In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects
In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects
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DOI：
10.1186/s12870-015-0616-0
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发表时间：
2015-10-06
影响因子：
5.3
通讯作者：
Haferkamp, Ilka
中科院分区：
文献类型：
--
作者：
Lorenz, Andre;Lorenz, Melanie;Haferkamp, Ilka
背景：哺乳动物和酵母中的腺嘌呤核苷酸/磷酸载体(APC)通常根据细胞需求调整线粒体腺嘌呤核苷酸池。它们催化腺嘌呤核苷酸--尤其是三磷酸腺苷-镁--和磷酸交换，它们的活性受钙的调节。我们目前对植物中相应蛋白质的了解相对有限。最近，来自拟南芥的三个可能的APC被证明恢复了APC酵母功能缺失突变体的特定生长表型，并在体外通过其N-末端EF-Hand基序与钙相互作用。结果：重组植物APC在功能上被重组为脂质体，并通过放射性底物转运测量确定了它们的生化特性。这三种植物APC都能进行ATP、ADP和磷酸盐交换，然而，如同源携带者所显示的那样，没有检测到对ATP-MG的高度偏好。相反，所获得的数据表明，在脂质体系统中，植物APC更倾向于以ATP-Ca为底物。此外，对一个具有代表性的突变体APC蛋白的研究表明，观察到的钙对ATP转运的影响主要/本质上不涉及钙与载体N-末端结构域的EF-Hand基序的结合。结论：生化特性表明，植物APC可以介导腺嘌呤核苷酸的净运输，因此，就像它们来自动物和酵母的悬垂物一样，可能参与线粒体腺嘌呤核苷酸池的改变。虽然ATP-Ca在体外被认为是植物APC的一个明显的输入底物，但在体内是否可以形成ATP-Ca从而进行相应的运输仍然是有争议的。
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 Ca2+-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.