Functional Studies of Split Arabidopsis Ca2+/H+ Exchangers

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

doi:10.1074/jbc.m109.070235

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 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 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.

Highlights

Ca2ϩ is both a signaling molecule and a component in providing the plant cell its structural strength [1,2,3]
Yeast co-expressing N-sCAX1 and C-terminal half of CAX3 (C-CAX3) could grow well on high Ca2ϩ medium. All other combinations, such as N-sCAX1ϩN-sCAX3, N-sCAX3ϩN-sCAX1, C-CAX3ϩCCAX1, N-sCAX3ϩC-CAX3, and N-sCAX3ϩC-CAX1 were unable to suppress the Ca2ϩ-sensitive phenotype, and expression of the N- or C-halves of the transporters alone were nonfunctional (Fig. 1A and data not shown). These observations suggested that N-sCAX1 could form an active Ca2ϩ transporter with either C-CAX1 or C-CAX3
Membrane vesicles from yeast expressing N-sCAX1ϩC-CAX3 showed significantly increased Ca2ϩ/Hϩ exchange activity compared with vector control (Fig. 1B) and sCAX3-expressing cells, but the activity was lower than from cells expressing sCAX1 and slightly lower than from cells expressing N-sCAX1ϩC-CAX3

Summary

Introduction

Ca2ϩ is both a signaling molecule and a component in providing the plant cell its structural strength [1,2,3]. CAX transporters can form homomeric complexes, as has been shown for Arabidopsis CAX1 and mung bean VCAX1 [9, 15] Using both plant and yeast assays, CAX1 and CAX3 combine to form functional transporters with novel transport properties [15]. Plant sucrose transporter members of the major facilitator superfamily possess two homologous, interacting domains [16] Both homoand heteromeric sucrose half proteins can functionally interact [16, 17]. CAX transporters appear to consist of two weakly homologous modules [18] that are likely to interact The analysis of these split CAX modules may allow us to further understand the mechanisms of protein-protein interaction between the CAX transporter isoforms. CAX proteins via the manipulation of split CAX halves will allow us to perform structure-function analyses and may afford a novel means to regulate CAX transporters

Methods

Results

Conclusion