[en] 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.
<br />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.
<br />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.
<br />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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Laurent, Clémentine ✱; Université de Liège > Département des sciences de la vie > Macromolécules biologiques
Lekeux, Gilles ✱; Université de Liège > Département des sciences de la vie > Centre d'ingénierie des protéines
Ukuwela, Ashwinie A
Xiao, Zhiguang
Charlier, Jean-Benoit
Bosman, Bernard ; Université de Liège > Département de Biologie, Ecologie et Evolution > Ecologie végétale et microbienne
Carnol, Monique ; Université de Liège > Département de Biologie, Ecologie et Evolution > Ecologie végétale et microbienne
Motte, Patrick ; Université de Liège > Département des sciences de la vie > Génomique fonctionnelle et imagerie moléculaire végétale
Damblon, Christian ; Université de Liège > Département de chimie (sciences) > Chimie biologique structurale
Galleni, Moreno ; Université de Liège > Département des sciences de la vie > Macromolécules biologiques
Hanikenne, Marc ; Université de Liège > Département des sciences de la vie > Génomique fonctionnelle et imagerie moléculaire végétale
✱ These authors have contributed equally to this work.
Language :
English
Title :
Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.
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