|Manohar, Murli -|
|Shigaki, Toshiro -|
|Hirschi, Kendal -|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 28, 2012
Publication Date: May 16, 2012
Citation: Manohar, M., Shigaki, T., Hirschi, K.D. 2012. Identification of microdomains involved in association of "Arabidopsis" Ca(2+)/H(+) exchangers [abstract]. Proceedings of the 4th Pan American Plant Membrane Biology Workshop, May 16-20, 2012, Asilomar, California. p. 22. Technical Abstract: In planta, high capacity tonoplast Ca2+/H+ antiport is mediated in part by a family of CAtion Exchangers (CAX). Each CAX can be divided into two weakly homologous halves (N- and C-) at the negatively charged loop between transmembrane (TM) 6 and TM7. Some CAX halves (N+C) co-expressed in yeast cells can function in Ca2+/H+ antiport, and function to suppress the calcium sensitivity of yeast cells defective in vacuolar Ca2+ sequestration. Our recent data suggest that the association of CAX halves exhibit altered substrate transport properties including enhanced sodium (Na+) transport. Here we are examining the dynamics of this association. We have identified five putative helix-helix interaction motifs (typically GxxxG) in CAX1 that may mediate the interaction among the half proteins. Some of these motifs appear to disrupt association between CAX half proteins without altering transport of the intact CAX transporter. Furthermore, we have also performed "domain swapping" of TMs of CAX1 with similar TMs from the closely related CAX transporters CAX2 to determine if these domains alter association among the CAXs. Although CAX2 has similar transport properties to CAX1, the N and C- halves of CAX2 do not interact with CAX1. A variant generated by replacing TM7 of CAX1 with TM7 of CAX2 was sufficient to abolish the interaction among the CAX1 halves but did not alter Ca2+/H+ antiport of the intact CAX1 variant. Our preliminary data suggest that we can engineer the interaction between split CAX in order to dissect the mechanisms of functional association between CAX halves. These finding could help in the engineering of novel CAX transporters. Furthermore, to gain insight into the role of these glycine residues in CAX structure, we are beginning to obtain structural data by X-ray crystallography.