Reference : Functional characterization of Arabidopsis HMA4 P-type ATPases
Dissertations and theses : Doctoral thesis
Life sciences : Biochemistry, biophysics & molecular biology
Functional characterization of Arabidopsis HMA4 P-type ATPases
Lekeux, Gilles mailto [Université de Liège - ULiège > Département des sciences de la vie > Centre d'ingénierie des protéines >]
Université de Liège, ​Liège, ​​Belgique
Doctorat en biochimie, biologie moléculaire et cellulaire, bioinformatique et modélisation
Galleni, Moreno mailto
Hanikenne, Marc mailto
Charlier, Paulette mailto
Feller, Georges mailto
González-Guerrero, Manuel mailto
Verbruggen, Nathalie mailto
Remacle, Claire mailto
Delaplace, Pierre mailto
[en] Transition metals play a vital role in the development and survival of photosynthetic organisms. Therefore, plants require a metal homeostasis network to control their inner metal concentration despite variation in metal supply. The HMA4 membrane protein has a central role in the zinc homeostasis network of the model plant Arabidopsis thaliana. This PIB-2 ATPase loads zinc (and cadmium) from the pericycle cells into the xylem in roots. It is involved in zinc hyperaccumulation as well as zinc and cadmium hypertolerance in the pseudometallophyte Arabidopsis halleri. In particular, the enhanced expression of HMA4 in A. halleri compared to A. thaliana triggers a higher translocation of metal from root to shoot.
As a PIB ATPase, the HMA4 architecture consists of a transmembrane domain, two cytoplasmic catalytic domains, the actuator domain and the ATP-binding domain divided in a nucleotide-binding domain and a phosphorylation domain, as well as N- and C-terminal cytosolic extensions. Thanks to high conservation level within the P-type ATPases, the role of the HMA4 cytoplasmic catalytic domains can be inferred from well characterized pumps of the family. In contrast, the function of its terminal cytosolic extensions as well as the metal permeation mechanism through the membrane remains elusive.
A combined in vivo and in vitro functional characterization pointed out the importance of high affinity Zn2+ binding to the HMA4 N- terminal extension C27CxxE31 motif and the C-terminal extension di-Cys motifs for the function of the protein in plants. This study also suggested the presence in the C-terminal extension of a signal for the HMA4 localization in the plasma membrane. Finally, the high divergence of coding sequence between the A. thaliana and A. halleri HMA4 C-terminal extensions does not result in major functional differences between the two proteins, at least when expressed in A. thaliana.
Moreover, a structural analysis by homology modeling of the HMA4 transmembrane region highlighted amino acids forming a metal permeation pathway, whose importance was subsequently investigated functionally through mutagenesis and complementation experiments in plants. The analysis newly identified amino acids whose mutation results in total or partial loss of the protein function. In addition, the comparison of zinc and cadmium accumulation in shoots of A. thaliana complemented lines revealed a number of HMA4 mutants exhibiting different abilities in zinc and cadmium translocation.
Altogether this work significantly advanced our understanding of the function of HMA4, a key protein of the zinc homeostasis network in plants.

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