Analysis of the Ca2+ domain in the Arabidopsis H+/Ca2+ antiporters CAX1 and CAX3

• Published in: Plant molecular biology 2002-10, Vol.50 (3), p.475
• Main Authors: Shigaki, Toshiro, Sreevidya, Coimbatore, Hirschi, Kendal D
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Amino acids; Antiporters - genetics; Antiporters - metabolism; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Binding Sites - genetics; Biological Transport; Calcium - metabolism; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Cation Transport Proteins; Gene Expression Regulation, Plant; Molecular Sequence Data; Mutation; Nicotiana - genetics; Nicotiana - metabolism; Plants, Genetically Modified; Protein Isoforms - genetics; Protein Isoforms - metabolism; Proteins; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Sequence Homology, Amino Acid; Vacuoles - metabolism; Yeast; Yeasts
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1023/A:1019880006606

Ca2+ levels in plants are controlled in part by H+/Ca2+ exchangers. Structure/function analysis of the Arabidopsis H+/cation exchanger, CAX1, revealed that a nine amino acid region (87-95) is involved in CAX1-mediated Ca2+ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca2+ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca2+ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca2+ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca2+ transport capability compared to CAX3-I. The entire Cad of CAX3 (87-95) inserted into CAX1 abolishes CAX1-mediated Ca2+ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca2+ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca2+ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues.