Abstract
Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4−/− mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.
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Acknowledgements
We would like to thank E. George, A. Donovan, K. Mansfield, L. Klickstein, D. Glass, R. Xavier and M. Fishman for their input and advice.
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W.E.D., B.N., E.T., S.L., Z.W., S.D’A., H.W., D.W., J.K., P.B., S.U. and L.O.M. performed cell biology experiments. J.N., J.T., D.S., and M.S. performed proteomic experiments. R.A.E., J.C., C.L., D.E.B. and M.S.K. performed protein crystallography studies and analysed the co-structural data. P.F., M.E.D. and F.H., performed high-throughput screens, I.C-T., E.H., A.H. and E.P.K. performed medicinal chemistry experiments. W.E.D., H.L., Q.F. and T.N. generated mice. W.E.D., B.N., R.A.V. and L.O.M. analysed mouse data and provided interpretation. B.N., I.C-T., S.B.H., J.B., J.T., C.J.W., V.E.M., J.A.P., D.B., P.M.F., M.A.L., X.M., L.G.H., B.D.M., T.N., M.S., K.M.K., E.P.K. and L.O.M. provided supervision. W.E.D., B.N. and L.O.M. wrote the manuscript with input from other co-authors. L.O.M. devised the concept and supervised the project.
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Supplementary Figure 1 PIK-III is a selective inhibitor of VPS34 in vitro and in mammalian cells.
(a) U2OS cells expressing GFP-FYVE were incubated with the indicated compounds (2 h), fixed, stained with Hoechst 33342 and imaged by automated epifluorescence microscopy. Representative images of GFP-FYVE are shown and scale bars are equivalent to 10 μm. GFP-positive puncta/cell were determined using an image analysis algorithm and these data were then used to determine IC50 values for each compound as shown in Fig. 1c. (b) U2OS cells expressing GFP-FOXO3A were incubated with the indicated compounds (2 h), fixed, stained with Hoechst 33342 and imaged by automated epifluorescence microscopy. Representative images of GFP-FOXO3A (green) and Hoechst 33342 nuclear staining (blue) are shown and scale bars are equivalent to 10 μm. The ratio of nuclear to cellular GFP fluorescence intensity was determined using an image analysis algorithm and these data were then used to determine IC50 values for each compound as shown in Fig. 1d.
Supplementary Figure 2 Validation of ATG7 conditional knockout DLD1 cells.
ATG7−/− DLD1 cells harbour a conditional knockout of ATG7, with exon 15 of the first allele being disrupted and exon 15 of the second allele being flanked with two loxP sites (see Experimental Procedures). ATG7+/+, ATG7−/+ or two ATG7flox/− DLD1 cell clones were infected with adenoviral-Cre recombinase (+ Adeno-Cre), lysed, and the levels of ATG7 and ATG5 evaluated using western blotting. Note the complete loss of ATG7 in the ATG7−/− clones treated with Adeno-Cre which results in full inhibition of ATG5-ATG12 conjugation. Cells treated with Adeno-Cre were used to generate data shown in Fig. 4d.
Supplementary Figure 3 The effect of autophagy disruption on NCOA4 mRNA.
(a) Total RNA was isolated from cells treated as in Fig. 4C and the relative levels of NCOA4 mRNA determined using quantitative RT-PCR. (b) Total RNA was isolated from cells treated as in Fig 4d and relative levels of NCOA4 and ATG7 mRNA determined using quantitative RT-PCR. Three different PCR probes (11/12, 14/15 and 15/16) were used to amplify ATG7 mRNA with the latter two flanking the targeted exon 15. (c) Total RNA was isolated from cells treated as in Fig. 4e and relative levels of NCOA4 mRNA determined using quantitative RT-PCR. Values are reported are two independent repeats from qPCR performed in quadruplicate.
Supplementary Figure 4 FTH1 and FTL interact with SBP-NCOA4 in DLD1 cells.
Steptavidin agarose beads were incubated with protein extracts from control or SBP-NCOA4-expressing DLD1 cells, bound proteins were eluted and analysed by LC-MS. Proteins enriched in the SBP-NCOA4 over control sample were determined as described in the methods and log10 fold ratios and adjusted P-Values are shown on the y and x axis, respectively. Note the robust enrichment of NCOA4, FTH1, FTL and TMSB10. The complete dataset is shown in Supplementary Table 3. P-values were calculated using a one-way T-test (arbitrarily set to 1 for non-significant single peptide quantitations) and adjusted using the Benjamini-Hochberg False Discovery Rate.
Supplementary Figure 5 Starvation-induced degradation of NCOA4, FTH1 and FTL does not require p62.
DLD1 cells transfected with the indicated siRNAs were left in rich medium (Fed) or nutrient starved (Starve) for the indicated times before cell lysis and western blot analysis.
Supplementary Figure 6 NCOA4 interacts specifically with FTH1.
(a) Endogenous NCOA4 was immunoprecipitated from FTH1−/− cells transfected with empty vector, N-SBP-FTH1 or N-SBP-FTL and the presence of FTH1 and FTL in the immunoprecipitates was determined by western blot. (b) Endogenous NCOA4 was immunoprecipitated from FTH1−/−/FTL−/− double knockout cells transfected with empty vector, N-SBP-FTH1 or N-SBP-FTL and the presence of FTH1 and FTL in the immunoprecipitates was determined by western blot. A specific interaction between NCOA4 and FTH1 only was detected. Asterisk indicates IgG band and arrow highlights location of FTL band.
Supplementary Figure 7 Uncropped scans of key western blots.
Area shown in figures is outlined in red and molecular weight markers are shown on the left.
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Dowdle, W., Nyfeler, B., Nagel, J. et al. Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo. Nat Cell Biol 16, 1069–1079 (2014). https://doi.org/10.1038/ncb3053
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DOI: https://doi.org/10.1038/ncb3053
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