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Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking

Nature Cell Biology volume 10, pages 776787 (2008) | Download Citation

Abstract

Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome–endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG–Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy — autophagosome formation and maturation — but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.

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References

  1. 1.

    The molecular machinery of autophagy: unanswered questions. J. Cell Sci. 118, 7–18 (2005).

  2. 2.

    Maturation of autophagic vacuoles in mammalian cells. Autophagy 1, 1–10 (2005).

  3. 3.

    & Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev. Cell 6, 463–477 (2004).

  4. 4.

    & Intra-endosomal membrane traffic. Trends Cell Biol. 16, 514–521 (2006).

  5. 5.

    & Rab proteins as membrane organizers. Nature Rev. Mol. Cell Biol. 2, 107–117 (2001).

  6. 6.

    et al. SKD1 AAA ATPase-dependent endosomal transport is involved in autolysosome formation. Cell Struct. Funct. 27, 29–37 (2002).

  7. 7.

    et al. Role of Hrs in maturation of autophagosomes in mammalian cells. Biochem. Biophys. Res. Commun. 360, 721–727 (2007).

  8. 8.

    et al. The VTI family of SNARE proteins is necessary for plant viability and mediates different protein transport pathways. Plant Cell 15, 2885–2899 (2003).

  9. 9.

    et al. Role for Rab7 in maturation of late autophagic vacuoles. J. Cell Sci. 117, 4837–4848 (2004).

  10. 10.

    et al. The apoptosis/autophagy paradox: autophagic vacuolization before apoptotic death. J. Cell Sci. 118, 3091–3102 (2005).

  11. 11.

    & Degradation of microinjected proteins: effects of lysosomotropic agents and inhibitors of autophagy. J. Cell Physiol. 116, 103–110 (1983).

  12. 12.

    et al. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct. Funct. 23, 33–42 (1998).

  13. 13.

    & Autophagic vacuoles rapidly fuse with pre-existing lysosomes in cultured hepatocytes. J. Cell Sci. 102 (Pt 3), 515–526 (1992).

  14. 14.

    , , & The autophagic and endocytic pathways converge at the nascent autophagic vacuoles. J. Cell Biol. 136, 61–70 (1997).

  15. 15.

    & Evidence for fusion between multilamellar endosomes and autophagosomes in HeLa cells. Eur. J. Cell Biol. 72, 307–313 (1997).

  16. 16.

    & Yeast homotypic vacuole fusion: a window on organelle trafficking mechanisms. Annu. Rev. Biochem. 69, 247–275 (2000).

  17. 17.

    , , , & The CORVET tethering complex interacts with the yeast Rab5 homolog Vps21 and is involved in endo-lysosomal biogenesis. Dev. Cell 12, 739–750 (2007).

  18. 18.

    , , & Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion. Mol. Cell 6, 661–671 (2000).

  19. 19.

    & The class C Vps complex functions at multiple stages of the vacuolar transport pathway. Traffic 2, 476–486 (2001).

  20. 20.

    , , & Vam2/Vps41p and Vam6/Vps39p are components of a protein complex on the vacuolar membranes and involved in the vacuolar assembly in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 272, 11344–11349 (1997).

  21. 21.

    , , , & A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion. Proc. Natl Acad. Sci. USA 97, 9402–9407 (2000).

  22. 22.

    , , & Sec17p and HOPS, in distinct SNARE complexes, mediate SNARE complex disruption or assembly for fusion. EMBO J. 24, 1775–1786 (2005).

  23. 23.

    , & New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion. J. Cell Biol. 151, 551–562 (2000).

  24. 24.

    , , & Rab conversion as a mechanism of progression from early to late endosomes. Cell 122, 735–749 (2005).

  25. 25.

    et al. Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules. J. Cell Sci. 118, 3663–3673 (2005).

  26. 26.

    et al. A dual function for Deep orange in programmed autophagy in the Drosophila melanogaster fat body. Exp. Cell Res. 312, 2018–2027 (2006).

  27. 27.

    et al. Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG. Nature Cell Biol. 8, 688–699 (2006).

  28. 28.

    et al. Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis. Nature Cell Biol. 9, 1142–1151 (2007).

  29. 29.

    et al. Identification of mouse Vps16 and biochemical characterization of mammalian class C Vps complex. Biochem. Biophys. Res. Commun. 311, 577–582 (2003).

  30. 30.

    et al. The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nature Cell Biol. 3, 675–678 (2001).

  31. 31.

    et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720–5728 (2000).

  32. 32.

    , , , & Manipulation of nonsense mediated decay identifies gene mutations in colon cancer cells with microsatellite instability. Oncogene 23, 639–645 (2004).

  33. 33.

    , & A putative zinc finger protein, Saccharomyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation. Mol. Cell Biol. 11, 5813–5824 (1991).

  34. 34.

    , , , & Reduced expression of vps11 causes less pigmentation in medaka, Oryzias latipes. Pigment Cell Res. 19, 628–634 (2006).

  35. 35.

    , , , & A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila. Mol. Cell 4, 479–486 (1999).

  36. 36.

    , , & Rab7 is required for the normal progression of the autophagic pathway in mammalian cells. J. Cell Sci. 117, 2687–2697 (2004).

  37. 37.

    , , , & Rab-interacting lysosomal protein (RILP): the Rab7 effector required for transport to lysosomes. EMBO J. 20, 683–693 (2001).

  38. 38.

    , , , & Structural basis for recruitment of RILP by small GTPase Rab7. EMBO J. 24, 1491–1501 (2005).

  39. 39.

    , , & Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep. 2, 330–335 (2001).

  40. 40.

    et al. Molecular characterization of mammalian homologues of class C Vps proteins that interact with syntaxin-7. J. Biol. Chem. 276, 29393–29402 (2001).

  41. 41.

    , , , & Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton. Mol. Biol. Cell 15, 1197–1210 (2004).

  42. 42.

    , & A multispecificity syntaxin homologue, Vam3p, essential for autophagic and biosynthetic protein transport to the vacuole. J. Cell Biol. 138, 517–529 (1997).

  43. 43.

    , , & Ultrastructural analysis of the autophagic process in yeast: detection of autophagosomes and their characterization. J. Cell Biol. 124, 903–913 (1994).

  44. 44.

    , , & Guanine nucleotide exchange on heterotrimeric Gi3 protein controls autophagic sequestration in HT-29 cells. J. Biol Chem. 271, 28593–28600 (1996).

  45. 45.

    et al. Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBO J. 13, 1287–1296 (1994).

  46. 46.

    , & Imaging of endosome fusion in BHK fibroblasts based on a novel fluorimetric avidin-biotin binding assay. Biophys. J. 69, 716–728 (1995).

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Acknowledgements

This work was partly supported by U.S. Public Health Service grants CA82057, CA91819, CA31363, CA106156, RR00168 (J.U.J.) and AI 42999, AI069345 (V.D.). C. L. is a Leukemia & Lymphoma Society Fellow. We thank B. Levine, M.J. Hardwick, S. Virgin, S. Field, T. Yoshimori and Y. Ohsumi for providing reagents, S.W. Richardson for helping with the in vitro endosome fusion assay, S. Gygi for mass spectrometry analysis and Michelle Connole for FACS analysis. Finally, we thank all lab members for their support and discussions.

Author information

Affiliations

  1. Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA 90033, USA.

    • Chengyu Liang
    • , Jong-soo Lee
    • , Kyung-Soo Inn
    • , Michaela U. Gack
    •  & Jae U. Jung
  2. Department of Microbiology and Molecular Genetics and Tumor Virology Division, New England Primate Research Center, Harvard Medical School, 1 Pine Hill Drive, Southborough, MA 01772, USA.

    • Chengyu Liang
    • , Jong-soo Lee
    • , Kyung-Soo Inn
    • , Michaela U. Gack
    • , Qinglin Li
    •  & Jae U. Jung
  3. Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.

    • Esteban A. Roberts
    • , Isabelle Vergne
    •  & Vojo Deretic
  4. University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.

    • Pinghui Feng
  5. Department of Biophysics and Biochemistry, Graduate School of Health Sciences, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-Ku, Tokyo 113-8519, Japan.

    • Chihiro Akazawa

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Contributions

C.L. performed all aspects of this study; L.S., K.I., M.G., Q.L. and P.F. assisted with the experimental design and in collecting the data; E.R., I.V. and V.D. assisted with the autophagic protein degradation and in vitro endosome fusion assay; C.A. provided Vps constructs and their antibodies; C.L. and J.J. organized this study and wrote the paper. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jae U. Jung.

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DOI

https://doi.org/10.1038/ncb1740

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