Location: Children's Nutrition Research CenterTitle: Functional studies of split "Arabidopsis" Ca(2+)/H(+) exchangers) Author
Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/9/2009
Publication Date: 12/4/2009
Publication URL: http://www.jbc.org/content/284/49/34075.long
Citation: Zhao, J., Connorton, J.M., Guo, YQ., Li, X., Shigaki, T., Hirschi, K.D., Pittman, J.K. 2009. Functional studies of split "Arabidopsis" Ca(2+)/H(+) exchangers. Journal of Biological Chemistry. 284(49):34075-34083. Interpretive Summary: Here we are trying to understand how plant calcium transporters have evolved. We have used yeast genetic manipulations and carefully designed experiments in an attempt to "evolve" plant transporters with new properties. This strategy could facilitate the engineering of novel transporters, which could eventually be used to improve crop nutrient content and increase plant productivity in marginal environmental conditions.
Technical Abstract: 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 using 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 Ca (2+) 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 Ca(2+) transport, whereas co-expressing C-terminal halves of CAX variants with CAX1 conferred salt tolerance but no apparent Ca(2+) transport. These findings demonstrate plasticity through hetero-CAX complex formation as well as a novel means to engineer CAX transport.