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Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis

Authors
  • Laurent, Clémentine1
  • Lekeux, Gilles1
  • Ukuwela, Ashwinie A.2
  • Xiao, Zhiguang2
  • Charlier, Jean-Benoit1
  • Bosman, Bernard3
  • Carnol, Monique3
  • Motte, Patrick1, 4
  • Damblon, Christian5
  • Galleni, Moreno1
  • Hanikenne, Marc1, 4
  • 1 University of Liège, Department of Life Sciences, Center for Protein Engineering (CIP), Liège, 4000, Belgium , Liège (Belgium)
  • 2 University of Melbourne, School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, Parkville, VIC, 3010, Australia , Parkville (Australia)
  • 3 University of Liège, Laboratory of Plant and Microbial Ecology, Department of Biology, Ecology, Evolution, Liège, 4000, Belgium , Liège (Belgium)
  • 4 University of Liège, PhytoSYSTEMS, Liège, 4000, Belgium , Liège (Belgium)
  • 5 University of Liège, Chimie Biologique Structurale, Department of Chemistry, Liège, Belgium , Liège (Belgium)
Type
Published Article
Journal
Plant Molecular Biology
Publisher
Springer Netherlands
Publication Date
Jan 21, 2016
Volume
90
Issue
4-5
Pages
453–466
Identifiers
DOI: 10.1007/s11103-016-0429-z
Source
Springer Nature
Keywords
License
Yellow

Abstract

PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases.

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