Identification of a crucial histidine involved in metal transport activity in the Arabidopsis cation/H+ exchanger CAX1

In plants, yeast, and bacteria, cation/H⁺ exchangers (CAXs) have been shown to translocate Ca²⁺ and other metal ions utilizing the H⁺ gradient. The best characterized of these related transporters is the plant vacuolar localized CAX1. We have used site-directed mutagenesis to assess the impact of altering the seven histidine residues to alanine within Arabidopsis CAX1. The mutants were expressed in a Saccharomyces cerevisiae strain that is sensitive to Ca²⁺ and other metals. By utilizing a yeast growth assay, the H338A mutant was the only mutation that appeared to alter Ca²⁺ transport activity. The CAX1 His³³⁸ residue is conserved among various CAX transporters and may be located within a filter for cation selection. We proceeded to mutate His³³⁸ to every other amino acid residue and utilized yeast growth assays to estimate the transport properties of the 19 CAX mutants. Expression of 16 of these His³³⁸ mutants could not rescue any of the metal sensitivities. However, expression of H338N, H338Q, and H338K allowed for some growth on media containing Ca²⁺. Most interestingly, H338N exhibited increased tolerance to Cd²⁺ and Zn²⁺. Endomembrane fractions from yeast cells were used to measure directly the transport of H338N. Although the H338N mutant demonstrated 25% of the wild type Ca²⁺/H⁺ transport, it showed an increase in transport for both Cd²⁺ and Zn²⁺ 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.