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Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1–phosphatidylinositol-3-kinase complex

Nature Cell Biology volume 11, pages 468–476 (2009)Cite this article

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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Figure 1: Identification of Beclin 1-interaction proteins from Becn1−/−;Becn1–EGFP/+ mice.
Figure 2: Atg14L positively regulates autophagy, and Beclin 1 and Atg14L synergistically promote double-membrane formation.
Figure 3: Rubicon is a negative regulator of autophagy.
Figure 4: Overexpressing Rubicon causes aberrant expansion of late endosomes/lysosomes.
Figure 5: Overexpressed Rubicon is localized on PI(3)P-enriched structures in a Beclin 1-independent manner.

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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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Author notes

  1. Yun Zhong and Qing Jun Wang: These authors contributed equally to the work.

Authors and Affiliations

  1. Departments of Neurology and Neuroscience, Mount Sinai School of Medicine, New York, 10029, NY, USA

    Qing Jun Wang, Xianting Li & Zhenyu Yue

  2. Laboratory of Molecular Biology, Howard Hughes Medical Institute, Rockefeller University, New York, 10065, NY, USA

    Yun Zhong & Nathaniel Heintz

  3. Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, 10065, NY, USA

    Qing Jun Wang & Brian T. Chait

  4. Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, 10461, NY, USA

    Ying Yan & Jonathan M. Backer

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  1. Yun Zhong
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Contributions

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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Correspondence to Zhenyu Yue.

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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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