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Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy

Nature volume 509, pages 105–109 (2014)Cite this article

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Abstract

Autophagy, the process by which proteins and organelles are sequestered in double-membrane structures called autophagosomes and delivered to lysosomes for degradation, is critical in diseases such as cancer and neurodegeneration1,2. Much of our understanding of this process has emerged from analysis of bulk cytoplasmic autophagy, but our understanding of how specific cargo, including organelles, proteins or intracellular pathogens, are targeted for selective autophagy is limited3. Here we use quantitative proteomics to identify a cohort of novel and known autophagosome-enriched proteins in human cells, including cargo receptors. Like known cargo receptors, nuclear receptor coactivator 4 (NCOA4) was highly enriched in autophagosomes, and associated with ATG8 proteins that recruit cargo–receptor complexes into autophagosomes. Unbiased identification of NCOA4-associated proteins revealed ferritin heavy and light chains, components of an iron-filled cage structure that protects cells from reactive iron species4 but is degraded via autophagy to release iron5,6 through an unknown mechanism. We found that delivery of ferritin to lysosomes required NCOA4, and an inability of NCOA4-deficient cells to degrade ferritin led to decreased bioavailable intracellular iron. This work identifies NCOA4 as a selective cargo receptor for autophagic turnover of ferritin (ferritinophagy), which is critical for iron homeostasis, and provides a resource for further dissection of autophagosomal cargo–receptor connectivity.

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Figure 1: Quantitative proteomics for identification of autophagosome-associated proteins.
Figure 2: NCOA4 associates with and co-localizes with ferritin.
Figure 3: NCOA4 mediates autophagic delivery of ferritin to lysosomes to control iron homeostasis.

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Processed proteomics data are available in Supplementary Information; raw data files have been deposited at Peptide Atlas under accession number PASS00440.

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Acknowledgements

We thank R. Everley and E. Huttlin for assistance with LC–MS/MS, A. Cuervo for assistance with autophagosome purification protocol development, Y. Liu for technical support with cell culture, A. White, J. Lydeard and S. Hayes for interaction proteomics support, M. Haigis for access to nitrogen cavitation equipment, and the Nikon Imaging and Electron Microscopy Centers (Harvard Medical School) for imaging support. We thank Millennium Pharmaceuticals for the GABARAPL2 antibody. We acknowledge support from National Institutes of Health grants GM070565 and GM095567 to J.W.H., and National Cancer Institute grant R01CA157490, American Cancer Society Research Scholar grant RSG-13-298-01-TBG and the Lustgarten Foundation to A.C.K. J.D.M. was supported by an American Board of Radiology Holman Research Pathway Post-doctoral Fellowship and an American Society of Radiation Oncology Junior Faculty Career Research Training Award (JF2013-2). J.W.H. is a consultant for Millennium: The Takeda Oncology Company and Biogen Idec. A.C.K. is a consultant for Forma Therapeutics.

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Authors and Affiliations

  1. Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, 02215, Massachusetts, USA

    Joseph D. Mancias, Xiaoxu Wang & Alec C. Kimmelman

  2. Department of Cell Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Joseph D. Mancias, Steven P. Gygi & J. Wade Harper

  3. Harvard Radiation Oncology Program, Boston, 02115, Massachusetts, USA

    Joseph D. Mancias

  4. Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, 02215, Massachusetts, USA

    Joseph D. Mancias

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  1. Joseph D. Mancias
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Contributions

J.D.M., J.W.H. and A.C.K. conceived the experiments. J.D.M. performed all experiments. X.W. assisted with cell line generation. S.P.G. provided proteomics software and analysis support. J.D.M., J.W.H. and A.C.K. analysed data and wrote the manuscript. All authors edited the manuscript.

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Correspondence to J. Wade Harper or Alec C. Kimmelman.

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Extended data figures and tables

Extended Data Figure 1 Autophagosome enrichment protocol validation.

a, Overlap of proteins identified in three prior autophagosome proteomics studies11,12,13 as evaluated by area-proportional Venn diagram. Proteins overlapping between data sets are noted. b, PANC-1 cells stably expressing GFP–LC3B have a high level of basal autophagy (left). One hour of wortmannin (Wort; 200 nM) treatment blocks autophagosome formation (middle) and 4 h of chloroquine treatment (CQ; 25 μM) causes accumulation of autophagosomes (right). c, Lysed PA-TU-8988T cells mixed with Nycodenz and placed at the bottom of a discontinuous density gradient with Nycodenz layers at the indicated concentrations (left). After 3 h centrifugation at the indicated speed, four bands appear at the indicated interfaces with enrichment of the indicated organelles in each interface (right). d, PA-TU-8988T cells treated with either wortmannin (1 h, 200 nM) or chloroquine (4 h, 25 μM) subjected to gradient centrifugation. A decreased amount of material is recovered from the A1 (autophagosome) interface due to the effect of wortmannin on autophagosome formation. e, Fluorescence microscopy of gradient load (LD) and autophagosome fraction (A1) from PA-TU-8988T cells stably expressing GFP–LC3B after either chloroquine or wortmannin treatment. f, Fluorescence microscopy of indicated fractions from density gradient of PA-TU-8988T cells stably expressing GFP–LC3B treated with chloroquine (A1 fraction image is also presented in e and gradient picture is also presented in c). g, PANC-1 autophagosome fractions analysed by immunoblotting using antibodies to LAMP2, VDAC1 and LC3B. LD is gradient load, A1 is autophagosome fraction from 15–20% nycodenz interface, A2 is the autophagolysosome fraction from the 20–24% nycodenz interface, L is the lysosome fraction from the 24–26% nycodenz interface, M is the mitochondrial fraction from the 26–50% nycodenz interface. h, PA-TU-8988T autophagosome fractions analysed as in g. i, PA-TU-8988T autophagosomes (A1 fraction) were incubated at 37 °C for 1 h ± Triton X-100 and centrifuged at high speed. The resulting pellet was resuspended in equal volume to supernatant and assayed by immunoblotting with antibodies to SQSTM1 and LC3B. j, Pearson correlation plot for overlapping candidates from MCF7 experiments (102 proteins, comparing Ex. 1 with Ex. 2). k, Electron micrographs of PA-TU-8988T gradient load (LD, left) and PA-TU-8988T autophagosome fraction (A1, right) at ×6,800 magnification. Scale bar, 500 nm. l, PA-TU-8988T gradient load (LD, left) and PA-TU-8988T autophagosome fraction (A1, right) at ×18,500 magnification. Scale bar, 500 nm. m, PA-TU-8988T autophagosome fraction (A1) at ×23,000 magnification. Scale bar, 100 nm. Arrowhead indicates double-membrane autophagosome, arrow indicates fused autophagolysosome.

Extended Data Figure 2 Autophagosome proteomics candidate list and validation in PA-TU-8988T cells.

a, Autophagosome proteomics class 1A candidate list. b, Overlap between class 1 candidates (MCF7 and PANC-1) and candidates from 4 h chloroquine SILAC PA-TU-8988T gradient autophagosome purification experiment. c, Data from PA-TU-8988T SILAC gradient autophagosome for class 1A candidates (and FTH1; see also Supplementary Table 6). d, Heat map of class 1A candidates (and FTH1) comparing PANC-1 4 h chloroquine and PA-TU-8988T 4 h chloroquine experiments.

Extended Data Figure 3 Candidates from autophagosome proteomics co-localize in autophagosomes.

Representative confocal images of PA-TU-8988T and U2OS cells expressing HA-tagged candidates after chloroquine treatment. Co-localization of HA-tagged candidates (green) with endogenous LC3B (red). Representative co-localization is marked by white arrows. Scale bar, 10 μm. MGRN1 is included as an example of one of the candidates that did not show co-localization.

Extended Data Figure 4 Immunopurification-based autophagosome proteomics.

a, Schematic for GFP immunoisolation of GFP–LC3B-labelled autophagosomes from PA-TU-8988T cells. b, Data from GFP immunoisolation for class 1A candidates (see also Supplementary Table 7).

Extended Data Figure 5 Comparative analysis of class 1 and 2 proteins with Dengjel et al. autophagosome proteomics.

a, Analysis and comparison of Dengjel et al.13 data and candidate list with data derived from MCF7 autophagosome proteomics experiments as detailed in Methods (see also Supplementary Table 8). Orange shading of gene symbols denotes proteins identified as MCF7 candidates. b–e, Analysis and comparison of Dengjel et al. data and candidate list with data derived from both MCF7 and PANC-1 autophagosome proteomics experiments (class 1 and 2 proteins) as detailed in Methods. Orange shading of gene symbols denotes proteins identified as class 1 or 2 candidates (see also Supplementary Table 8).

Extended Data Figure 6 NCOA4 co-localizes with LC3B and GABARAPL2 in autophagosomes.

a, GFP–NCOA4 (green) co-localizes with mCherry–LC3B (red) in chloroquine-treated cells. Scale bar, 20 μm. b, GFP–NCOA4 (green) co-localizes with mCherry–GABARAPL2 (red) in chloroquine-treated cells. Scale bar, 20 μm. c, GFP–NCOA4 (green) co-localizes with endogenous GABARAPL2 (red) in chloroquine-treated cells. Scale bar, 20 μm. d, GFP–NCOA4 (green) does not co-localize with endogenous mannose 6-phosphate receptor (M6PR) (red) in chloroquine-treated cells. Scale bar, 20 μm. e, HERC2 does not co-localize in autophagosomes. Immunostaining of U2OS cells subjected to chloroquine treatment, endogenous LCB3 (green) and endogenous HERC2 (red). Scale bar, 20 μm.

Extended Data Figure 7 Ferritin undergoes primarily lysosomal mediated degradation.

a, PA-TU-8988T cells were cultured with FAC for 24 h, washed, followed by chelation with three chelators (DFO, BPS, DFP) plus or minus lysosomal protease inhibitors (E-64d and pepstatin A) or proteasomal inhibitor, bortezomib (lane 5) for 8 h. Lysates were immunoblotted using antibodies to ACTB and FTH1. b, U2OS cells were cultured with FAC for 24 h, washed, followed by chelation with two chelators (DFO, DFX) plus or minus lysosomal protease inhibitors (E-64d and pepstatin A) and analysed as in a. c, PA-TU-cells transfected with luciferase control siRNA or validated siRNA to ATG5 were cultured with FAC, washed and subjected to DFO chelation for 9 h. Lysates were immunoblotted using antibodies to FTH1, ATG5 and ACTB. d, RNAi-mediated knockdown of NCOA4 in PA-TU-8988T cells. PA-TU-8988T cells stably expressing a control shRNA (shGFP) and two independent shRNAs to NCOA4 (shNCOA4-1 and shNCOA4-2) were lysed and analysed by immunoblotting with two different antibodies to NCOA4 and ACTB as a loading control. Middle panel shows immunoblot probed with NCOA4 antibody from Bethyl Laboratories (#A302-272A). A non-specific band migrates just below the NCOA4-specific band. Top panel shows immunoblot probed with NCOA4 antibody from Sigma (SAB1404569). e, U2OS cells stably expressing shGFP, shNCOA4-1 or shNCOA4-2 were analysed by immunoblotting as in d. f, NCOA4 depletion rescues ferritin degradation upon 9 h iron chelation in U2OS cells. Relative FTH1 levels (n = 3) for each chelator are quantified. Bars and error bars represent mean values and s.d., respectively. **P < 0.01 and *P < 0.02 comparing FTH1 levels between different cell lines to shGFP control (one-sided t-test). g, U2OS cells stably expressing shGFP, shNCOA4-1 or shNCOA4-2 were cultured with FAC for 24 h, washed, and subjected to DFO chelation plus or minus lysosomal protease inhibitors. Lysates were immunoblotted using antibodies to NCOA4, FTH1 and ACTB.

Extended Data Figure 8 NCOA4 mediates autophagic delivery of ferritin to lysosomes.

a, PA-TU-8988T cells were cultured in the presence or absence of FAC for 24 h, washed, and subjected to DFO chelation plus or minus lysosomal protease inhibitors (E-64d and pepstatin A). Cells were fixed and immunostained using antibodies to ferritin (red) and LAMP2 (green). Higher magnification views of the boxed areas are shown in the insets. Scale bar, 10 μm. b, U2OS cells treated and analysed as in a. c, Immunostaining of U2OS cells expressing a control shRNA (shGFP) and two independent shRNAs to NCOA4 (shNCOA4-1 and shNCOA4-2) subjected to DFO chelation in the presence of lysosomal protease inhibitors for 9 h. Scale bar, 10 μm. Punctate ferritin fraction was quantified from >75 cells per cell line from two independent experiments (number of U2OS cells quantified is as follows: shGFP, 133 cells; shNCOA4-1, 103 cells; and shNCOA4-2, 79 cells). Bars and error bars represent mean values and s.d., respectively. ***P < 0.001 using a one-sided t-test. d, IMR-90 cells were treated and immunostained as in c. Scale bar, 50 μm. Punctate ferritin fraction was quantified from >25 cells per cell line in two independent experiments and from more than ten microscopy fields (number of IMR-90 cells quantified is as follows: shGFP, 29 cells; shNCOA4-1, 26 cells; and shNCOA4-2, 31 cells). Bars and error bars represent mean values and s.d., respectively. ***P < 0.001 using a one-sided t-test. e, HPDE cells were treated and immunostained as in c. Scale bar, 10 μm. Quantification was not possible due to the high background signal in shGFP control cells.

Extended Data Figure 9 A non-degradable murine NCOA4 rescues RNAi-mediated NCOA4 knockdown.

a, PA-TU-8988T cells stably expressing either a control MSCV empty vector or the murine homologue of NCOA4 (selected with blasticidin) as well as stably expressing shRNAs (shGFP, shNCOA4-1, shNCOA4-2, selected with puromycin) were cultured in the presence or absence of FAC for 24 h, washed, and subjected to DFO chelation in the presence of lysosomal protease inhibitors (E-64d and pepstatin A). Cells were fixed and immunostained using antibodies to ferritin (red) and LAMP2 (green). Scale bar, 10 μm. Punctate ferritin fraction was quantified from ≥100 cells per cell line (number of PA-TU-8988T cells quantified is as follows: PA-TU-8988T-control MSCV-shGFP, 100 cells; PA-TU-8988T-control-MSCV-shNCOA4-1, 125 cells; PA-TU-8988T-control-MSCV-shNCOA4-2, 132 cells; PA-TU-8988T-mouse-NCOA4-shGFP, 151 cells; PA-TU-8988T-mouse-NCOA4-shNCOA4-1, 153 cells; and PA-TU-8988T-mouse-NCOA4-shNCOA4-2, 172 cells). Bars and error bars represent mean values and s.d., respectively. ***P < 0.001 using a one-sided t-test. b, RNAi-mediated knockdown of NCOA4 in PA-TU-8988T cells. PA-TU-8988T cells stably expressing either a control MSCV empty vector or the murine homologue of NCOA4 (selected with blasticidin) as well as stably expressing a control shRNA (shGFP) and two independent shRNAs to NCOA4 (shNCOA4-1 and shNCOA4-2) were lysed and analysed by immunoblotting with an antibody to NCOA4 and ACTB as a loading control. Light and dark exposures are shown. A non-specific band migrates just below the NCOA4-specific band. c, Expression of murine HA–NCOA4 protein in PA-TU-8988T cells. Cell lysates were probed with HA antibody.

Extended Data Figure 10 NCOA4 mediates autophagic delivery of ferritin to lysosomes to control iron homeostasis.

a, Immunostaining of PA-TU-8988T cells transfected with luciferase control siRNA or two independent siRNAs to NCOA4 and subjected to DFO chelation in the presence of lysosomal protease inhibitors for 9 h. Scale bar, 20 μm. b, Immunostaining of PA-TU-8988T cells transfected with luciferase control siRNA or two independent siRNAs to HERC2 and subjected to DFO chelation in the presence of lysosomal protease inhibitors for 9 h. Scale bar, 20 μm. c, PA-TU-8988T cells were transfected with luciferase control siRNA or two independent siRNAs to HERC2. Lysates were immunoblotted using antibodies to HERC2 and ACTB. d, siRNA-mediated knockdown of NCOA4 in U2OS, IMR-90 and PA-TU-8988T cells leads to increases in IRP2, FTH1 and TFRC levels. Cells were transfected with luciferase control siRNA or two independent siRNAs to NCOA4. Lysates were immunoblotted using antibodies to NCOA4, IRP2, TFRC, FTH1 and ACTB. Light and dark exposures are shown for TFRC. e, NCOA4 expression levels are affected by iron levels and NCOA4 levels affect ferritin levels in response to iron load. PA-TU-8988T cells stably expressing either a control MSCV empty vector or mouse NCOA4 were cultured in the presence of FAC for the indicated times. Cells were lysed and analysed by immunoblotting with antibodies to NCOA4, FTH1 and ACTB as a loading control. A non-specific band migrates just below the NCOA4-specific band.

Supplementary information

Supplementary Table 1

This file contains the autophagosome proteomics dataset key. (XLSX 38 kb)

Supplementary Table 2

This file contains the PANC1 4 hr CQ experiment SILAC profiles. (XLSX 354 kb)

Supplementary Table 3

This file contains the PANC1 experiments 1-3 SILAC profiles. (XLSX 1304 kb)

Supplementary Table 4

This file contains the MCF7 experiments 1-2 SILAC profiles. (XLSX 883 kb)

Supplementary Table 5

This file contains the PANC1 and MCF7 overlap and Class 1 and 2 Candidate List. (XLSX 48 kb)

Supplementary Table 6

This file contains the 8988T 4 hr CQ experiment SILAC profiles. (XLSX 377 kb)

Supplementary Table 7

This file contains the 8988T GFP-LC3B immunoisolation semi-quantitative comparison. (XLSX 417 kb)

Supplementary Table 8

This file contains the comparison of MCF7 and PANC1 experiments with candidate lists from Dengjel et al. (XLSX 176 kb)

Supplementary Table 9

This file contains the NCOA4 AP-MS data from 3 cell lines, FTH1 AP-MS from 293T. (XLSX 256 kb)

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Mancias, J., Wang, X., Gygi, S. et al. Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature 509, 105–109 (2014). https://doi.org/10.1038/nature13148

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