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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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Affiliations

  1. Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA

    • William E. Dowdle
    • , Jane Nagel
    • , Shanming Liu
    • , Ellen Triantafellow
    • , Suchithra Menon
    • , Zuncai Wang
    • , Ayako Honda
    • , Ivan Cornella-Taracido
    • , Edmund Harrington
    • , Peter Fekkes
    • , Hong Lei
    • , Qing Fang
    • , Mary Ellen Digan
    • , Debra Burdick
    • , Andrew F. Powers
    • , Henry Wang
    • , Dmitri Wiederschain
    • , Jenny Kuerth
    • , Philip Bergman
    • , David Schwalb
    • , Jason Thomas
    • , Savuth Ugwonali
    • , Fred Harbinski
    • , John Tallarico
    • , Christopher J. Wilson
    • , Vic E. Myer
    • , Jeffery A. Porter
    • , Peter M. Finan
    • , Mark A. Labow
    • , Xiaohong Mao
    • , Lawrence G. Hamann
    • , Reginald A. Valdez
    • , Thomas Nicholson
    • , Markus Schirle
    • , Erin P. Keaney
    •  & Leon O. Murphy
  2. Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland

    • Beat Nyfeler
    • , Stephen B. Helliwell
    • , Simon D’Aquin
    •  & Julie Bastien
  3. Novartis Institutes for BioMedical Research, 4560 Horton Street Emeryville, California 94608, USA

    • Robert A. Elling
    • , Gwynn Pardee
    • , John Cantwell
    • , Catherine Luu
    • , Dirksen E. Bussiere
    •  & Mark S. Knapp
  4. Department of Genetics and Complex Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115, USA

    • Brendan D. Manning

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Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Leon O. Murphy.

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https://doi.org/10.1038/ncb3053

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