Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis
- Harvard Medical School, United States
- Dana-Farber Cancer Institute, United States
- Beth Israel Deaconess Medical Center, United States
- Brigham and Women's Hospital, United States
- Harvard Stem Cell Institute, United States
- Broad Institute of MIT and Harvard, United States
Figures
Figure 1 with 1 supplement
NCOA4 recognizes FTH1 via a conserved C-terminal domain.
(A) Schematic of NCOA4 truncation constructs. (B) Horse spleen apoferritin was mixed with in vitro translated MYC-NCOA4 fragments, immunoprecipitated with anti-MYC (MYC-IP) and immunoblotted with …
Figure 1—figure supplement 1
NCOA4 interacts with ferritin via a C-terminal domain and promotes lysosomal ferritin accumulation upon iron depletion.
(A) GST pulldown assay of recombinant NCOA4-GST fragments as indicated mixed with ferritin. Bound ferritin was analyzed by immunoblotting with FTH1 antibody. Load is 5% of ferritin input. NCOA4-GST …
Figure 2 with 1 supplement
FTH1R23 is essential for NCOA4 binding and ferritinophagy.
(A) GST pulldown assay of recombinant NCOA4383−522-GST mixed with recombinant FTH1-only or FTL-only complexes. Bound FTH1 or FTL was analyzed by 4–20% SDS-PAGE and Coomassie blue staining. Load is …
Figure 2—figure supplement 1
FTH1 R23A forms functional ferritin cages and abrogates NCOA4 binding.
(A) Size exclusion chromatogram of recombinant wild-type FTH1-only complexes with main peak noted at approximately 14 ml corresponding to an apparent 450 kDa molecular weight. (B) Size exclusion …
Figure 3 with 1 supplement
Iron-dependent NCOA4 turnover via the HERC2 E3 Ubiquitin Ligase.
(A) Endogenous IP of NCOA4 from 293T cells treated with DFO or FAC for 6 hr. Lysate input for control IgG IP is the untreated sample. HERC2 and NCOA4 binding was evaluated by immunoblot. (B) …
Figure 3—figure supplement 1
HERC2 regulates NCOA4 turnover.
(A) FLAG affinity purification of stably expressed NCOA4-FLAG-HA (CTAP) from 293T cells treated as indicated for 9 hr (100 μM DFO, 30 μM DFX (Deferasirox), 0.05 mg/ml FAC, biological replicates of …
Figure 4 with 1 supplement
NCOA4 is an iron-binding protein.
(A) GST pulldown assay of recombinant NCOA4-GST fragments as indicated mixed with recombinant HERC22540−2700. Bound HERC2 was analyzed by 4–20% SDS-PAGE and Coomassie blue staining. Load is 5% of …
Figure 4—figure supplement 1
Evaluating NCOA4 iron binding.
(A) NCOA4383−509 and HERC22553−2639 were expressed in E. coli and the purified protein solution is depicted with NCOA4383−509 solution having a brown color suggestive of an iron-containing protein. …
Figure 5
Knockdown of ncoa4 impacts erythropoiesis in zebrafish and tissue culture cells.
(A, B) Expression of HGBG (A), HGBA (B) determined by quantitative RT-PCR in K562 cells expressing a control (shGFP) or two independent shRNAs targeting NCOA4. Hemin treatment (25 μM, 72 hr) as …
Figure 6
Iron Levels Regulate NCOA4-mediated ferritinophagy.
A model of NCOA4 ferritinophagy regulation (see the text for details).
