Bernard Bosman

Functional analysis of the three HMA4 copies of the metal hyperaccumulator Arabidopsis halleri

Highlight Expression of the Arabidopsis halleri hyperaccumulation gene HMA4 in A. thaliana reveals functional differentiation among the three AhHMA4 copies and non-polar localization of the AhHMA4 protein in root pericycle cells.

Zinc triggers a complex transcriptional and post-transcriptional regulation of the metal homeostasis gene FRD3 in Arabidopsis relatives

Highlight High expression of the FRD3 gene in the metal hyperaccumulator Arabidopsis halleri stems from complex zinc-regulated transcriptional and post-transcriptional mechanisms present in the non-hyperaccumulating Arabidopsis thaliana.

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

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.

di-Cysteine motifs in the C-terminus of plant HMA4 proteins confer nanomolar affinity for zinc and are essential for HMA4 function in vivo

Functional analysis of the HMA4 protein C-terminal extension reveals a similar and key function of high affinity zinc binding by di-Cys motifs in both Arabidopsis thaliana and A. halleri.