AtHMA1 is a thapsigargin-sensitive Ca²⁺/heavy metal pump

The Arabidopsis thaliana AtHMA1 protein is a member of the PIB-ATPase family, which is implicated in heavy metal transport. However, sequence analysis reveals that AtHMA1 possesses a predicted stalk segment present in SERCA (sarcoplasmic/ endoplasmic reticulum Ca²⁺ ATPase)-type pumps that is involved in inhibition by thapsigargin. To analyze the ion specificity of AtHMA1, we performed functional complementation assays using mutant yeast strains defective in Ca²⁺ homeostasis or heavy metal transport. The heterologous expression of AtHMA1 complemented the phenotype of both types of mutants and, interestingly, increased heavy metal tolerance of wildtype yeast. Biochemical analyses were performed to describe the activity of AtHMA1 in microsomal fractions isolated from complemented yeast. Zinc, copper, cadmium, and cobalt activate the ATPase activity of AtHMA1, which corroborates the results of metal tolerance assays. The outcome establishes the role of AtHMA1 in Cd²⁺ detoxification in yeast and suggests that this pump is able to transport other heavy metals ions. Further analyses were performed to typify the active Ca²⁺ transport mediated by AtHMA1. Ca²⁺ transport displayed high affinity with an apparent K_m of 370 nM and a V _max of 1.53 nmol mg^-1 min^-1. This activity was strongly inhibited by thapsigargin (IC₅₀ = 16.74 nM), demonstrating the functionality of its SERCA-like stalk segment. In summary, these results demonstrate that AtHMA1 functions as a Ca²⁺/heavy metal pump. This protein is the first described plant P-type pump specifically inhibited by thapsigargin.