A combined zinc/cadmium sensor and zinc/cadmium export regulator in a heavy metal pump

Abstract

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn(2+) nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn(2+) and Cd(2+) chelator with capacity to bind 10 Zn(2+) ions per C terminus. When AtHMA4 is expressed in a Zn(2+)-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn(2+) and Cd(2+) tolerance and lowered Zn(2+) and Cd(2+) content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn(2+) and Cd(2+) chelator (sensor) and as a regulator of the efficiency of Zn(2+) and Cd(2+) export. The identification of a post-translational handle on Zn(2+) and Cd(2+) transport efficiency opens new perspectives for regulation of Zn(2+) nutrition and tolerance in eukaryotes.

FIGURE 1.

Overview of the AtHMA4 Zn²⁺ pump. A, schematic representation of AtHMA4. B, amino acid sequence of the C-terminal domain of AtHMA4.

FIGURE 2.

The C terminus of AtHMA4 serves as a high capacity chelator of Zn²⁺ and Cd²⁺. A, recombinant purified C terminus of AtHMA4 (aa 716–1173) binds Zn²⁺ and Cd²⁺. A purified maltose-binding protein was used as negative control. From left, Coomassie Brilliant Blue-stained gel, Western blot immunostained with penta-His antibody, and protein blots stained with dithizone for measuring Zn²⁺ and Cd²⁺ binding to the proteins. Before staining, the proteins were subjected to renaturation in the presence or absence of Zn²⁺ and Cd²⁺. B, Zn²⁺ binding to AtHMA4 C terminus quantified by SEC-ICP-MS. Peaks of Ni²⁺ and Zn²⁺ bound to the AtHMA4 C terminus (⁴⁸SO peak) are shown. C, expression of the isolated AtHMA4 C terminus (C_t; aa 716–1173) and the C terminus lacking the His stretch at the terminal end (C_t-Δ18; aa 716–1155) in zrc1 cot1. D, anti-HMA4 antibody-immunostained protein blots with yeast total protein extracted from cells transformed with either empty vector, or expressing full-length AtHMA4, the C-terminal domain (C_t; aa 716–1173) or the C terminus lacking the His stretch at the terminal end (C_t-Δ18; aa 716–1155).

FIGURE 3.

The C-terminal His stretch of AtHMA4 is not essential for pump activity. A, AtHMA4 and a mutant lacking the C-terminal His stretch (Δ18) were expressed in zrc1 cot1, and yeast growth was measured on Zn²⁺-containing media. B, three different suppressor mutants resulting in C-terminal deletion mutants of AtHMA4 (Δ204, Δ359, and Δ369) were identified in the Athma4Δ18 sequence.

FIGURE 4.

Sequential deletions in the C-terminal of AtHMA4 results in a gradual increase in pump efficiency. AtHMA4, Athma4Δ18, and 13 C-terminal deletion mutants lacking 1–13 cysteine pairs (Δ32-Δ459) were expressed in zrc1 cot1, and yeast growth was compared on Zn²⁺-containing media.

FIGURE 5.

Zinc content is reduced in yeast cells expressing C-terminally truncated AtHMA4. A, localization of AtHMA4, Athma4Δ18, and Athma4Δ459 in transgenic yeast. The pumps were tagged with GFP to their C terminus and expressed in zrc1 cot1. A and B, free GFP; C and D, AtHMA4; E and F, Athma4Δ18; and G and H, Athma4Δ459. A, C, E, and G, GFP fluorescence; B, D, F, and H, Nomarsky image; bar = 2 μm. B and C, zinc content in transgenic yeast measured by ICP-Optical Emission Spectroscopy (B) or ICP-MS (C). The yeast cells were grown in 100 μm ZnCl₂ for 15 h. Data are means ± S.E. (n = 8–9). Significant difference compared with vector control was analyzed by Student's t test. *, p < 0.05; and ***, p < 0.001. DW, dry weight.

FIGURE 6.

Athma4Δ18, Athma4Δ459, and AtHMA4 C terminus increase growth of zrc1 cot1 yeast cells on cadmium compared with AtHMA4. Cadmium content in Athma4Δ459- and Athma4Δ18-expressing yeast is decreased compared with AtHMA4, whereas cells expressing AtHMA4 C terminus contain higher amounts of cadmium compared with vector control. A, AtHMA4, Athma4Δ18, Athma4Δ459, and AtHMA4 C-terminus were expressed in the zrc1 cot1 strain, and yeast growth was compared on increasing concentrations of CdCl₂. B, cadmium content in transgenic zrc1 cot1 cells measured by ICP-MS. Yeast cells were grown in 1 μm CdCl₂ for 15 h. Data are means ± S.E. (n = 8–9). Significant difference compared with vector control was analyzed by Student's t test. *, p < 0.05; and ***, p < 0.001. DW, dry weight.

FIGURE 7.

AtHMA4 and the two C-terminal deletion mutants Athma4Δ18 and Athma4Δ268 are expressed and functional in the membranes of the Zn²⁺-sensitive yeast strain zrc1 cot1. A, expression analysis of the pumps in yeast microsomes detected by Coomassie Brilliant Blue staining (upper panel) and by immunoblotting using a specific antibody against the C terminus of AtHMA4 (lower panel). B, phosphorylation of AtHMA4, Athma4Δ268, and Athma4D401A from γ-³²P-labeled ATP in the presence of added Zn²⁺. C, phosphorylation of AtHMA4, Athma4Δ18, Athma4Δ268, and Athma4D401A from γ-³²P-labeled ATP in the absence of added Zn²⁺ and with increasing amounts of EGTA.

FIGURE 8.

Kinetics of phosphorylation and dephosphorylation of AtHMA4 and the C-terminal mutant Athma4Δ268. A, Zn²⁺-dependent phosphorylation. Phosphorylation was initiated in the presence of 500 μm EGTA with addition of Zn²⁺ as indicated. B, ATP-Mg-dependent phosphorylation. Phosphorylation was initiated at conditions giving a maximal steady-state phosphorylation level above, with addition of ATP as indicated so that the ratio of hot to cold ATP remained the same for all measurements. C, time course of the phosphorylation reaction. D, dephosphorylation in the presence of ATP. Dephosphorylation was initiated after phosphorylation for 30 s by addition of a chase solution producing final concentrations of 5 mm EGTA and 0.5 mm unlabeled ATP. E, dephosphorylation in the presence of ADP. Dephosphorylation was initiated after phosphorylation for 30 s by addition of a chase solution producing final concentrations of 5 mm EGTA and 0.5 mm unlabeled ADP. Values reported are the mean ± S.E. for at least three independent measurements.