Decoupling ferritin synthesis from free cytosolic iron results in ferritin secretion

Abstract

Ferritin is a multisubunit protein that is responsible for storing and detoxifying cytosolic iron. Ferritin can be found in serum but is relatively iron poor. Serum ferritin occurs in iron overload disorders, in inflammation, and in the genetic disorder hyperferritinemia with cataracts. We show that ferritin secretion results when cellular ferritin synthesis occurs in the relative absence of free cytosolic iron. In yeast and mammalian cells, newly synthesized ferritin monomers can be translocated into the endoplasmic reticulum and transits through the secretory apparatus. Ferritin chains can be translocated into the endoplasmic reticulum in an in vitro translation and membrane insertion system. The insertion of ferritin monomers into the ER occurs under low-free-iron conditions, as iron will induce the assembly of ferritin. Secretion of ferritin chains provides a mechanism that limits ferritin nanocage assembly and ferritin-mediated iron sequestration in the absence of the translational inhibition of ferritin synthesis.

Figure 1. Ferritin is secreted by mouse bone marrow macrophages

A.ffe/+ mouse bone marrow macrophages were incubated with or without iron (FAC, 10 μM Fe) for 24 hours followed by incubation without iron for an additional 18 hrs (+/−). Media and cells were harvested and ferritin content determined by ELISA. The data are expressed as ng ferritin per culture. B. Wild-type (C3H) mouse bone marrow macrophages were incubated with iron (FAC, 10 μM Fe) for 24 hours. After iron was removed (+/−) cells were incubated in presence or absence of hepcidin (1 μg/ml media) for four hrs. Ferritin levels were analyzed as in A. C. Wild-type (C3H) macrophages were incubated with iron (FAC, 10 μM Fe). After 24 hr media was collected and cells were lysed. Ferritin levels were detected by Western blot using a rabbit anti-L-ferritin antibody or a rabbit anti-H-ferritin antibody followed by a peroxidase-conjugated goat anti-rabbit IgG. D. Serum was obtained from WT (C3H), ffe/+ and HAMP^−/− mice. Ferritin levels were measured by as described in C.

Figure 2. Newly synthesized ferritin is secreted by the classic secretory apparatus

A. Wild-type (C3H) mouse bone marrow macrophages were incubated with iron (FAC, 10 μM Fe) for 18 hours followed by incubation in presence or absence of cycloheximide (75 μg/ml media) for six hours. Ferritin levels were analyzed using an ELISA. The data are expressed as ng ferritin per culture. B. Wild-type (C3H) mouse bone marrow macrophages were incubated with iron for 24 hours followed by incubation in presence or absence of BFA (5 μg/ml media) for two or four hours. Media and cells were harvested and ferritin content determined by ELISA. C. Wild-type (C3H) mouse bone marrow macrophages were transfected with either control (NS, non-specific) or oligonucleotides specific for mouse Mon1a, incubated 24 hrs followed by incubation with iron (FAC, 10 μM Fe). After 24 hr iron was removed (+/−), cell incubated for eighteen hr and media and cells were harvested. Ferritin levels were measured by ELISA. The efficiency of Mon1a depletion was assessed by Western blot analysis using antibodies to Mon1a and tubulin. Error bars represent the standard error of the mean of three independent experiments.

Figure 3. Ferritin expressed in yeast is secreted in the media

A. Wild type (Wt) and B. sec61-2 strains were transformed with a pGAL-H-ferritin plasmid. Cells were grown in medium with or without galactose for 20 hr at either 22°C or 37°C. Media was collected, cells were harvested and ferritin levels determined by ELISA.

Figure 4. Iron depletion induces ferritin secretion

A. mRNA extraction from wild type mouse bone marrow macrophages was performed as described in the Experimental procedures. The mRNA was used in vitro to synthesize ferritin in the presence of a reticulocytes lysate containing ³⁵S-methionine and in the presence or absence of canine pancreatic microsomal membranes. The in vitro translation was performed in the presence or absence of iron (FAC, 10 μM Fe). In vitro translates were treated with or without PK in the presence or absence of 1% Triton X-100 and the synthesized ferritin was immunoprecipitated using mouse anti-ferritin (Sigma). Immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. B. mRNA from wild type mouse bone marrow macrophages was treated as in A, Cp immunoprecipitated and the immunoprecipitates treated plus or minus Endo H. Immunoprecipitates were separated on SDS-PAGE followed by autoradiography. C. L-ferritin mRNA was transcribed in vitro as described in experimental procedures and in vitro translation was performed as in A in presence or absence of iron (FAC, 10 μM Fe) for 30 minutes. Cycloheximide was added to inhibit further translation and canine pancreatic microsomes were added. The synthesized ferritin was treated as in A and immunoprecipitated ferritin detected by autography.

Figure 5. Iron induces the formation of ferritin-nanocage

A. Cytosolic ferritin and membrane-associated ferritin synthesized in vitro in the presence or absence of iron were applied to a Superdex 200 FPLC column in the presence of 1.0% Triton X-100. Fractions were collected and examined for ferritin by Western blot analysis. B. Ferritin synthesized in the absence of membranes in vitro was incubated with or without iron (FAC, 10 μM Fe) and ferritin assembly analyzed as in A using fractions 5 and 75. C. In vitro synthesized ferritin as in B was incubated with different amounts of iron (FAC, 5, 10 and 30 μM Fe) and ferritin assembly was analyzed as in A using fractions 5 and 75.

Figure 6. The amino terminus of ferritin is required for secretion

A. L-ferritin mRNA containing the coding sequence for a FLAG tag at the amino-terminal was transcribed in vitro as described in Experimental procedures. The mRNA was used in vitro to synthesize ferritin in presence of a reticulocyte lysate containing ³⁵S-methionine, iron (FAC, 10 μM Fe) and in presence or absence of canine pancreatic microsomal membranes. The in vitro translation was performed in the presence or absence of iron (FAC, 10 μM Fe). In vitro translates were treated with or without PK in the presence or absence of 1% Triton X-100 and the synthesized ferritin was immunoprecipitated using mouse anti-FLAG (Sigma). Immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. B. L-ferritin mRNA containing the coding sequence for a FLAG tag at the C-terminal was transcribed and translated in vitro and analyzed as in A. C. HEK293T cells were transfected with empty plasmid (pCMV), plasmid containing L-ferritin (pCMV-Ft), plasmid containing L-ferritin with an amino terminal FLAG tag (pCMV-FLAG-Ft), L-ferritin with a carboxyl-terminal FLAG tag (pCMV-Ft-FLAG) or mutant T30I L-ferritin (pCMV-Ft-T30I). Twenty four hours after transfection, media were collected and cells harvested. Ferritin levels were measured by ELISA. D. Mutant T30I L-ferritin mRNA was transcribed and translated in vitro as in A. and ferritin analyzed by SDS-PAGE and autoradiography. E. Full length L-ferritin-GFP, L-ferritin (WT-GFP), mutants 15AA+GFP (truncated mutant expressing the first 15AA of L-ferritin), and 25AA+GFP (truncated mutant expressing the first 25AA of L-ferritin) were transfected in HEK293T cells. After 24 hours, ferritin localization was assayed by epifluorescence microscopy and media and cells were harvested and ferritin content was analyzed by Western blot using rabbit anti-GFP followed by peroxidase-conjugated goat anti-rabbit IgG and ELISA.

Figure 7. Cytosolic iron chelation enhances ferritin secretion

A. Wild-type (C57/BL6) mouse fibroblasts were incubated with iron (FAC, 10 μM Fe). After 24 hr media was collected and cells were incubated with new media containing iron with or without 100 μM desferasirox for two hr. Ferritin levels in cells and media were analyzed using an ELISA. B. HEK 293T cells were transformed with a plasmid expressing a short-lived YFP under the control of the CMV promoter and cultured for 18 hours. Iron was added to the cells and cell samples taken to assay cellular ferritin by ELISA GFP, tubulin by Western blot analysis and YFP mRNA by RT-PCR. The upper graph represents changes in 5'IRE-YFP and cellular ferritin over time with the amount of IRE-YFP normalized to tubulin at each time point (Arbitrary Units). 5'IRE-YFP mRNA was expressed as % YFP mRNA seen with 100% representing the amount of mRNA seen after 18hrs transfection. C. In low iron conditions IRP-1/2 is bound to the 5'-IRE of ferritin mRNA. Under high iron conditions IRP-1/2 is released from the IRE, IRP-1 is populated with an Fe-S cluster, IRP2 is degraded and ferritin mRNA is translated. As iron is reduced in the cytosol, ferritin is still translated but iron levels remain low and ferritin is secreted through the secretory apparatus prior to the resynthesis of IRP-2.