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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

Impact of rising EGTA concentrations on adenine nucleotide transport of full-length and N-terminally truncated AtAPC2. Transport via recombinant AtAPC2 (a) and via the mutated version lacking its N-terminal domain (b). Import of [α32P]-ATP into Pi loaded proteoliposomes (black squares) and of [α32P]-ADP into ATP loaded vesicles (gray circles) was allowed for 5 min. Transport without EGTA was set to 100 % and transport in presence of rising concentrations of externally added EGTA (0 - 500 μM) was calculated accordingly. Data represent net values of ATP/Pi and ADP/ATP import minus the respective control (non-loaded vesicles) of three independent replicates. Standard errors are given
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Fig4: Impact of rising EGTA concentrations on adenine nucleotide transport of full-length and N-terminally truncated AtAPC2. Transport via recombinant AtAPC2 (a) and via the mutated version lacking its N-terminal domain (b). Import of [α32P]-ATP into Pi loaded proteoliposomes (black squares) and of [α32P]-ADP into ATP loaded vesicles (gray circles) was allowed for 5 min. Transport without EGTA was set to 100 % and transport in presence of rising concentrations of externally added EGTA (0 - 500 μM) was calculated accordingly. Data represent net values of ATP/Pi and ADP/ATP import minus the respective control (non-loaded vesicles) of three independent replicates. Standard errors are given

Mentions: The cation chelator EGTA efficiently chelates Ca2+ (with significant higher affinity than to Mg2+) and accordingly should remove residual Ca2+ from the medium. We thus used addition of EGTA to the transport medium to investigate whether and how Ca2+ depletion affects carrier activities. ATP/Pi exchange of full-length AtAPC2 becomes significantly reduced by addition of 10 μM EGTA and further increase of its concentration causes total inhibition (Fig. 4a, black squares). Interestingly, a similar inhibitory effect was also observed for the truncated carrier version (Fig. 4b, black squares). Given that the N-terminal domain forms a lid that virtually closes the translocation pathway when free Ca2+ is missing, efficient Ca2+-removal should impede transport activity of AtAPC2 but not of the “un-capped” mutant. Moreover, ADP/ATP exchange of both, full-length and truncated, AtAPC2 variants remained more or less unaltered by EGTA addition (Fig. 4a and b, gray circles). Accordingly, Ca2+ removal from the medium did not cause inhibition of the overall transport capacity by deactivation of the reconstituted carrier.Fig. 4


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)

Impact of rising EGTA concentrations on adenine nucleotide transport of full-length and N-terminally truncated AtAPC2. Transport via recombinant AtAPC2 (a) and via the mutated version lacking its N-terminal domain (b). Import of [α32P]-ATP into Pi loaded proteoliposomes (black squares) and of [α32P]-ADP into ATP loaded vesicles (gray circles) was allowed for 5 min. Transport without EGTA was set to 100 % and transport in presence of rising concentrations of externally added EGTA (0 - 500 μM) was calculated accordingly. Data represent net values of ATP/Pi and ADP/ATP import minus the respective control (non-loaded vesicles) 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

Fig4: Impact of rising EGTA concentrations on adenine nucleotide transport of full-length and N-terminally truncated AtAPC2. Transport via recombinant AtAPC2 (a) and via the mutated version lacking its N-terminal domain (b). Import of [α32P]-ATP into Pi loaded proteoliposomes (black squares) and of [α32P]-ADP into ATP loaded vesicles (gray circles) was allowed for 5 min. Transport without EGTA was set to 100 % and transport in presence of rising concentrations of externally added EGTA (0 - 500 μM) was calculated accordingly. Data represent net values of ATP/Pi and ADP/ATP import minus the respective control (non-loaded vesicles) of three independent replicates. Standard errors are given
Mentions: The cation chelator EGTA efficiently chelates Ca2+ (with significant higher affinity than to Mg2+) and accordingly should remove residual Ca2+ from the medium. We thus used addition of EGTA to the transport medium to investigate whether and how Ca2+ depletion affects carrier activities. ATP/Pi exchange of full-length AtAPC2 becomes significantly reduced by addition of 10 μM EGTA and further increase of its concentration causes total inhibition (Fig. 4a, black squares). Interestingly, a similar inhibitory effect was also observed for the truncated carrier version (Fig. 4b, black squares). Given that the N-terminal domain forms a lid that virtually closes the translocation pathway when free Ca2+ is missing, efficient Ca2+-removal should impede transport activity of AtAPC2 but not of the “un-capped” mutant. Moreover, ADP/ATP exchange of both, full-length and truncated, AtAPC2 variants remained more or less unaltered by EGTA addition (Fig. 4a and b, gray circles). Accordingly, Ca2+ removal from the medium did not cause inhibition of the overall transport capacity by deactivation of the reconstituted carrier.Fig. 4

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