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

Abstract

The PIB ATPase heavy metal ATPase 4 (HMA4) has a central role in the zinc homeostasis network of Arabidopsis thaliana. This membrane protein loads metal from the pericycle cells into the xylem in roots, thereby allowing root to shoot metal translocation. Moreover, HMA4 is key for zinc hyperaccumulation as well as zinc and cadmium hypertolerance in the pseudometallophyte Arabidopsis halleri. The plant-specific cytosolic C-terminal extension of HMA4 is rich in putative metal-binding residues and has substantially diverged between A. thaliana and A. halleri. To clarify the function of the domain in both species, protein variants with truncated C-terminal extension, as well as with mutated di-Cys motifs and/or a His-stretch, were functionally characterized. We show that di-Cys motifs, but not the His-stretch, contribute to high affinity zinc binding and function in planta. We suggest that the HMA4 C-terminal extension is at least partly responsible for protein targeting to the plasma membrane. Finally, we reveal that the C-terminal extensions of both A. thaliana and A. halleri HMA4 proteins share similar function, despite marginally different zinc-binding capacity.

Fig. 1.

Overview of the HMA4 variant constructs for complementation experiments in plants. The expression cassettes are schematically represented. The nucleotide sequences encoding A. thaliana (dark gray) or A. halleri (light gray) native or mutant HMA4 proteins are preceded by the A. thaliana HMA4 promoter (pAt, 2595 bp). Numbers correspond to the amino acid position in the HMA4 protein. Ah, A. halleri; At, A. thaliana; AtAhHMA4 and AhAtHMA4, swapped C-terminal extensions; HA: His- → Ala-stretch; CCAA: di-Cys → di-Ala motifs; CHA: HA and CCAA mutations combined; Ctrunc: fully truncated C-terminal extension.

Fig. 2.

Complementation of the A. thaliana hma2hma4 zinc deficiency phenotype. HMA4 variants were expressed in hma2hma4 plants under the control of the AtHMA4 promoter. The plant phenotypes are shown after 6 weeks of growth on soil without zinc supplementation. Non-transformed hma2hma4 plants or expressing the native HMA4 proteins were respectively used as negative and positive controls. Images are representative of multiple observations of four to eight independent homozygous T3 lines for each genotype. Ah, A. halleri; At, A. thaliana; AtAhHMA4 and AhAtHMA4, swapped C-terminal extensions; HA: His- → Ala-stretch; CCAA: di-Cys → di-Ala motifs; CHA: HA and CCAA mutations combined; Ctrunc: fully truncated C-terminal extension.

Fig. 3.

Zinc accumulation in complemented plants grown on soil. Non-transformed hma2hma4 mutant (white) and expressing the native or mutant A. thaliana (dark gray) or A. halleri (light gray) HMA4 proteins under the control of the AtHMA4 promoter were grown for 6 weeks on soil without zinc supplementation. Zinc concentrations were measured in shoot tissues collected from two plants per line. Values are relative to lines expressing the native AtHMA4 proteins and are means±SEM of four to eight homozygous T3 independent lines for each genotype. The data were analysed with one-way ANOVA followed by Tukey’s multiple comparison test. Statistically significant differences (P<0.05) between means are indicated by different letters. Ah, A. halleri; At, A. thaliana; AtAhHMA4 and AhAtHMA4, swapped C-terminal extensions; HA: His- → Ala-stretch; CCAA: di-Cys → di-Ala motifs; CHA: HA and CCAA mutations combined; Ctrunc: fully truncated C-terminal extension.

Fig. 4.

Zinc accumulation and expression of zinc deficiency response genes in complemented plants grown in hydroponic conditions. Non-transformed hma2hma4 mutant (white) and expressing the native or mutant A. halleri HMA4 proteins (medium gray) under the control of AtHMA4 promoter were grown for the last 3 weeks before harvest in Hoagland hydroponic medium containing 0.2 µM zinc. Zinc concentrations were measured in shoot (A) and root (B) tissues collected from two plants per line. Values are relative to lines expressing the native AhHMA4 proteins and are means±SEM of two independent lines from three biological replicates. Transcript levels were quantified from plant tissues collected from two plants per line. Relative transcript levels (RTL) of IRT3 (C) and ZIP9 (D) in shoots are mean±SEM of two independent lines from two biological replicates. The data were analysed with one-way ANOVA followed by Tukey’s multiple comparison test. Statistically significant differences (P<0.05) between means within each figure panel are indicated by different letters. Ah: A. halleri; HA: His-stretch→Ala-stretch; CCAA: di-Cys→di-Ala motifs.

Fig. 5.

HMA4 variant localization in A. thaliana. The AtHMA4CCAA (B), AtHMA4CHA (C), AtHMA4Ctrunc (D), AhHMA4CCAA (E), AhHMA4CHA (F), and AhHMA4Ctrunc (G) variants fused to GFP and expressed under control of the copy 2 AhHMA4 promoter in Col-0 were imaged by confocal microscopy in roots of 18-day-old T1 seedlings. Non-transformed Col-0 seedlings (A) were used as negative controls. The images are, for each genotype, representative of three to six independent lines. Scale bars 25 µm. Ah, A. halleri; At, A. thaliana; CCAA: di-Cys→di-Ala motifs; CHA: HA: His- → Ala-stretch; CCAA: di-Cys → di-Ala motifs; CHA: HA and CCAA mutations combined; Ctrunc: fully truncated C-terminal extension.

Fig. 6.

Quantification of Zn²⁺ binding affinity and stoichiometry of isolated proteins AtHMA4c (A), AhHMA4c (B), and AtHMA4CCAAc (C) with Par probe at either 22 µM (open circles) or 111 µM (solid circles). (a, b) Variation of A₅₀₀ with average zinc binding stoichiometry of proteins; (c, d) Variation of log [Zn²⁺] with average zinc binding stoichiometry of proteins. The experiments were conducted by titration of each protein (1.0 µM) in MOPS buffer (50 mM, pH 7.3) containing NaCl (100 mM) and TCEP (1 mM) in the presence of excess Par ligand.