Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis
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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.
KeywordsMetal P-type ATPase Metal binding domain Zinc transport Structure–function analysis Arabidopsis
We thank Dr. C. Nouet, S. Fanara, M. Schloesser and M.C. Requier for technical support. Prof. A. Wedd is thanked for his constructive comments and suggestions. We thank Dr. M. Haydon for the kind gift of hma2hma4 seeds. Funding was provided by the “Fonds de la Recherche Scientifique–FNRS” (FRFC-2.4583.08, PDR-T.0206.13) (MH, MG), the University of Liège (SFRD-12/03) (MH), the Belgian Program on Interuniversity Poles of Attraction (IAP no. P6/19) and the Australian Research Council (Grant DP130100728) (AAU, ZX). MH is Research Associate of the FNRS. Doctoral fellowships were funded by the FNRS (CL) and the “Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture” (GL, JBC).
MG and MH conceived and directed the study. MH, MG, CD and ZX designed experiments. CL, GL, JBC, BB, AAU, ZX and CD performed experiments. CL, MH, CD, GL, BB, AAU and ZX analysed the data. MH, MG, CD, AAU, ZX, PM and MC contributed reagents/materials/analysis tools. MH, CL and MG wrote the paper and all authors commented on the manuscript.
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