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J. Biol. Chem., Vol. 283, Issue 15, 9633-9641, April 11, 2008

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AtHMA1 Is a Thapsigargin-sensitive Ca²⁺/Heavy Metal Pump^*

Ignacio Moreno, Lorena Norambuena, Daniel Maturana, Mauricio Toro^¶, Cecilia Vergara, Ariel Orellana^||, Andrés Zurita-Silva^¶, and Viviana R. Ordenes^¶1

From the ^¶Laboratorio de Biología Molecular Vegetal, Centro de Estudios Avanzados en ZonasÁridas (CEAZA), La Serena 1720170, Chile, Laboratorio de Fisiología Celular, Facultad de Ciencias, Universidad de Chile, Santiago 7800024, Chile, Laboratorio de Biología Molecular Vegetal, Facultad de Ciencias, Universidad de Chile, Santiago 1720170, Chile, and ^||Millenium Nucleus in Plant Cell Biotechnology, Centro de Biotecnología Vegetal, Universidad Andrés Bello, Santiago 8370146, Chile

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 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 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 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.

Received for publication, January 28, 2008

^* This work was supported mainly by Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) Grant 3040057 and in part by FONDECYT Grant 1040681 and by Grant PCB P06-065-F from Millennium Nucleus in Plant Cell Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Laboratorio de Biología Molecular Vegetal CEAZA, Universidad de La Serena, Campus Andres Bello, Casilla 599, La Serena 1720170, Chile. Tel.: 56-51-334855; Fax: 56-51-334741; E-mail: viviana.ordenes{at}ceaza.cl.

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