Crucial function of vertebrate glutaredoxin 3 (PICOT) in iron homeostasis and hemoglobin maturation

Abstract

The mechanisms by which eukaryotic cells handle and distribute the essential micronutrient iron within the cytosol and other cellular compartments are only beginning to emerge. The yeast monothiol multidomain glutaredoxins (Grx) 3 and 4 are essential for both transcriptional iron regulation and intracellular iron distribution. Despite the fact that the mechanisms of iron metabolism differ drastically in fungi and higher eukaryotes, the glutaredoxins are conserved, yet their precise function in vertebrates has remained elusive. Here we demonstrate a crucial role of the vertebrate-specific monothiol multidomain Grx3 (PICOT) in cellular iron homeostasis. During zebrafish embryonic development, depletion of Grx3 severely impairs the maturation of hemoglobin, the major iron-consuming process. Silencing of human Grx3 expression in HeLa cells decreases the activities of several cytosolic Fe/S proteins, for example, iron-regulatory protein 1, a major component of posttranscriptional iron regulation. As a consequence, Grx3-depleted cells show decreased levels of ferritin and increased levels of transferrin receptor, features characteristic of cellular iron starvation. Apparently, Grx3-deficient cells are unable to efficiently use iron, despite unimpaired cellular iron uptake. These data suggest an evolutionarily conserved role of cytosolic monothiol multidomain glutaredoxins in cellular iron metabolism pathways, including the biogenesis of Fe/S proteins and hemoglobin maturation.

FIGURE 1:

Depletion of Grx3 in zebrafish embryos causes loss of hemoglobin. (A) In situ hybridization of a zebrafish embryo at 48 hpf with a zfGrx3-specific DNA probe. The arrow indicates the region most likely constituting the intermediate cell mass, the site of embryonic erythropoiesis. (B) Western blot quantification of Grx3, relative to the loading control lamin, from wild-type and a pool of ∼250 morpholino-injected embryos. (C–G) Positive hemoglobin staining of zebrafish embryos at 48 hpf depends on the presence of zfGrx3. Diaminofluorene staining of (C) wild-type embryos, (D) zfGrx3-knockdown embryos, and (E) embryos injected with both morpholino and capped mRNA coding for zfGrx3. (F–G) o-Dianisidine staining and β-globin–specific in situ hybridization of (F) wild-type and (G) zfGrx3-knockdown embryos. One typical embryo is shown to exemplify each phenotype.

FIGURE 2:

Zebrafish Grx3 function in iron homeostasis is independent of IRP1. (A) Detection of ALAS2 levels in wild-type embryos and embryos depleted for Grx3. Diaminofluorene staining of hemoglobin at 48 hpf in (B) wild-type embryos (n = 30), (C) embryos depleted for IRP1 (n = 95), (D) embryos depleted for both IRP1 and zfGrx3 (n = 40), and (E) embryos depleted for IRP1 and overexpressing zfGrx3 by injection of zfGrx3 mRNA (n = 39). Knockdown of IRP1 and zfGrx3 was induced by injection of specific morpholinos into single-cell eggs. (F) Quantification of the experiments in B–E relative to wild-type fish.

FIGURE 3:

Activities of total aconitase and cytochrome c oxidase are decreased in Grx3-depleted zebrafish embryos. Enzymatic activities of (A) aconitase, (B) cytochrome c oxidase, and (C) malate dehydrogenase (MDH) in pooled wild-type control (wt) and Grx3 morpholino-injected (mo) zebrafish embryos at 48 hpf. Because extracts of the entire embryos were analyzed, aconitase activity represents the sum of cytosolic and mitochondrial isoforms. MDH is an iron-independent control protein. All activities were normalized to the values of the respective wt samples. The 100% aconitase activity corresponds to 26.3 ± 0.4 mU/mg (of total protein), 100% cytochrome c oxidase activity to 136.4 ± 11.2 mU/mg, and 100% MDH activity to 0.98 ± 0.05 U/mg (n = 3–4).

FIGURE 4:

Grx3 depletion in HeLa cells impairs cytosolic iron-dependent processes despite an increased iron uptake. (A) Representative Western blots of control cells, transfected with scrambled siRNA (si-scr), and Grx3-depleted HeLa cells (si-Grx3) stained for Grx3, α-tubulin (TubA), IRP1 and IRP2, transferrin receptor, ferritin (Ft), and GPAT. (B) Densitometric quantification of Western blots as depicted in A from five independent experiments. (C) The enzyme activity of cytosolic aconitase was measured in the soluble fraction of a cell extract (see Supplemental Figure S4 for the efficiency of fractionation). Decrease in activity indicates impaired maturation of IRP1. Aconitase activity was normalized with respect to the control samples. The 100% activity corresponds to 47.8 ± 1.7 mU/mg of total cytosolic protein (n = 5). (D) Depletion of Grx3 impairs the IRE binding capacity of IRP1. Binding of both IRP1 and IRP2 to ³²P-labeled IRE of human ferritin mRNA was analyzed by RNA electrophoretic mobility shift assay. The IRE-binding capacity of IRP1 was measured after IRP2 supershift upon addition of IRP2 antiserum and calculated as the ratio of the respective binding activity without and with 1.7% (vol/vol) β-ME. Data were normalized to the respective ratio of control cells. The IRE-binding activity of IRP2 was measured after IRP1 supershift upon addition of IRP1 antiserum. The assay was performed in the presence of 0.3% (vol/vol) β-ME to increase the signal-to-noise ratio (Zumbrennen et al., 2009), and results were normalized to the respective values of control cells. (E) Uptake of transferrin-bound ⁵⁵Fe into Grx3-depleted and scrambled siRNA-treated HeLa cells was allowed for 4 h and estimated by scintillation counting of crude cell extracts. The 100% values typically represent ∼10,000 cpm.

FIGURE 5:

Grx3 depletion affects the subcellular distribution of the transferrin receptor and ferritin. HeLa cells transfected with scrambles control siRNA (si-scr) and Grx3 siRNA were fixated and stained for TfR (left) and ferritin (right) by immunofluorescence. Five layers in the volume (z = 0.5 μm) were scanned by confocal microscopy. For TfR, the focus was placed in close vicinity to the site of attachment of the cell to the glass slide, that is, on the cell membrane. For the analysis of ferritin distribution, the focus was placed ∼1 μm ahead of the glass slide substrate within the cytosol.

FIGURE 6:

Grx3 depletion in HeLa cells only slightly affects the activities of mitochondrial iron-dependent enzymes. Activities of iron-dependent mitochondrial proteins in Grx3-depleted HeLa cells were measured in crude mitochondrial fractions or in total cell extracts (for ferrochelatase). (A) Cytochrome c oxidase activity, dependent on heme synthesis. (B) Ferrochelatase activity, Fe/S cluster dependent. (C) SDH activity, heme and Fe/S cluster dependent. (D) Mitochondrial aconitase, dependent on the insertion of a Fe/S cluster. For the efficiency of subcellular fractionation see Supplemental Figure S4. All activities were normalized to the scrambled siRNA-treated (si-scr) control samples. The 100% cytochrome c oxidase activity corresponds to 24.1 ± 2.1 mU/mg (of total mitochondrial protein), 100% SDH activity to 1.99 ± 0.14 U/mg, and 100% aconitase activity to 54.1 ± 3.5 mU/mg (n = 3–5).