Metal tolerance protein MTP6 affects mitochondrial iron and manganese homeostasis in cucumber

Abstract

Members of the cation diffusion facilitator (CDF) family have been identified in all kingdoms of life. They have been divided into three subgroups, namely Zn-CDF, Fe/Zn-CDF, and Mn-CDF, based on their putative specificity to transported metal ions. The plant metal tolerance protein 6 (MTP6) proteins fall into the Fe/Zn-CDF subgroup; however, their function in iron/zinc transport has not yet been confirmed. Here, we characterized the MTP6 protein from cucumber, Cucumis sativus. When expressed in yeast and in protoplasts isolated from Arabidopsis cells, CsMTP6 localized in mitochondria and contributed to the efflux of Fe and Mn from these organelles. Immunolocalization of CsMTP6 in cucumber membranes confirmed this association with mitochondria. Root expression and protein levels of CsMTP6 were significantly up-regulated in conditions of Fe deficiency and excess, but were not affected by Mn availability. These results indicate that MTP6 proteins contribute to the distribution of Fe and Mn between the cytosol and mitochondria of plant cells, and are regulated by Fe to maintain mitochondrial and cytosolic iron homeostasis under varying conditions of Fe availability.

Fig. 1.

Sequence analysis of cucumber CsMTP6. The transmembrane topology of CsMTP6 was predicted by HMMTOP. The positions of CDF signature sequence, mitochondrial targeting peptide, and HD motif 2 are indicated by dashed arrows. The two motifs specific for the Fe/Zn-subgroup, HSVSD (motif HD 1 in the CDF signature sequence) and HHRAD (motif HD 2) are highlighted in boxes. The amino acids of the HD motifs are marked in bold and the conservative histidine and aspartate are underlined.

Fig. 2.

Subcellular localization of CsMTP6. (A) Mitochondrial localization of CsMTP6 in protoplasts prepared from suspensions of Arabidopsis cells: 1, GFP fluorescence of protoplast expressing CsMTP6-GFP; 2, MitoTracker red fluorescence; 3, merged image; 4, transmission image of the same protoplast. The scale bar is 10 µm. (B) Immunolocalization of CsMTP6 in cucumber root cells. SDS-PAGE followed by western blot analysis of plasma membrane (PM), tonoplast (Ton), and mitochondrial (Mit) and plastidial (Plast) fractions. The fractions (15 μg of protein per lane) were blotted with the primary antibodies to identify marker enzymes for the plasma membrane (PM-ATPase), tonoplast (V-ATPase), plastids (Toc75), mitochondria (COXII), and to localize CsMTP6. L, protein ladder.

Fig. 3.

Complementation assay and localization of CsMTP6 in yeast cells defective in vacuolar and mitochondrial iron transporters. (A) Representative images (from four replicates) of serial dilutions of the wild-type (WT) and yeast mutants sensitive to high iron (Δccc1) or low iron (Δmrs3mrs4 and Δmmt1/mmt2) transformed with the empty vector or with CsMTP6. Yeast were grown in SC/Glu–Ura media supplemented with 100 µM BPS (low Fe), 3 mM FeSO₄-EDTA, or in the control SC/Glu–Ura media. (B) CsMTP6-GFP localization in Δccc1, Δmrs3mrs4 and Δmmt1/mmt2 cells. Yeast cultured overnight in control liquid SC/Glu–Ura media were diluted to OD₆₀₀ 0.1 and then grown in liquid SC/Glu–Ura media supplemented with 3 mM FeSO₄-EDTA (Δccc1) or 100 µM BPS (low Fe, Δmrs3mrs4 and Δmmt1/mmt2) for 8–10 h. 1, Transmission image of the cells expressing CsMTP6-GFP; 2, GFP fluorescence; 3, MitoTracker red fluorescence; 4, merged image. Scale bars are 5 μm.

Fig. 4.

Cytosolic and mitochondrial Fe content in yeast expressing CsMTP6. (A) Western blot analysis of CsMTP6-GFP and c-GDO with a carboxyl-FLAG epitope in wild-type (WT) yeast and transformants using antibodies against GFP and FLAG; vec, empty vector. (B) c-GDO activity in WT and yeast mutant cells expressing CsMTP6-GFP and GDO-FLAG. c-GDO activity is expressed as nmol of substrate converted min^–1 mg^–1 of protein. Data are means (±SD) of four individual transformants. (C) Mitochondrial Fe content in yeast expressing CsMTP6. Data are means (±SD) of three separate experiments. Significant differences were determined using Tukey’s test.

Fig. 5.

Effect of CsMTP6 expression on yeast sensitivity to Co, Cu, and H₂O₂. (A) Representative images (from four replicates) of serial dilutions of the wild-type (WT) and Δmmt1Δmmt2 and Δaft1 mutants transformed with the empty vector or CsMTP6 and grown on either SC/Glu–Ura medium supplemented with 2 mM CoCl₂ or control SC/Glu–Ura medium. (B) Mitochondrial Co content in yeast expressing CsMTP6. Data are means (±SD) from three separate experiments. (C) Representative images (from four replicates) of serial dilutions of the WT and Δmmt1Δmmt2 mutants transformed with the empty vector or CsMTP6 and grown on either SC/Glu–Ura medium supplemented with 1.5 mM CuCl₂ or control SC/Glu–Ura medium. (D) Representative images (from four replicates) of serial dilutions of the WT and Δfet5Δsmf3 mutants transformed with the empty vector or CsMTP6 and grown on either SC/Glu–Ura medium supplemented with 0.0075% H₂O₂ or control SC/Glu–Ura medium. (E) Mitochondrial Cu content in yeast expressing CsMTP6. Data are means Cu l(±SD) from three separate experiments.

Fig. 6.

Effect of CsMTP6 expression on the Mn sensitivity, mitochondrial Mn content, and Mn-SOD activity of yeast. (A) Representative images (from four replicates) of serial dilutions of the wild-type (WT) and K667 mutants transformed with the empty vector or CsMTP6 and grown on either SC/Glu–Ura medium supplemented with MnSO₄ or control SC/Glu–Ura medium. (B) Mitochondrial content of Mn in yeast expressing CsMTP6. Data are means (±SD) from separate experiments. (C) Mitochondrial Mn-SOD activity in yeast expressing CsMTP6. Data are means (±SD) from three separate experiments, expressed as enzyme units (U). Significant differences were determined using Tukey’s test. (D) Representative images (from four replicates) of serial dilutions of the WT and Δsmf1 mutants transformed with the empty vector or CsMTP6 and grown on either SC/Glu–Ura medium supplemented with EGTA (low Mn medium) or control SC/Glu–Ura medium.

Fig. 7.

Effects of CsMTP6 expression on mitochondrial Fe and Mn contents and Mn-SOD activity in Arabidopsis protoplasts. (A) Western blot analysis of the purity of mitochondrial and plastidial fractions prepared from protoplasts expressing CsMTP6 using primary antibodies raised against GFP (to detect CsMTP6-GFP), and marker proteins for plastids (Toc75) and mitochondria. The mitochondria and plastid fractions were prepared from protoplasts by discontinuous density gradient centrifugation in Percoll. (B) Fe and (C) Mn content in the mitochondrial fraction prepared from Arabidopsis protoplasts expressing CsMTP6 or transformed with the empty vector. Data are means (±SD) from three separate experiments. (D) Mitochondrial Mn-SOD activity in protoplasts expressing CsMTP6. Data are means (±SD) from three separate experiments, expressed as enzyme units (U). Significant differences were determined using Student’s t-test.

Fig. 8.

The levels of CsMTP6 transcript and protein under different Fe and Mn availability. (A) Real-time analysis of CsMTP6 expression in the roots of 2-week-old cucumber seedlings growing under control Fe (75 µM), control Mn (0.1 µM), excess Fe (1 mM), excess Mn (10 µM), Fe deficiency, or Mn deficiency. Data are mean CsMTP6 transcript levels (±SD) relative to the constitutively expressed reference gene CACS calculated from the arithmetic means of ΔCp values obtained in three independent experiments. (B) Western blot analysis of CsMTP6 protein levels in mitochondria isolated from roots of cucumbers grown under different Fe and Mn treatments for 2 weeks. Immunoblots of the same mitochondria were probed with antibodies against mitochondrial COXII cytochrome oxidase to ensure equal loading of membrane protein (10 µg) onto SDS-PAGE. Significant differences were determined using Tukey’s test.