on [45Ca] transport into Pi loaded (dark gray bars) and non-loaded (light gray bars) AtAPC2 proteoliposomes. Transport media contained 20 μM [45Ca] and were additionally supplemented with 100 μM non-labeled ATP and the indicated concentrations of MgCl2. For determination of the Mg2+-effects on Ca2+ transport via AtAPC2 uptake was allowed for 10 min (given as nmol mg protein−1 h−1). Data represent mean values of three independent replicates. Standard errors are indicated

Mentions: Ca2+ uptake into Pi loaded vesicles (Fig. 6a and b, black rhombs) always exceeded the corresponding rates obtained with non-loaded proteoliposomes (Fig. 6a and b, gray squares) indicating that Ca2+ accumulation is directly connected to the antiport activity of the carrier. The full-length protein exhibits higher Ca2+ transport rates and also the back-ground values of the non-loaded vesicles are enhanced when compared to the truncated version (compare Fig. 6a and b). So far it cannot be discriminated whether - albeit EGTA treatment - a certain amount of Ca2+ still binds to the N-terminal domain of recombinant AtAPC2 or/and the functionality of the truncated protein is generally slightly impaired.Fig. 6


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)

Determination of Ca2+ transport via AtAPC2. Time dependent uptake of [45Ca] via full-length AtAPC2 (a) and via N-terminally truncated AtAPC2 (b) reconstituted into Pi (black rhombs) and non-loaded liposomes (gray squares). (c) Effects of rising MgCl2 concentrations on [45Ca] transport into Pi loaded (dark gray bars) and non-loaded (light gray bars) AtAPC2 proteoliposomes. Transport media contained 20 μM [45Ca] and were additionally supplemented with 100 μM non-labeled ATP and the indicated concentrations of MgCl2. For determination of the Mg2+-effects on Ca2+ transport via AtAPC2 uptake was allowed for 10 min (given as nmol mg protein−1 h−1). Data represent mean values of three independent replicates. Standard errors are indicated
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Determination of Ca2+ transport via AtAPC2. Time dependent uptake of [45Ca] via full-length AtAPC2 (a) and via N-terminally truncated AtAPC2 (b) reconstituted into Pi (black rhombs) and non-loaded liposomes (gray squares). (c) Effects of rising MgCl2 concentrations on [45Ca] transport into Pi loaded (dark gray bars) and non-loaded (light gray bars) AtAPC2 proteoliposomes. Transport media contained 20 μM [45Ca] and were additionally supplemented with 100 μM non-labeled ATP and the indicated concentrations of MgCl2. For determination of the Mg2+-effects on Ca2+ transport via AtAPC2 uptake was allowed for 10 min (given as nmol mg protein−1 h−1). Data represent mean values of three independent replicates. Standard errors are indicated
Mentions: Ca2+ uptake into Pi loaded vesicles (Fig. 6a and b, black rhombs) always exceeded the corresponding rates obtained with non-loaded proteoliposomes (Fig. 6a and b, gray squares) indicating that Ca2+ accumulation is directly connected to the antiport activity of the carrier. The full-length protein exhibits higher Ca2+ transport rates and also the back-ground values of the non-loaded vesicles are enhanced when compared to the truncated version (compare Fig. 6a and b). So far it cannot be discriminated whether - albeit EGTA treatment - a certain amount of Ca2+ still binds to the N-terminal domain of recombinant AtAPC2 or/and the functionality of the truncated protein is generally slightly impaired.Fig. 6

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.