Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

Nature Cell Biology volume 17, pages 759–770 (2015)Cite this article

Subjects

Abstract

Chaperone-mediated autophagy (CMA) selectively degrades a subset of cytosolic proteins in lysosomes. A potent physiological activator of CMA is nutrient deprivation, a condition in which intracellular triglyceride stores or lipid droplets (LDs) also undergo hydrolysis (lipolysis) to generate free fatty acids for energetic purposes. Here we report that the LD-associated proteins perilipin 2 (PLIN2) and perilipin 3 (PLIN3) are CMA substrates and their degradation through CMA precedes lipolysis. In vivo studies revealed that CMA degradation of PLIN2 and PLIN3 was enhanced during starvation, concurrent with elevated levels of cytosolic adipose triglyceride lipase (ATGL) and macroautophagy proteins on LDs. CMA blockage both in cultured cells and mouse liver or expression of CMA-resistant PLINs leads to reduced association of ATGL and macrolipophagy-related proteins with LDs and the subsequent decrease in lipid oxidation and accumulation of LDs. We propose a role for CMA in LD biology and in the maintenance of lipid homeostasis.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: LAMP-2A-deficient cells accumulate LDs.
Figure 2: PLIN2 and PLIN3 are CMA substrates.
Figure 3: PLIN2 and PLIN3 interact with CMA chaperone hsc70.
Figure 4: Post-translational modifications of PLINs and their degradation by CMA.
Figure 5: Cells expressing CMA-mutant PLIN2 accumulate LDs.
Figure 6: Failure to remove PLINs by CMA blocks lipolysis.
Figure 7: Failure to remove PLINs by CMA blocks macrolipophagy.
Figure 8: Scheme of the interplay of CMA with cytosolic and lysosomal lipolysis through PLIN2 and PLIN3 degradation.

Similar content being viewed by others

References

  1. Mizushima, N. & Komatsu, M. Autophagy: renovation of cells and tissues. Cell 147, 728–741 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Yang, Z. & Klionsky, D. J. Mammalian autophagy: core molecular machinery and signaling regulation. Curr. Opin. Cell Biol. 22, 124–131 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Singh, R. & Cuervo, A. M. Autophagy in the cellular energetic balance. Cell Metab. 13, 495–504 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Dice, J. F. Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trends Biochem. Sci. 15, 305–309 (1990).

    Article  CAS  PubMed  Google Scholar 

  5. Chiang, H. L., Terlecky, S. R., Plant, C. P. & Dice, J. F. A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science 246, 382–385 (1989).

    Article  CAS  PubMed  Google Scholar 

  6. Cuervo, A. M. & Dice, J. F. A receptor for the selective uptake and degradation of proteins by lysosomes. Science 273, 501–503 (1996).

    Article  CAS  PubMed  Google Scholar 

  7. Bandyopadhyay, U., Kaushik, S., Varticovski, L. & Cuervo, A. M. The chaperone-mediated autophagy receptor organizes in dynamic protein complexes at the lysosomal membrane. Mol. Cell. Biol. 28, 5747–5763 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Agarraberes, F. A., Terlecky, S. R. & Dice, J. F. An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation. J. Cell Biol. 137, 825–834 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Salvador, N., Aguado, C., Horst, M. & Knecht, E. Import of a cytosolic protein into lysosomes by chaperone-mediated autophagy depends on its folding state. J. Biol. Chem. 275, 27447–27456 (2000).

    CAS  PubMed  Google Scholar 

  10. Arias, E. & Cuervo, A. M. Chaperone-mediated autophagy in protein quality control. Curr. Opin. Cell Biol. 23, 184–189 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. Kaushik, S. & Cuervo, A. M. Chaperone-mediated autophagy: a unique way to enter the lysosome world. Trends Cell Biol. 22, 407–417 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Cuervo, A. M., Knecht, E., Terlecky, S. R. & Dice, J. F. Activation of a selective pathway of lysosomal proteolysis in rat liver by prolonged starvation. Am. J. Physiol. 269, C1200–C1208 (1995).

    Article  CAS  PubMed  Google Scholar 

  13. Kiffin, R., Christian, C., Knecht, E. & Cuervo, A. M. Activation of chaperone-mediated autophagy during oxidative stress. Mol. Biol. Cell 15, 4829–4840 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Dohi, E. et al. Hypoxic stress activates chaperone-mediated autophagy and modulates neuronal cell survival. Neurochem. Int. 60, 431–442 (2012).

    Article  CAS  PubMed  Google Scholar 

  15. Rodriguez-Navarro, J. A. et al. Inhibitory effect of dietary lipids on chaperone-mediated autophagy. Proc. Natl Acad. Sci. USA 109, E705–E714 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Cuervo, A. M. & Dice, J. F. Age-related decline in chaperone-mediated autophagy. J. Biol. Chem. 275, 31505–31513 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Kiffin, R. et al. Altered dynamics of the lysosomal receptor for chaperone-mediated autophagy with age. J. Cell Sci. 120, 782–791 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Singh, R. et al. Autophagy regulates lipid metabolism. Nature 458, 1131–1135 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ouimet, M. et al. Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase. Cell Metab. 13, 655–667 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Walther, T. C. & Farese, R. V. Jr Lipid droplets and cellular lipid metabolism. Annu. Rev. Biochem. 81, 687–714 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lass, A., Zimmermann, R., Oberer, M. & Zechner, R. Lipolysis—a highly regulated multi-enzyme complex mediates the catabolism of cellular fat stores. Prog. Lipid Res. 50, 14–27 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Schneider, J. L., Suh, Y. & Cuervo, A. M. Deficient chaperone-mediated autophagy in liver leads to metabolic dysregulation. Cell Metab. 20, 417–432 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Quiroga, A. D. & Lehner, R. Liver triacylglycerol lipases. Biochim. Biophys. Acta 1821, 762–769 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Greenberg, A. S. et al. The role of lipid droplets in metabolic disease in rodents and humans. J. Clin. Invest. 121, 2102–2110 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Massey, A. C., Kaushik, S., Sovak, G., Kiffin, R. & Cuervo, A. M. Consequences of the selective blockage of chaperone-mediated autophagy. Proc. Natl Acad. Sci. USA 103, 5805–5810 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Katayama, H., Kogure, T., Mizushima, N., Yoshimori, T. & Miyawaki, A. A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem. Biol. 18, 1042–1052 (2011).

    Article  CAS  PubMed  Google Scholar 

  27. Ranall, M. V., Gabrielli, B. G. & Gonda, T. J. High-content imaging of neutral lipid droplets with 1,6-diphenylhexatriene. Biotechniques 51, 35–42 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Masuda, Y. et al. ADRP/adipophilin is degraded through the proteasome-dependent pathway during regression of lipid-storing cells. J. Lipid Res. 47, 87–98 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Xu, G. et al. Post-translational regulation of adipose differentiation-related protein by the ubiquitin/proteasome pathway. J. Biol. Chem. 280, 42841–42847 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Greussing, R., Unterluggauer, H., Koziel, R., Maier, A. B. & Jansen-Durr, P. Monitoring of ubiquitin-proteasome activity in living cells using a Degron (dgn)-destabilized green fluorescent protein (GFP)-based reporter protein. J. Vis. Exp. 69, e3327 (2012).

    Google Scholar 

  31. Cuervo, A. M., Dice, J. F. & Knecht, E. A population of rat liver lysosomes responsible for the selective uptake and degradation of cytosolic proteins. J. Biol. Chem. 272, 5606–5615 (1997).

    Article  CAS  PubMed  Google Scholar 

  32. Aniento, F., Roche, E., Cuervo, A. M. & Knecht, E. Uptake and degradation of glyceraldehyde-3-phosphate dehydrogenase by rat liver lysosomes. J. Biol. Chem. 268, 10463–10470 (1993).

    CAS  PubMed  Google Scholar 

  33. Koga, H., Kaushik, S. & Cuervo, A. M. Altered lipid content inhibits autophagic vesicular fusion. FASEB J. 24, 3052–3065 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Anguiano, J. et al. Chemical modulation of chaperone-mediated autophagy by retinoic acid derivatives. Nat. Chem. Biol. 9, 374–382 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bostrom, P. et al. Cytosolic lipid droplets increase in size by microtubule-dependent complex formation. Arterioscler. Thromb. Vasc. Biol. 25, 1945–1951 (2005).

    Article  PubMed  Google Scholar 

  36. Targett-Adams, P. et al. Live cell analysis and targeting of the lipid droplet-binding adipocyte differentiation-related protein. J. Biol. Chem. 278, 15998–16007 (2003).

    Article  CAS  PubMed  Google Scholar 

  37. Lizaso, A., Tan, K. T. & Lee, Y. H. β-adrenergic receptor-stimulated lipolysis requires the RAB7-mediated autolysosomal lipid degradation. Autophagy 9, 1228–1243 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Dupont, N. et al. Neutral lipid stores and lipase PNPLA5 contribute to autophagosome biogenesis. Curr. Biol. 24, 609–620 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wang, H. et al. Unique regulation of adipose triglyceride lipase (ATGL) by perilipin 5, a lipid droplet-associated protein. J. Biol. Chem. 286, 15707–15715 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Listenberger, L. L., Ostermeyer-Fay, A. G., Goldberg, E. B., Brown, W. J. & Brown, D. A. Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover. J. Lipid Res. 48, 2751–2761 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Kovsan, J., Ben-Romano, R., Souza, S. C., Greenberg, A. S. & Rudich, A. Regulation of adipocyte lipolysis by degradation of the perilipin protein: nelfinavir enhances lysosome-mediated perilipin proteolysis. J. Biol. Chem. 282, 21704–21711 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Ogasawara, J. et al. Oligonol-induced degradation of perilipin 1 is regulated through lysosomal degradation machinery. Nat. Prod. Commun. 7, 1193–1196 (2012).

    CAS  PubMed  Google Scholar 

  43. Xu, G., Sztalryd, C. & Londos, C. Degradation of perilipin is mediated through ubiquitination-proteasome pathway. Biochim. Biophys. Acta 1761, 83–90 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Cermelli, S., Guo, Y., Gross, S. P. & Welte, M. A. The lipid-droplet proteome reveals that droplets are a protein-storage depot. Curr. Biol. 16, 1783–1795 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Fujimoto, T. & Ohsaki, Y. Cytoplasmic lipid droplets: rediscovery of an old structure as a unique platform. Ann. N. Y. Acad. Sci. 1086, 104–115 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Parent, R., Qu, X., Petit, M. A. & Beretta, L. The heat shock cognate protein 70 is associated with hepatitis C virus particles and modulates virus infectivity. Hepatology 49, 1798–1809 (2009).

    Article  CAS  PubMed  Google Scholar 

  47. Turro, S. et al. Identification and characterization of associated with lipid droplet protein 1: a novel membrane-associated protein that resides on hepatic lipid droplets. Traffic 7, 1254–1269 (2006).

    Article  CAS  PubMed  Google Scholar 

  48. Binns, D. et al. An intimate collaboration between peroxisomes and lipid bodies. J. Cell Biol. 173, 719–731 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Blanchette-Mackie, E. J. & Scow, R. O. Movement of lipolytic products to mitochondria in brown adipose tissue of young rats: an electron microscope study. J. Lipid Res. 24, 229–244 (1983).

    CAS  PubMed  Google Scholar 

  50. Wang, H. et al. Perilipin 5, a lipid droplet-associated protein, provides physical and metabolic linkage to mitochondria. J. Lipid Res. 52, 2159–2168 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Bartz, R. et al. Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation. J. Proteome Res. 6, 3256–3265 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Goldstein, J. L., Basu, S. K. & Brown, M. S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 98, 241–260 (1983).

    Article  CAS  PubMed  Google Scholar 

  53. Boland, B. et al. Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer’s disease. J. Neurosci. 28, 6926–6937 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Massey, A. C., Follenzi, A., Kiffin, R., Zhang, C. & Cuervo, A. M. Early cellular changes after blockage of chaperone-mediated autophagy. Autophagy 4, 442–456 (2008).

    Article  CAS  PubMed  Google Scholar 

  55. Kaushik, S., Massey, A. C. & Cuervo, A. M. Lysosome membrane lipid microdomains: novel regulators of chaperone-mediated autophagy. EMBO J. 25, 3921–3933 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Bolte, S. & Cordelieres, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Kaushik, S. & Cuervo, A. M. Methods to monitor chaperone-mediated autophagy. Methods Enzymol. 452, 297–324 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Institutes of Health AG021904, AG031782, AG038072, DK098408 and the generous support of Robert and Renée Belfer. We thank B. Patel for performing the electron microscopy, R. Singh for assistance in biochemical lipid assays and the Analytical Imaging Facility for support with live-cell imaging.

Author information

Authors and Affiliations

  1. Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA

    Susmita Kaushik & Ana Maria Cuervo

  2. Institute for Aging Studies, Albert Einstein College of Medicine, Bronx, New York 10461, USA

    Susmita Kaushik & Ana Maria Cuervo

Authors
  1. Susmita Kaushik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ana Maria Cuervo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.K. designed and performed the experiments, analysed and interpreted the data, and contributed to writing the manuscript; A.M.C. coordinated the study, contributed to designing and interpretation of the experiments and to writing the manuscript.

Corresponding author

Correspondence to Ana Maria Cuervo.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 LAMP-2A-deficient cells accumulate LD.

(a) MitoTracker and MitoTracker CMXRos staining in CTR and L2A(−) cells untreated or treated with OL. Graph: average percentage colocalization between the two fluorophores. n = 4 independent experiments with 40 cells per condition in each experiment. (b) CTR and L2A(−) expressing mtKeima untreated or treated with OL. FCCP is shown as a positive control. Graph: average number/cell. n = 9 fields with 1350 cells per condition from 3 independent experiments. (c) Oil Red O staining in WT and L2AKO liver sections. Graphs: LD number, LD size and percentage of cytosolic area occupied by LD. n = 6 micrographs from 3 animals in each group. (d) LD size for the cells shown in Fig. 1e. n = 7 independent experiments with 40 cells per condition in each experiment. (e,f) DPH staining in CTR and L2A(−) cells untreated or treated with OL, or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: average LD number/cell. n = 5 independent experiments with 40 cells per condition in each experiment. (g) BODIPY 493/503 staining in OL-treated CTR and L2A(−) cells untreated or treated with etomoxir. Graph: average LD number/cell. n = 5 independent experiments with 40 cells per condition in each experiment. (h) BODIPY 493/603 staining in CTR and L2A(−) cells maintained in the indicated conditions. Graph: average LD number/cell. n = 18 fields with 2700 cells per condition from 3 independent experiments. (i) Immunoblot for hLAMP2 in CTR and L2A(−) cells transfected with hL2A. (j) Immunostaining for hLAMP2 in CTR and L2A(−) cells transfected with hL2A and treated with OL. Values are mean ± SEM. Differences are significant for ∗P < 0.05,∗∗P < 0.01,∗∗∗P < 0.001 using Student’s t-test. Uncropped images of blots are shown in Supplementary Fig. 8. Source data is available in Supplementary Table 1.

Supplementary Figure 2 PLIN2 and PLIN3 are CMA substrates.

(a) Immunoblot for indicated proteins in total cell lysates from CTR and L2A(−) cells. Two sets of cells are shown. (b) BODIPY 493/503 staining in control cells untreated or treated with ammonium chloride and leupeptin (NL), lactacystin (Lacta) or MG132. Graph: average LD number/cell. n = 5 independent experiments with 40 cells per condition in each experiment. (c) Immunoblot for indicated proteins in total cell lysates from CTR and L2A(−) cells treated or not with OL and lactacystin (Lacta). n = 2 independent experiments. (d,e) CTR and L2A(−) cells expressing DGN or DGN-FS treated or not with OL and Lacta. Graph: average fluorescence intensity/cell. n = 10 fields with 1500 cells per condition from 3 independent experiments. (f,g) Coimmunostaining for PLIN3 and LAMP1 in CTR and L2A(−) cells treated or not with OL, followed by treatment with lysosomal inhibitors (NL). Top: Colocalized pixels in white. Bottom: Merged image of the boxed area at higher magnification. Graph: percentage colocalization of PLIN3 with LAMP1. n = 5 (L2A(−)) and 6 (CTR) independent experiments with 40 cells per condition in each experiment. (h) Immunoblot for indicated proteins of HOM, CMA+ and CMA− lysosomes isolated from fed or starved (Stv) livers of rat untreated or treated with leupeptin (Leup). These blots contributed to the quantification shown in Fig. 2g. All values are mean ± SEM. Differences are significant for ∗P < 0.05,∗∗P < 0.01,∗∗∗P < 0.001 using Student’s t-test. Uncropped images of blots are shown in Supplementary Fig. 8. Source data is available in Supplementary Table 1.

Supplementary Figure 3 PLIN2 and PLIN3 associate with hsc70.

(a) Coimmunostaining for PLIN3 and hsc70 in CTR and L2A(−) cells treated or not with OL. Colocalized pixels are in white. Boxed areas are shown at higher magnification. Bottom: graph: percentage colocalization of PLIN3 (bottom) with hsc70. n = 5 independent experiments with 40 cells per condition in each experiment. (b) Coimmunostaining for PLIN2 and hsc70 in CTR and L2A(−) cells treated as indicated. Colocalized pixels are in white. Graph: percentage colocalization. n = 4 independent experiments with 40 cells per condition in each experiment. (c,d) Immunoblot for indicated proteins of immunoprecipitates (IP) of hsc70 from total extracts of CTR and L2A(−) cells treated or not with OL. Representative blots of n = 3 independent experiments. (e) Immunoblot for indicated proteins of IP of GAPDH (top) and IkB (bottom) from total extracts of CTR and L2A(−) cells. (f) Immunoblot for indicated proteins of IP of PLIN2 (top) and PLIN3 (bottom) from total extracts of CTR and L2A(−) cells treated with the indicated increasing concentrations of OL. These blots are an extension of those shown in Fig. 3e, f to show the dose-dependence effect. Representative blots of n = 3 (PLIN3) and 4 (PLIN2) independent experiments. Values are mean ± SEM. Differences are significant for ∗P < 0.05,∗∗P < 0.01,∗∗∗P < 0.001 using Student’s t-test. Uncropped images of blots are shown in Supplementary Fig. 8. Source data is available in Supplementary Table 1.

Supplementary Figure 4 Inhibition of PLIN2 degradation by CMA leads to lipid droplet accumulation.

(a) Immunostaining for hsc70 in OL-treated cells expressing WT or MT PLIN2-GFP. Right: 3D-reconstruction of the marked regions of the images shown on left. See also Supplementary Video 1 . (b) Right: Serial Z-sections of the marked region of CTR cells expressing MT PLIN2-GFP shown on left.

Supplementary Figure 5 Altered cytosolic and lysosomal lipolysis in CMA-incompetent cells.

(a) Coimmunostaining for PLIN2 and ATGL in CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented (OL > S+) or serum-deprived (OL > S−) medium after OL treatment. Insets: Higher magnification areas. Right: Images of the same fields with colocalized pixels shown in white. Graph: percentage colocalization of PLIN2 with ATGL. n = 5 independent experiments with 40 cells per condition in each experiment. (b) Costaining for BODIPY493/503 and ATGL in CTR and L2A(−) cells incubated or not in OL > S+ regular or low glucose media. Bottom: Higher magnification areas. Graph: percentage colocalization of BODIPY with ATGL. n = 5 independent experiments with 40 cells per condition in each experiment. (c) Immunoblot for ATGL of homogenates (HOM) and lipid droplets (LD) isolated from starved wild-type (+) or L2A knockout (−) mice livers (these 3 additional blots support the reproducibility of the blot shown in Fig. 6i). (d) BODIPY493/503 staining in CTR and L2A(−) cells untreated or treated with OL, or treated with 3-methyladenine (3MA) or lysosomal inhibitors ammonium chloride and leupeptin (NL). Higher magnification insets and quantification are shown in Fig. 7a. (e) Immunoblot for LC3 of total cell lysates from CTR and L2A(−) cells treated with OL in the presence or absence of 3-methyladenine (3MA). n = 3 independent experiments. (f) Immunostaining for LC3 in OL-treated CTR and L2A(−) cells. Graph: average number of LC3 puncta/cell. n = 6 independent experiments with 30 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for ∗∗P < 0.01,∗∗∗P < 0.001 using Student’s t-test. Uncropped images of blots are shown in Supplementary Fig. 8. Source data is available in Supplementary Table 1.

Supplementary Figure 6 Failure to remove PLINs by CMA alters association of macroautophagy proteins with LD.

(a,b) Costaining for BODIPY 493/503 and the indicated macroautophagy proteins in CTR cells treated or not with OL. Colocalized pixels are in white. Graph: Percentage colocalization with BODIPY. n = 3 independent experiments with 40 cells per condition in each experiment. (c–f) Costaining for BODIPY493/503 and indicated macroautophagy proteins in CTR and L2A(−) cells treated with OL. Right in c: Colocalized pixels of the boxed area at higher magnification. e,f: Colocalized pixels are shown. Graphs: percentage colocalization with BODIPY. n = 6 (d), 7 (e), 3 (f) independent experiments with 30 (d) 40 (e), 10 (f) cells per condition in each experiment. Values are mean ± SEM. Differences are significant for ∗P < 0.05,∗∗P < 0.01,∗∗∗P < 0.001 using Student’s t-test. Source data is available in Supplementary Table 1.

Supplementary Figure 7 Failure to remove PLINs by CMA blocks macrolipophagy.

(a) Live-cell imaging of cells cotransfected with wild-type (WT) or CMA-mutant (MT) PLIN2-GFP and dsRed-LC3 and treated with OL. Sequential frames at 30s intervals are shown. Arrows: LD. See also Supplementary Video 4 . (b) Immunostaining for ATG5 (top) or Beclin1 (bottom) in cells expressing WT or MT PLIN2-GFP treated with OL. Higher magnification show colocalized pixels in white. Graph: percentage colocalization PLIN2-GFP. n = 5 independent experiments with 40 cells per condition in each experiment. (c) Immunostaining for LC3 in OL-treated cells expressing WT or MT PLIN2-GFP. Right: 3D-reconstruction of the images shown on left. See also Supplementary Video 5 . (d) Electron microscopy of CTR or L2A(−) cells maintained in serum-deprived media with or without OL. Several fields are shown. Insets on the right show details of different morphological features previously attributed to macrolipophagy18 and that include: LD (blue arrows; no limiting membrane); where a limiting membrane appears to be forming on the LD surface (yellow arrows); vesicles (putative autolysosomes) containing cargo compatible with lipids (red arrows; limiting membrane) and LD with membranes that originate from their surface toward the core of the LD (green arrows), previously described to correspond to LC3-positive limiting membranes1. Micrographs originate from 3 independent experiments. Values are mean ± SEM. Differences are significant for ∗∗∗P < 0.001 using Student’s t-test. Source data is available in Supplementary Table 1.

Supplementary Figure 8 Images of the uncropped immunoblots with molecular weight markers of the blot data shown in the main and the Supplementary Figures.

Boxes indicate cropped regions. Molecular weight markers are color coded according to the key shown on the bottom right.

Supplementary information

Supplementary Information

Supplementary Information (PDF 5572 kb)

3D-reconstruction of WT or MT PLIN2 and hsc70.

3D-reconstruction of fluorescence stacks of NIH3T3 cells transfected with wild type (WT) or CMA-motif mutated (MT) PLIN2-GFP and immunostained for hsc70. Left: progression from bottom to top. Right: planar rotation. (AVI 5457 kb)

Dynamics of WT or MT PLIN2 in NIH3T3 cells.

Time-lapse microscopy of NIH3T3 cells transfected with wild type (WT) or CMA-motif mutated (MT) PLIN2-GFP. Green channel shown. Arrows point to lipid droplets. (AVI 556 kb)

Dynamics of WT or MT PLIN2 and LAMP1 in NIH3T3 cells.

Time-lapse microscopy of NIH3T3 cells co-transfected with wild type (WT) or CMA-motif mutated (MT) PLIN2-GFP and with LAMP1-RFP. Merged channels are shown. Right shows details at higher magnification. Arrows point to lipid droplets. (AVI 1202 kb)

Dynamics of WT or MT PLIN2 and LC3 in NIH3T3 cells.

Time-lapse microscopy of NIH3T3 cells co-transfected with wild type (WT) or CMA-motif mutated (MT) PLIN2-GFP and with dsRed-LC3. Merged channels are shown. Right shows details at higher magnification. (AVI 624 kb)

3D-reconstruction of WT or MT PLIN2 and LC3.

3D-reconstruction of fluorescence stacks of NIH3T3 cells transfected with wild type (WT) or CMA-motif mutated (MT) PLIN2-GFP and immunostained for LC3. Left: progression from bottom to top. Right: planar rotation. (AVI 9631 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kaushik, S., Cuervo, A. Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis. Nat Cell Biol 17, 759–770 (2015). https://doi.org/10.1038/ncb3166

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3166

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing