title: AtHMA1 is a thapsigargin-sensitive Ca2+/heavy metal pump.
abstract: The Arabidopsis thaliana AtHMA1 protein is a member of the P(IB)-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 Ca(2+) 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 Ca(2+) 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 wild-type 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 Cd(2+) detoxification in yeast and suggests that this pump is able to transport other heavy metals ions. Further analyses were performed to typify the active Ca(2+) transport mediated by AtHMA1. Ca(2+) 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 (IC(50) = 16.74 nm), demonstrating the functionality of its SERCA-like stalk segment. In summary, these results demonstrate that AtHMA1 functions as a Ca(2+)/heavy metal pump. This protein is the first described plant P-type pump specifically inhibited by thapsigargin.
authors.affiliation: Laboratorio de Biología Molecular Vegetal, Centro de Estudios Avanzados en Zonas Aridas (CEAZA), La Serena 1720170, Chile.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Moreno
authors.forename: Lorena
authors.initials: L
authors.lastname: Norambuena
authors.forename: Daniel
authors.initials: D
authors.lastname: Maturana
authors.forename: Mauricio
authors.initials: M
authors.lastname: Toro
authors.forename: Cecilia
authors.initials: C
authors.lastname: Vergara
authors.forename: Ariel
authors.initials: A
authors.lastname: Orellana
authors.forename: Andrés
authors.initials: A
authors.lastname: Zurita-Silva
authors.forename: Viviana R
authors.initials: VR
authors.lastname: Ordenes
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005816:Genetic Complementation Test; D006706:Homeostasis / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D019216:Metals, Heavy / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D019284:Thapsigargin / Q000494:pharmacology*
chemical_list: D029681:Arabidopsis Proteins; D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D019216:Metals, Heavy; D019284:Thapsigargin; D000251:Adenosine Triphosphatases; C509978:HMA1 protein, Arabidopsis; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium