Abstract
Beclin 1, a mammalian autophagy protein that has been implicated in development, tumour suppression, neurodegeneration and cell death, exists in a complex with Vps34, the class III phosphatidylinositol-3-kinase (PI(3)K) that mediates multiple vesicle-trafficking processes including endocytosis and autophagy. However, the precise role of the Beclin 1–Vps34 complex in autophagy regulation remains to be elucidated. Combining mouse genetics and biochemistry, we have identified a large in vivo Beclin 1 complex containing the known proteins Vps34, p150/Vps15 and UVRAG, as well as two newly identified proteins, Atg14L (yeast Atg14-like) and Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein). Characterization of the new proteins revealed that Atg14L enhances Vps34 lipid kinase activity and upregulates autophagy, whereas Rubicon reduces Vps34 activity and downregulates autophagy. We show that Beclin 1 and Atg14L synergistically promote the formation of double-membraned organelles that are associated with Atg5 and Atg12, whereas forced expression of Rubicon results in aberrant late endosomal/lysosomal structures and impaired autophagosome maturation. We hypothesize that by forming distinct protein complexes, Beclin 1 and its binding proteins orchestrate the precise function of the class III PI(3)K in regulating autophagy at multiple steps.
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Acknowledgements
This work was supported by National Institutes of Health Grants RNS055683A (Z.Y.), RR00862 and RR022220 (B.T.C.) and by the Howard Hughes Medical Institute (N.H.). We thank A. Tolkovsky for GFP–LC3 HeLa cells, C. Münz for GFP–LC3 MLE12 cells, T. Johansen for mCherry–EGFP–LC3 plasmid and X. Jiang for EGFP–Atg12 and EGFP–Atg5 constructs. We thank W. Yang for help with cell culture and generation of stable cells, and H. Shio, E. Sphicas and A. North in the Bio-Imaging Resource Center at The Rockefeller University for help with microscopy.
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Z.Y. and Q.J.W. conceived the project. Z.Y. coordinated all efforts in the study; Z.Y., Q.J.W., Y.Z., N.H. and B.T.C. planned the project; Y.Z. and Q.J.W. performed most of the assays; X.L. assisted with p40 (phox)-PX–EGFP localization and Vps34 kinase analyses; Y.Y. and J.M.B. developed the Myc–Vps34–Vps15 constructs and Vps34 kinase assay protocol; Z.Y., Q.J.W. and Y.Z. wrote the paper.
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Zhong, Y., Wang, Q., Li, X. et al. Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1–phosphatidylinositol-3-kinase complex. Nat Cell Biol 11, 468–476 (2009). https://doi.org/10.1038/ncb1854
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DOI: https://doi.org/10.1038/ncb1854
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