Limits...
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 and ADP 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 CaCl2 was set to 100 % and transport in presence of rising concentrations of externally added CaCl2 (0 - 500 μM) was calculated accordingly. Data represent net values of ATP/Pi and ADP/ATP uptake 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
getmorefigures.php?uid=PMC4595200&req=5

Fig3: Ca2+-impact on ATP and ADP 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 CaCl2 was set to 100 % and transport in presence of rising concentrations of externally added CaCl2 (0 - 500 μM) was calculated accordingly. Data represent net values of ATP/Pi and ADP/ATP uptake minus the respective control (non-loaded vesicles) of three independent replicates. Standard errors are given

Mentions: Determination of Ca2+ impact on transport activity showed that ATP/Pi exchange via the mutated carrier was considerably stimulated by increasing Ca2+ concentrations (~3-fold). Moreover, the degree of Ca2+-dependent stimulation and the general course of the corresponding transport basically resembled that of the full-length protein (Fig. 3, black squares). Investigation of ADP uptake into ATP loaded liposomes revealed slight transport stimulation of the full-length protein by low Ca2+ concentrations (~35 % at 50 to 100 μM Ca2+), which approached saturation at higher concentrations (+60 %) (Fig. 3a, gray circles), whereas the corresponding transport of the truncated carrier version remained rather unaffected by moderate Ca2+ concentrations (+/− 10 % until 200 μM Ca2+) and became stimulated only at higher Ca2+ concentrations (+50 %) (Fig. 3b, gray circles).Fig. 3


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 and ADP 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 CaCl2 was set to 100 % and transport in presence of rising concentrations of externally added CaCl2 (0 - 500 μM) was calculated accordingly. Data represent net values of ATP/Pi and ADP/ATP uptake 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

Fig3: Ca2+-impact on ATP and ADP 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 CaCl2 was set to 100 % and transport in presence of rising concentrations of externally added CaCl2 (0 - 500 μM) was calculated accordingly. Data represent net values of ATP/Pi and ADP/ATP uptake minus the respective control (non-loaded vesicles) of three independent replicates. Standard errors are given
Mentions: Determination of Ca2+ impact on transport activity showed that ATP/Pi exchange via the mutated carrier was considerably stimulated by increasing Ca2+ concentrations (~3-fold). Moreover, the degree of Ca2+-dependent stimulation and the general course of the corresponding transport basically resembled that of the full-length protein (Fig. 3, black squares). Investigation of ADP uptake into ATP loaded liposomes revealed slight transport stimulation of the full-length protein by low Ca2+ concentrations (~35 % at 50 to 100 μM Ca2+), which approached saturation at higher concentrations (+60 %) (Fig. 3a, gray circles), whereas the corresponding transport of the truncated carrier version remained rather unaffected by moderate Ca2+ concentrations (+/− 10 % until 200 μM Ca2+) and became stimulated only at higher Ca2+ concentrations (+50 %) (Fig. 3b, gray circles).Fig. 3

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