Edmond Clare, Shigaki Toshiro, Ewert Sophie, Nelson Matthew D, Connorton James M, Chalova Vesela, Noordally Zeenat, Pittman Jon K
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.
Biochem J. 2009 Feb 15;418(1):145-54. doi: 10.1042/BJ20081814.
Internal compartmentalization of metals is an important metal tolerance mechanism in many organisms. In plants and fungi, sequestration into the vacuole is a major detoxification mechanism for metals. Cation transport into the vacuole can be mediated by CAX (cation exchanger) transporters. The Arabidopsis thaliana AtCAX2 transporter was shown previously to transport Ca(2+), Cd(2+) and Mn(2+). To assess the conservation of the functional and regulatory characteristics of CAX2-like transporters in higher plants, we have characterized AtCAX2 orthologues from Arabidopsis (AtCAX5), tomato (LeCAX2) and barley (HvCAX2). Substrate specificity and regulatory activity were assessed using a yeast heterologous-expression assay. Each CAX could transport Ca(2+) and Mn(2+) into the yeast vacuole, but they each had different cation transport kinetics. Most notably, there was variation in the regulation of the transporters. As found with AtCAX2 previously, only expression of an N-terminally truncated form of AtCAX5 in yeast was able to mediate Ca(2+) and Mn(2+) transport, indicating that activity may be controlled by an autoregulatory region at the N-terminus. In contrast, either full-length or truncated LeCAX2 could efficiently transport Ca(2+), although Mn(2+) transport was controlled by the N-terminus. HvCAX2 did not appear to possess an N-terminal regulatory domain. Expression of AtCAX2 was not significantly modulated by metal stress; however, AtCAX5 and HvCAX2 were transcriptionally up-regulated by high Mn(2+) treatment, and by Ca(2+) and Na(+) stress respectively. It is therefore apparent that, despite the high sequence identity between plant CAX2 orthologues, there is significant diversity in their functional characteristics, particularly with regard to regulatory mechanisms.
金属的细胞内区室化是许多生物体中一种重要的金属耐受机制。在植物和真菌中，金属螯合到液泡中是主要的解毒机制。阳离子转运到液泡中可由CAX（阳离子交换体）转运蛋白介导。先前已证明拟南芥AtCAX2转运蛋白可转运Ca(2+)、Cd(2+)和Mn(2+)。为了评估高等植物中CAX2样转运蛋白功能和调控特性的保守性，我们对来自拟南芥（AtCAX5）、番茄（LeCAX2）和大麦（HvCAX2）的AtCAX2直系同源物进行了表征。使用酵母异源表达试验评估底物特异性和调控活性。每个CAX都能将Ca(2+)和Mn(2+)转运到酵母液泡中，但它们各自具有不同的阳离子转运动力学。最值得注意的是，转运蛋白的调控存在差异。如先前在AtCAX2中发现的那样，只有酵母中AtCAX5的N端截短形式的表达能够介导Ca(2+)和Mn(2+)的转运，这表明活性可能由N端的一个自调控区域控制。相比之下，全长或截短的LeCAX2都能有效地转运Ca(2+)，尽管Mn(2+)的转运受N端控制。HvCAX2似乎不具有N端调控结构域。AtCAX2的表达不受金属胁迫的显著调节；然而，AtCAX5和HvCAX2分别在高浓度Mn(2+)处理以及Ca(2+)和Na(+)胁迫下转录上调。因此很明显，尽管植物CAX2直系同源物之间具有高度的序列同一性，但它们的功能特性存在显著差异，特别是在调控机制方面。
