Dissecting the role of the N-terminal metal-binding domains in activating the yeast copper ATPase in vivo

Authors

  • Isabelle Morin,

    1.  CEA, DSV, iRTSV, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France
    2.  CNRS, UMR 5249, Grenoble, France
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  • Simon Gudin,

    1.  CEA, DSV, iRTSV, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France
    2.  CNRS, UMR 5249, Grenoble, France
    3.  Université Joseph Fourier, Grenoble, France
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  • Elisabeth Mintz,

    1.  CEA, DSV, iRTSV, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France
    2.  CNRS, UMR 5249, Grenoble, France
    3.  Université Joseph Fourier, Grenoble, France
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  • Martine Cuillel

    1.  CEA, DSV, iRTSV, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France
    2.  CNRS, UMR 5249, Grenoble, France
    3.  Université Joseph Fourier, Grenoble, France
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  • Present address
    Comparative Genomics Centre, James Cook University, Townsville, Australia

M. Cuillel, CEA/iRTSV/LCBM, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France
Fax: +334 38 78 54 87
Tel: +334 38 78 96 51
E-mail: martine.cuillel@cea.fr

Abstract

In yeast, copper delivery to the trans-Golgi network involves interactions between the metallo-chaperone Atx1 and the N-terminus of Ccc2, the P-type ATPase responsible for copper transport across trans-Golgi network membranes. Disruption of the Atx1–Ccc2 route leads to cell growth arrest in a copper-and-iron-limited medium, a phenotype allowing complementation studies. Coexpression of Atx1 and Ccc2 mutants in an atx1Δccc2Δ strain allowed us to study in vivo Atx1–Ccc2 and intra-Ccc2 domain–domain interactions, leading to active copper transfer into the trans-Golgi network. The Ccc2 N-terminus encloses two copper-binding domains, M1 and M2. We show that in vivo Atx1–M1 or Atx1–M2 interactions activate Ccc2. M1 or M2, expressed in place of the metallo-chaperone Atx1, were not as efficient as Atx1 in delivering copper to the Ccc2 N-terminus. However, when the Ccc2 N-terminus was truncated, these independent metal-binding domains behaved like functional metallo-chaperones in delivering copper to another copper-binding site in Ccc2 whose identity is still unknown. Therefore, we provide evidence of a dual role for the Ccc2 N-terminus, namely to receive copper from Atx1 and to convey copper to another domain of Ccc2, thereby activating the ATPase. At variance with their prokaryotic homologues, Atx1 did not activate the Ccc2-derived ATPase lacking its N-terminus.

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