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Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation

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

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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.

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Figure 1: Identification of genes regulating ALR by proteomic analysis and SAMCell-RNAi screening.
Figure 2: Clathrin regulates ALR.
Figure 3: PIP5K1B is required for the initiation of ALR.
Figure 4: Phosphatidylinositol-4-phosphate 5-kinase (PIP5K1A) is required for proto-lysosome budding during ALR.
Figure 5: AP2 is required for ALR.
Figure 6: AP4 is required for ALR.
Figure 7: Clathrin mediates autolysosome membrane budding.
Figure 8: Starvation induces LC3 puncta formation in clathrin knockdown cells.

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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.

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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

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  1. Yueguang Rong
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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.

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Correspondence to Jianzhong Xi or Li Yu.

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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

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