Abstract
In plants, high capacity tonoplast cation/H(+) antiport is mediated in part by a family of CAX (cation exchanger) transporters. Functional association between CAX1 and CAX3 has previously been inferred; however, the nature of this interaction has not been established. Here we analyze the formation of "hetero-CAX" complexes and their transport properties. Co-expressing both CAX1 and CAX3 mediated lithium and salt tolerance in yeast, and these phenotypes could not be recapitulated by expression of deregulated versions of either transporter. Coincident expression of Arabidopsis CAX1 and CAX3 occurs during particular stress responses, flowering, and seedling growth. Analysis of cax1, cax3, and cax1/3 seedlings demonstrated similar stress sensitivities. When plants expressed high levels of both CAXs, alterations in transport properties were evident that could not be recapitulated by high level expression of either transporter individually. In planta coimmunoprecipitation suggested that a protein-protein interaction occurred between CAX1 and CAX3. In vivo interaction between the CAX proteins was shown using a split ubiquitin yeast two-hybrid system and gel shift assays. These findings demonstrate cation exchange plasticity through hetero-CAX interactions.
Highlights
Calcium (Ca2ϩ) is a cofactor for many enzymes, a vital signaling molecule and a structural component in providing the plant cell its strength [1, 2]
CAX3, which is most similar to CAX1 (77% identical at the amino acid level), is at best a weak vacuolar Ca2ϩ transporter when expressed in yeast cells [10, 11]
Microsomal proteins were incubated with 20 l of monoclonal mouse IgG against HA (Covance, Berkeley, CA) or 50 l of CAX1 peptide antibody raised in rabbit against the N terminus of CAX1 [12], or 50 l of V-ATPase B subunit antibody [24], 20 l of vacuolar TPC1 antibody [25], TIP1;2 [26], or AVP1 (Arabidopsis vacuolar Hϩ-pyrophosphatase antibody) [27] raised against rabbit IgG in CIP buffer (50 mM Tris-HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 mM NaCl) with protein inhibitor mixture (Sigma)
Summary
The cax, cax, cax, and cax1/cax (cax1/3) lines are in the Arabidopsis ecotype Col-0 [11, 12]. For seed germination test, seed lots for Col-0, cax, cax, cax, and cax1/cax seeds were treated as described previously [17] and sown on one-half strength MS medium [18] agar plates (1.5% sucrose, 0.8% agar, pH 5.6) with or without CaCl2 (50 and 100 mM) or LiCl (0, 6, 10, and 15 mM). Histochemical assays for CAX::GUS activity were performed according to the protocol described previously [11]. These CAX::GUS seeds were germinated on one-half strength MS medium [18] and monitored daily. (MATa cnb1::LEU1 pmc1::TRP1 vcx1⌬ ade can100 his311 leu112 trp ura3-1) was used for most yeast assays [19]. CAX1, CAX3, sCAX (where the first 36 amino acids of CAX1 and CAX3 have been removed), and sCAX1H338N have been described previously [21, 22]
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