Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation (original) (raw)

Nature Cell Biology volume 14, pages 924–934 (2012)Cite this article

Subjects

Abstract

Autophagy is a lysosome-based degradation pathway. During autophagy, lysosomes fuse with autophagosomes to form autolysosomes. Following starvation-induced autophagy, nascent lysosomes are formed from autolysosomal membranes through an evolutionarily conserved cellular process, autophagic lysosome reformation (ALR), which is critical for maintaining lysosome homeostasis. Here we report that clathrin and phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) regulate ALR. Combining a screen of candidates identified through proteomic analysis of purified ALR tubules, and large-scale RNAi knockdown, we unveiled a tightly regulated molecular pathway that controls lysosome homeostasis, in which clathrin and PtdIns(4,5)P2 are the central components. Our functional study demonstrates the central role of clathrin and its associated proteins in cargo sorting, phospholipid conversion, initiation of autolysosome tubulation, and proto-lysosome budding during ALR. Our data not only uncover a molecular pathway by which lysosome homeostasis is maintained through the ALR process, but also reveal unexpected functions of clathrin and PtdIns(4,5)P2 in lysosome homeostasis.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 12 print issues and online access

$209.00 per year

only $17.42 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

References

  1. Klionsky, D. J. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat. Rev. Mol. Cell Biol. 8, 931–937 (2007).
    Article CAS Google Scholar
  2. Mizushima, N. Autophagy: process and function. Genes Dev. 21, 2861–2873 (2007).
    Article CAS Google Scholar
  3. Kraft, C. & Martens, S. Mechanisms and regulation of autophagosome formation. Curr. Opin. Cell Biol.http://dx.doi.org/10.1016/j.ceb.2012.05.001 (2012).
  4. Klionsky, D. J. & Emr, S. D. Autophagy as a regulated pathway of cellular degradation. Science 290, 1717–1721 (2000).
    Article CAS Google Scholar
  5. Yu, L. et al. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 465, 942–946 (2010).
    Article CAS Google Scholar
  6. Rong, Y. et al. Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvation. Proc. Natl Acad. Sci. USA 108, 7826 (2011).
    Article CAS Google Scholar
  7. Zhang, H. et al. Genome-wide functional screening of miR-23b asa pleiotropic modulator suppressing cancer metastasis. Nat. Commun. 2, 554 (2011).
    Article Google Scholar
  8. Pawlowski, N. Dynamin self-assembly and the vesicle scission mechanism: how dynamin oligomers cleave the membrane neck of clathrin-coated pits during endocytosis. Bioessays 32, 1033–1039 (2010).
    Article CAS Google Scholar
  9. Kirchhausen, T. Clathrin. Annu. Rev. Biochem. 69, 699–727 (2000).
    Article CAS Google Scholar
  10. Mettlen, M., Loerke, D., Yarar, D., Danuser, G. & Schmid, S. L. Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis. J. Cell Biol. 188, 919–933 (2010).
    Article CAS Google Scholar
  11. Raiborg, C. et al. Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes. Nat. Cell Biol. 4, 394–398 (2002).
    Article CAS Google Scholar
  12. Popoff, V. et al. The retromer complex and clathrin define an early endosomal retrograde exit site. J. Cell Sci. 120, 2022–2031 (2007).
    Article CAS Google Scholar
  13. Shi, A. et al. Regulation of endosomal clathrin and retromer-mediated endosome to Golgi retrograde transport by the J-domain protein RME-8. EMBO J. 28, 3290–3302 (2009).
    Article CAS Google Scholar
  14. Di Paolo, G. & De Camilli, P. Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657 (2006).
    Article CAS Google Scholar
  15. Divecha, N. & Irvine, R. F. Phospholipid signaling. Cell 80, 269–278 (1995).
    Article CAS Google Scholar
  16. Funakoshi, Y., Hasegawa, H. & Kanaho, Y. Activation mechanisms of PIP5K isozymes by the small GTPase ARF6. Adv. Enzyme Regul. 50, 72–80 (2010).
    Article Google Scholar
  17. van den Bout, I. & Divecha, N. PIP5K-driven PtdIns(4,5)P2 synthesis: regulation and cellular functions. J. Cell Sci. 122, 3837–3850 (2009).
    Article CAS Google Scholar
  18. Boronenkov, I. V. & Anderson, R. A. The sequence of phosphatidylinositol-4-phosphate 5-kinase defines a novel family of lipid kinases. J. Biol. Chem. 270, 2881–2884 (1995).
    Article CAS Google Scholar
  19. Rohde, G., Wenzel, D. & Haucke, V. A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis. J. Cell Biol. 158, 209–214 (2002).
    Article CAS Google Scholar
  20. Levine, T. P. & Munro, S. Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components. Curr. Biol. 12, 695–704 (2002).
    Article CAS Google Scholar
  21. Lehto, M. & Olkkonen, V. M. The OSBP-related proteins: a novel protein family involved in vesicle transport, cellular lipid metabolism, and cell signalling. Biochim. Biophys. Acta 1631, 1–11 (2003).
    Article CAS Google Scholar
  22. Ford, M. G. J. et al. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science 291, 1051–1055 (2001).
    Article CAS Google Scholar
  23. Smythe, E., Carter, L. L. & Schmid, S. L. Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors. J. Cell Biol. 119, 1163–1171 (1992).
    Article CAS Google Scholar
  24. McNiven, M. A. & Thompson, H. M. Vesicle formation at the plasma membrane and trans-golgi network: the same but different. Science 313, 1591–1594 (2006).
    Article CAS Google Scholar
  25. Schmid, S. L. Clathrin-coated vesicle formation and protein sorting: an integrated process. Annu. Rev. Biochem. 66, 511–548 (1997).
    Article CAS Google Scholar
  26. Traub, L. M. et al. AP-2-containing clathrin coats assemble on mature lysosomes. J. Cell Biol. 135, 1801–1814 (1996).
    Article CAS Google Scholar
  27. Popoff, V. et al. Analysis of articulation between clathrin and retromer in retrograde sorting on early endosomes. Traffic 10, 1868–1880 (2009).
    Article CAS Google Scholar
  28. Ravikumar, B., Moreau, K., Jahreiss, L., Puri, C. & Rubinsztein, D. C. Plasma membrane contributes to the formation of pre-autophagosomal structures. Nat. Cell Biol. 12, 747–757 (2011).
    Article Google Scholar
  29. Volpicelli-Daley, L. A. et al. Phosphatidylinositol-4-phosphate 5-kinases and phosphatidylinositol 4,5-bisphosphate synthesis in the brain. J. Biol. Chem. 285 (2010).

Download references

Acknowledgements

We are grateful to Olympus China, Nikon Instruments (Shanghai) and the Tsinghua Cell Biology Core Facility for providing technical support, and to Q. Dong, Y. Li and L. Huang for assistance with microscopy, TEM and image processing. We thank J-J. Liu for helpful discussions and J. Lippincott-Schwartz and J. Bonifacino (Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, USA) for constructs and antibodies. This research was supported by 973 Program grants 2010CB833704 and 2011CB910100, National Science Foundation grants 31030043 and 30971484, and Tsinghua University grants 2010THZ0 and 2009THZ03071 to L.Y., and NSFC grant 81030040, MOST grant 2008ZX09401—002, 2011CB809106 to J.X.

Author information

Authors and Affiliations

  1. State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University-Peking University Center for Life Sciences, School of Life Science, Tsinghua University, Beijing 100084, China
    Yueguang Rong, Mei Liu, Liang Ma, Wanqing Du, Zhen Cao & Li Yu
  2. College of Engineering, Peking University, Beijing 100871, China
    Hanshuo Zhang & Jianzhong Xi
  3. College of Biological Sciences, China Agricultural University, Beijing 100193, China
    Yuan Tian
  4. Cell Biology Core Facility, Tsinghua University, Beijing 100084, China
    Ying Li
  5. School of Life Sciences, Peking University, Beijing 100871, China
    He Ren & Chuanmao Zhang
  6. Proteomics Facility, National Institute of Biological Sciences, Beijing 102206, China
    Lin Li & She Chen

Authors

  1. Yueguang Rong
    You can also search for this author inPubMed Google Scholar
  2. Mei Liu
    You can also search for this author inPubMed Google Scholar
  3. Liang Ma
    You can also search for this author inPubMed Google Scholar
  4. Wanqing Du
    You can also search for this author inPubMed Google Scholar
  5. Hanshuo Zhang
    You can also search for this author inPubMed Google Scholar
  6. Yuan Tian
    You can also search for this author inPubMed Google Scholar
  7. Zhen Cao
    You can also search for this author inPubMed Google Scholar
  8. Ying Li
    You can also search for this author inPubMed Google Scholar
  9. He Ren
    You can also search for this author inPubMed Google Scholar
  10. Chuanmao Zhang
    You can also search for this author inPubMed Google Scholar
  11. Lin Li
    You can also search for this author inPubMed Google Scholar
  12. She Chen
    You can also search for this author inPubMed Google Scholar
  13. Jianzhong Xi
    You can also search for this author inPubMed Google Scholar
  14. Li Yu
    You can also search for this author inPubMed Google Scholar

Contributions

L.Y. and Y.R. conceived and designed the experiments. J.X. designed SAMCell base screening and H.Z. manufactured the SAMCell chip. L.L. and S.C. carried out the mass spectrometric analysis. Y.R, M.L., Y.T. and Z.C. carried out screening. Y.R carried out the functional study with help from M.L. L.M, Y.T., H.R. and C.Z. performed the FEISEM in manuscript revision experiments. Y.L. carried out the embedding and ultrathin sectioning for TEM experiments. W.D. carried out the in vitro staining experiments. L.Y. and Y.R. wrote the manuscript.

Corresponding authors

Correspondence toJianzhong Xi or Li Yu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

About this article

Cite this article

Rong, Y., Liu, M., Ma, L. et al. Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation.Nat Cell Biol 14, 924–934 (2012). https://doi.org/10.1038/ncb2557

Download citation