Submitted to: American Society of Plant Biologists Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2005
Publication Date: 7/16/2005
Citation: Shigaki, T., Barkla, B.J., Miranda-Vergara, M.C., Zhao, J., Pantoja, O., Hirschi, K.D. 2005. Identification of a crucial histidine involved in metal transport activity in the Arabidopsis cation/H(+) exchanger CAX1 [abstract]. The 2005 Annual Meeting of the American Society of Plant Biologists Meeting, July 16-20, 2005, Seattle, Washington. Abstract #89, p. 113. Interpretive Summary:
Technical Abstract: In plants, yeast and bacteria, cation/H(+) exchangers (CAXs), have been shown to translocate Ca(2+) and other metals. The best characterized of these related transporters is the plant vacuolar-localized CAX1. We used site-directed mutagenesis to assess the impact of altering the seven histidine residues to alanine within A"t"CAX1. The mutants were expressed in a "Saccharomyces cerevisiae" strain that is sensitive to Ca(2+) and other metals. Utilizing a yeast growth assay, the H338A mutant was the only mutation that appeared to alter Ca(2+) transport. The CAX1 His(338) residue is conserved among various CAX transporters and may be located within a filter for cation selection. We proceeded to mutate His(338) to every other amino acid residue and estimate the transport properties of the 19 CAX mutants. Expression of 16 of these mutants could not rescue any of the metal sensitivities. However, expression of H338N, H338Q, and H338K allowed for some growth on media containing Ca(2+). Interestingly, H338N exhibited increased tolerance to Cd(2+) and Zn(2+). Vacuolar membrane fractions from yeast cells were used to directly measure the transport properties of the H338N mutant. While the H338N mutant demonstrated 25% of the wild-type Ca(2+)/H(+) transport, it showed an increase in transport for both Cd(2+) and Zn(2+), reflected in a decrease in the K[m] for these substrates. This study provides insights into the CAX cation filter and novel mechanisms by which metals may be partitioned across membranes.