Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells

Abstract

The Ca 21 -transport activity and intracellular localization of the translation product of cDNA for mung bean Ca 21 /H 1 antiporter (VCAX1) were examined. When the cDNA was expressed in Saccharomyces cerevisiae that lacked its own genes for vacuolar Ca 21 -ATPase and the antiporter, VCAX1 complemented the active Ca 21 transporters, and the microsomal membranes from the transformant showed high activity of the Ca 21 /H 1 antiporter. Treatment of the vacuolar membranes with a cross-linking reagent resulted in a clear band of the dimer detected with antibody specific for VCAX1p. The antibody was also used for immunolocalization of the antiporter in fractions obtained by sucrose-density-gradient centrifugation of the microsomal fraction from mung bean. The immunostained band was detected in the vacuolar membrane fraction and the slightly heavy fractions that exhibited activity of the Golgi marker enzyme. A fusion protein of VCAX1p and green fluorescent protein was expressed in tobacco cells. The green fluorescence was clearly observed on the vacuolar membrane and, in some cases, in the small vesicles. [10] revealed that the antiporter is a highly hydrophobic protein with an acidic motif in the centre. The deduced sequences have relatively high identity between the two plant species, although another type of Ca 21 /H 1 antiporter (CAX2p) has been cloned for A. thaliana in addition to the high-capacity antiporter (CAX1p) [9]. The Ca 21 /H 1 antiporter has been detected not only in the vacuolar membrane but also in the plasma [11,12] and plastid [13] membranes. At present, the intracellular distribution of each molecular species is not clear. Without knowing the exact intracellular localization of the antiporter, we cannot understand its cellular function and the contribution of vacuoles to homeostasis in plant cells. The physiological significance of the presence of two different antiporters in plant cells also needs to be clarified.In this study we focused our attention on the function of the translation product of cDNA for the antiporter from mung bean, VCAX1 [10], and its localization in plant cells. By heterologous expression in S. cerevisiae, we directly confirmed that the cDNA (VCAX1) encodes a functional Ca 21 /H 1 antiporter. A specific high-affinity antibody could be obtained by immunization of the recombinant protein purified from VCAX1-transformed S. cerevisiae. Using this antibody, we determined the intracellular location of VCAX1p by two different systems: immunological detection in organelles fractionated from mung bean hypocotyls; fluorescent microscopic observation of the fusion protein of VCAX1p with a green fluorescent protein (GFP) expressed in tobacco cells. In addition to the Ca 21 -transport activity and intracellular localization of VCAX1p, the multimeric structure is also discussed.

Residues in Internal Repeats of the Rice Cation/H+ Exchanger Are Involved in the Transport and Selection of Cations

“…2). Although the detailed amino acid sequence is not conserved among CAXs, this acidic region is thought to be involved in the capture and selection of metal ions (7,18).…”

Deletion of a Histidine-rich Loop of AtMTP1, a Vacuolar Zn2+/H+ Antiporter of Arabidopsis thaliana, Stimulates the Transport Activity

“…Zn 2ϩ Transport Assay-Zn 2ϩ uptake into vacuolar membrane vesicles was measured using the filtration method (27). Assays were performed at 25°C in aliquots of medium containing of 300 mM sorbitol, 5 mM MES-Tris, pH 6.9, 25 mM KCl, 1 mM dithiothreitol, 5 mM MgCl 2 , 200 M NaN 3 , 100 M vanadate, 3 mM ATP-Tris, and 5 M 65 ZnCl 2 .…”

Cloning and Characterization of CXIP1, a Novel PICOT Domain-containing Arabidopsis Protein That Associates with CAX1

“…Initially, plant H ϩ /Ca 2ϩ antiporter genes were cloned by their ability to suppress the Ca 2ϩ -hypersensitive phenotype of a Saccharomyces cerevisiae mutant (4,6). These genes are termed CAX for cation exchangers, and CAX1 from Arabidopsis thaliana is a high capacity Ca 2ϩ transporter.…”