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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

Time dependent ATP transport via AtAPC1-3. Transport of 50 μM [α32P]-ATP into Pi (a, c, e) and into ATP (b, d, f) loaded proteoliposomes with reconstituted AtAPC1 (a, b), AtAPC2 (c, d) and AtAPC3 (e, f). ATP uptake was measured in absence (black rhombs) and presence (gray circles) of 500 μM externally applied MgCl2. Non-loaded liposomes (non-filled rhombs; negative control) showed only marginal accumulation of radioactivity and the corresponding rates were unaffected by MgCl2 addition. Data represent mean values of at least three independent replicates, standard errors are given
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Fig1: Time dependent ATP transport via AtAPC1-3. Transport of 50 μM [α32P]-ATP into Pi (a, c, e) and into ATP (b, d, f) loaded proteoliposomes with reconstituted AtAPC1 (a, b), AtAPC2 (c, d) and AtAPC3 (e, f). ATP uptake was measured in absence (black rhombs) and presence (gray circles) of 500 μM externally applied MgCl2. Non-loaded liposomes (non-filled rhombs; negative control) showed only marginal accumulation of radioactivity and the corresponding rates were unaffected by MgCl2 addition. Data represent mean values of at least three independent replicates, standard errors are given

Mentions: Import measurements were performed on proteoliposomes either harboring or lacking selected possible counter exchange substrates in the lumen (Fig. 1, Additional file 2: Figure S2). This allowed investigation of in vitro transport activities and hence functionality of the reconstituted proteins as well as of the catalyzed transport mode. All recombinant plant APCs mediated time dependent uptake of [α32P]-ATP into phosphate (Pi) loaded liposomes (Fig. 1a, c, e, black rhombs) and no comparable accumulation of radioactivity was observable with corresponding vesicles lacking Pi in the lumen (Fig. 1a, c, e, open rhombs). This observation already demonstrates that plant APCs can act as antiporters; ATP/Pi exchange by the different APC isoforms was linear for at least 5 min. Maximal uptake via AtAPC1 of ~ 6 nmol/mg protein was reached after 10 to 15 min (Fig. 1a, black rhombs), whereas AtAPC2 and AtAPC3 show marginally or considerably higher transport rates that approached a maximum of ~ 9 nmol/mg protein and ≥ 17 nmol/mg protein after 20 min, respectively (Fig. 1c and e, black rhombs).Fig. 1


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)

Time dependent ATP transport via AtAPC1-3. Transport of 50 μM [α32P]-ATP into Pi (a, c, e) and into ATP (b, d, f) loaded proteoliposomes with reconstituted AtAPC1 (a, b), AtAPC2 (c, d) and AtAPC3 (e, f). ATP uptake was measured in absence (black rhombs) and presence (gray circles) of 500 μM externally applied MgCl2. Non-loaded liposomes (non-filled rhombs; negative control) showed only marginal accumulation of radioactivity and the corresponding rates were unaffected by MgCl2 addition. Data represent mean values of at least 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

Fig1: Time dependent ATP transport via AtAPC1-3. Transport of 50 μM [α32P]-ATP into Pi (a, c, e) and into ATP (b, d, f) loaded proteoliposomes with reconstituted AtAPC1 (a, b), AtAPC2 (c, d) and AtAPC3 (e, f). ATP uptake was measured in absence (black rhombs) and presence (gray circles) of 500 μM externally applied MgCl2. Non-loaded liposomes (non-filled rhombs; negative control) showed only marginal accumulation of radioactivity and the corresponding rates were unaffected by MgCl2 addition. Data represent mean values of at least three independent replicates, standard errors are given
Mentions: Import measurements were performed on proteoliposomes either harboring or lacking selected possible counter exchange substrates in the lumen (Fig. 1, Additional file 2: Figure S2). This allowed investigation of in vitro transport activities and hence functionality of the reconstituted proteins as well as of the catalyzed transport mode. All recombinant plant APCs mediated time dependent uptake of [α32P]-ATP into phosphate (Pi) loaded liposomes (Fig. 1a, c, e, black rhombs) and no comparable accumulation of radioactivity was observable with corresponding vesicles lacking Pi in the lumen (Fig. 1a, c, e, open rhombs). This observation already demonstrates that plant APCs can act as antiporters; ATP/Pi exchange by the different APC isoforms was linear for at least 5 min. Maximal uptake via AtAPC1 of ~ 6 nmol/mg protein was reached after 10 to 15 min (Fig. 1a, black rhombs), whereas AtAPC2 and AtAPC3 show marginally or considerably higher transport rates that approached a maximum of ~ 9 nmol/mg protein and ≥ 17 nmol/mg protein after 20 min, respectively (Fig. 1c and e, black rhombs).Fig. 1

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