Metal-sensing properties of the disordered loop from the Arabidopsis metal transceptor IRT1

Abstract

The plant iron-regulated transporter 1 (IRT1) iron transporter is a plasma membrane protein that takes up iron in the root under iron-limited conditions. Besides its primary metal substrate iron, IRT1 transports other divalent metals that overaccumulate in plants when soil iron is low and IRT1 is highly expressed. We previously reported that the intracellular regulatory loop between transmembrane helices TM4 and TM5 is involved in the post-translational regulation of IRT1 by its non-iron metal substrates. Upon excess of zinc, IRT1 undergoes phosphorylation by CIPK23 followed by its ubiquitination by IDF1 to target IRT1 for vacuolar degradation. This zinc-dependent down-regulation of IRT1 requires the presence of four histidine (H) residues in the IRT1 loop, which directly bind zinc. However, how selective metal binding is achieved and how this allows downstream regulation to take place is largely not known. Here, we characterized the metal-binding properties and structure of the IRT1 loop to better understand the molecular basis of non-iron metal sensing and signaling. Using a combination of circular dichroism and NMR, we reveal that zinc and manganese bind to the IRT1 loop with nanomolar range affinity and that metal binding does not trigger structuration of the loop. We validate that zinc and manganese binding is mediated by four H residues and identify aspartic acid (D) residue D173 as helping in metal co-ordination and participating to metal sensing and metal-dependent degradation of IRT1 in plants. Altogether, our data provide further understanding of how IRT1 regulatory loop senses high cytosolic divalent metal concentrations to regulate metal uptake in plants.

Keywords: Arabidopsis; iron; metals; plants; sensing; transport.

Figure 1. The IRT1 regulatory loop is disordered.

(A) Far-UV circular dichroism spectra (CD) of the IRT1 regulatory loop. Equivalents of Zn²⁺ (0–40 eq) were added to IRT1 peptide, and CD spectra were recorded from 190 to 240 nm. Corrections of the final peptide concentrations were done with each addition of Zn²⁺. The data presented are an average of 128 scans. (B) One-dimensional (1D) ¹H NMR spectra of the IRT1 loop recorded in the absence (black) and presence (blue) of Zn²⁺. Only the amide and the aromatic protons are shown. IRT1, iron-regulated transporter 1.

Figure 2. Zinc directly binds to histidine residues in the IRT1 regulatory loop.

Fingerprint region of the ¹H-1H NOESY spectrum recorded on the IRT1 loop in the presence or absence of Zn²⁺. Several intramolecular and intermolecular NH-CHβ signals are shown for the wildtype IRT1 loop (A) and two mutant peptides (B,C); and intramolecular CHα-CHβ signals for the wildtype IRT1 loop (D) and two mutant peptides (E, F). The sequences of the domain into which mutations have been introduced are shown above each spectrum, and the mutations are highlighted in green. The spectra recorded in the absence of Zn²⁺ are shown in black, and those recorded in the presence of two molar equivalents of Zn²⁺ are shown in red. Asterisks (*) indicate impurities in the sample. Annotations with a dash indicate cross-peak between inter-residue, that is NHi-CHβi-1, and without dash cross-peaks between intra-residue, that is HNi-Hβi. IRT1, iron-regulated transporter 1.

Figure 3. Zinc binding affinities of the IRT1 regulatory loop.

(A) Microscale thermophoresis (MST) analyses of zinc binding to the wildtype IRT1 loop (IRT1; dark blue), double mutant with histidine residues 162 and 164 mutated to alanine (H162A/H164A; green), double mutant with histidine residues 166 and 168 mutated to alanine (H166A/H168A; orange), and quadruple mutant with histidine residues 162, 164, 166, and 168 mutated to alanine (4HA; light blue). Dots represent the average dose response of at least six technical replicates derived from two biological replicates. Errors bars and MST binding parameters are shown in online supplementary figure 5. (B) MST analyses of zinc binding to the wildtype IRT1 loop (IRT1; dark blue), single mutant with aspartic acid 173 mutated to asparagine (D173N; yellow), double mutant with histidine residues H162 and H164 mutated to alanine (H162A/H164A; green), and triple mutant with histidine residues 162 and 164 mutated to alanine and aspartic acid 173 mutated to asparagine (H166A/H168A/D173N; red). Dots represent the average dose response of at least six technical replicates derived from two biological replicates. Errors bars and MST binding parameters are shown in online supplementary figure 5. IRT1, iron-regulated transporter 1.

Figure 4. Manganese binding affinities of the IRT1 regulatory loop.

(A) Microscale thermophoresis (MST) analyses of manganese binding to the wildtype IRT1 loop (IRT1; dark pink), double mutant with histidine residues 162 and 164 mutated to alanine (H162A/H164A; green), double mutant with histidine residues 166 and 168 mutated to alanine (H166A/H168A; orange), and quadruple mutant with histidine residues 162, 164, 166, and 168 mutated to alanine (4HA; light pink). Dots represent the average dose response of at least six technical replicates derived from two biological replicates. Errors bars and MST binding parameters are shown in online supplementary figure 6 . (B) MST analyses of manganese binding to the wildtype IRT1 loop (IRT1; dark pink), single mutant with aspartic acid 173 mutated to asparagine (D173N; yellow), double mutant with histidine residues H162 and H164 mutated to alanine (H162A/H164A; green), and triple mutant with histidine residues 162 and 164 mutated to alanine and aspartic acid 173 mutated to asparagine (H162A/H164A/D173N; red). Dots represent the average dose response of at least six technical replicates derived from two biological replicates. Errors bars and MST binding parameters are shown in online supplementary figure 6 . IRT1, iron-regulated transporter 1.

Figure 5. Residue D173 is involved in IRT1 endocytosis in response to non-iron metal excess.

(A) Representative confocal microscopy images of epidermal cells from Nicotiana benthamiana leaves transiently expressing 35::IRT1-mCitrine (IRT1) or mutated versions 35S::IRT1_D173Q-mCitrine, 35S::IRT1_H162A/H164A-mCitrine (H162A/H164A), and 35S::IRT1_{H162A/H164A/D173Q}-mCitrine (H162A/H164A/D173Q) after 3 h of control treatment (without non-iron metals; − metals) or after non-iron metal excess treatment (+ ++ metals). Showed maximum projection of 10–15 optical sections taken using 1 µm z-distance. Scale bars, 20 μm. (B) Quantification of the ratio of the plasma membrane to intracellular signals and (C) quantification of intracellular particles per µm² from cells exposed to metal excess relative to cells exposed to control solution of plants treated as described in A). Error bars represent SD (n = 25–50) from three independent experiments. ‘ns’ indicate no significant differences, and asterisks indicate significant differences (one-way ANOVA, Tukey post-test, ****P<0.0001). (D) Trimolecular fluorescence complementation (TriFC) assay with CIPK23 fused to the mCit_C fragment and ALFA tag-bearing IRT1 variants (from left to right, IRT1, D173Q, H162A/H164A, and H62A/H64A/D173Q) co-expressed with ALFA-NB C-terminally fused to the mCit_N fragment. CIPK24, which does not interact with IRT1, is used as negative control. Representative images of two biological replicates are shown. Scale bars, 10 µm. (E) Quantification of the reconstituted mCitrine signal upon association of CIPK23 or CIPK24 to IRT1, IRT1_D173Q, IRT1_H162A/H164A, or IRT1_{H162A/H164A/D173Q}. ‘ns’ indicate no significant differences, and asterisks indicate significant differences (one-way ANOVA, Tukey post-test, ****P<0.0001). IRT1, iron-regulated transporter 1.

Figure 6. Zinc and manganese ion co-ordination by the IRT1 regulatory loop. Alphafold3 prediction for the IRT1 loop co-ordinating Zn²⁺ ions (gray spheres) with residues H162, H164, H166, H168, and D173.

Slashed green lines represent metal co-ordination within 3.5 Å distance. Analyses performed with UScf. ChimeraX application. IRT1, iron-regulated transporter 1.