Functional studies of split Arabidopsis Ca2+/H+ exchangers

Jian Zhao, James M. Connorton, Ying Qing Guo, Xiangkai Li, Toshiro Shigaki, Kendal D. Hirschi, Jon K. Pittman

    Research output: Contribution to journalArticlepeer-review


    In plants, high capacity tonoplast cation/H+ antiport is mediated in part by a family of cation exchanger (CAX) transporters. Functional association between CAX1 and CAX3 has previously been shown. In this study we further examine the interactions between CAX protein domains through the use of nonfunctional halves of CAX transporters. We demonstrate that a protein coding for an N-terminal half of an activated variant of CAX1 (sCAX1) can associate with the C-terminal half of either CAX1 or CAX3 to form a functional transporter that may exhibit unique transport properties. Using yeast split ubiquitin, in planta bimolecular fluorescence complementation, and gel shift experiments, we demonstrate a physical interaction among the half proteins. Moreover, the half-proteins both independently localized to the same yeast endomembrane. Co-expressing variants of N- and C-terminal halves of CAX1 and CAX3 in yeast suggested that the N-terminal region mediates Ca2+ transport, whereas the C-terminal half defines salt tolerance phenotypes. Furthermore, in yeast assays, auto-inhibited CAX1 could be differentially activated by CAX split proteins. The N-terminal half of CAX1 when co-expressed with CAX1 activated Ca2+ transport, whereas co-expressing C-terminal halves of CAX variants with CAX1 conferred salt tolerance but no apparent Ca2+ transport. These findings demonstrate plasticity through hetero-CAX complex formation as well as a novel means to engineer CAX transport.
    Original languageEnglish
    Pages (from-to)34075-34083
    Number of pages8
    JournalJournal of Biological Chemistry
    Issue number49
    Publication statusPublished - 4 Dec 2009


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