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Autophagy pp 535-540 | Cite as

Measuring Nonselective and Selective Autophagy in the Liver

  • Takashi UenoEmail author
  • Masaaki KomatsuEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)

Abstract

Administration of leupeptin, a specific inhibitor of lysosomal cysteine proteinases, to starved rats or mice inhibits autolysosomal protein degradation and results in accumulation of autolysosomes in their livers. Immunoblotting of liver homogenates to examine autophagic flux in vivo reveals elevated levels of the selective autophagy substrate p62 and the autophagosomal membrane protein LC3-II in the livers of leupeptin-treated animals. Percoll density gradient centrifugation can be used to isolate autolysosomes from the livers of untreated and leupeptin-treated animals. Moreover, autolysosomes can be examined for the presence of sequestered cytoplasmic proteins as well as degradation intermediates.

Key words

Liver Leupeptin Autolysosome Percoll Selective autophagy Nonselective autophagy 

Notes

Acknowledgments

M.K. is supported by Grant-in-Aid for Scientific Research on Innovative Areas (25111006 and 25111001, to M.K) and Japan Society for the Promotion of Science (an A3 foresight program). T.U. is supported by High Technology Research Center Grant, Strategic Research Foundation at Private Universities. T.U. and M.K. are supported by Grant-in-Aid for Scientific Research on Priority Areas (18076005) and the Takeda Science Foundation.

References

  1. 1.
    Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo AA et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12:1–222CrossRefGoogle Scholar
  2. 2.
    Kirschke H, Langner J, Wiederanders B, Ansorge S, Bohley P (1977) Cathepsin L. A new proteinase from rat-liver lysosomes. Eur J Biochem 74:293–301CrossRefGoogle Scholar
  3. 3.
    Seglen PO, Grinde B, Solheim AE (1979) Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. Eur J Biochem 95:215–225CrossRefGoogle Scholar
  4. 4.
    Aronson NN Jr, Dennis PA, Dunn WA (1981) Metabolism of leupeptin and its effect on autophagy in the perfused rat liver. Acta Biol Med Ger 40:1531–1538PubMedGoogle Scholar
  5. 5.
    Tanaka K, Ikegaki N, Ichihara A (1981) Effects of leupeptin and pepstatin on protein turnover in adult rat hepatocytes in primary culture. Arch Biochem Biophys 208:296–304CrossRefGoogle Scholar
  6. 6.
    Furuno K, Ishikawa T, Kato K (1982) Appearance of autolysosomes in rat liver after leupeptin treatment. J Biochem 91:1485–1494CrossRefGoogle Scholar
  7. 7.
    Kominami E, Hashida S, Khairallah EA, Katunuma N (1983) Sequestration of cytoplasmic enzymes in an autophagic vacuole-lysosomal system induced by injection of leupeptin. J Biol Chem 258:6093–6100PubMedGoogle Scholar
  8. 8.
    Ishikawa T, Furuno K, Kato K (1983) Ultrastructural studies on autolysosomes in rat hepatocytes after leupeptin treatment. Exp Cell Res 144:15–24CrossRefGoogle Scholar
  9. 9.
    Kopitz J, Kisen GO, Gordon PB, Bohley P, Seglen PO (1990) Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes. J Cell Biol 111:941–953CrossRefGoogle Scholar
  10. 10.
    Ueno T, Ishidoh K, Mineki R, Tanida I, Murayama K, Kadowaki M et al (1999) Autolysosomal membrane-associated betaine homocysteine methyltransferase. Limited degradation fragment of a sequestered cytosolic enzyme monitoring autophagy. J Biol Chem 274:15222–15229CrossRefGoogle Scholar
  11. 11.
    Ueno T, Muno D, Kominami E (1991) Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes isolated from leupeptin-administered rat liver. J Biol Chem 266:18995–18999PubMedGoogle Scholar
  12. 12.
    Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E (2005) Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy 1:84–91CrossRefGoogle Scholar
  13. 13.
    Matsumoto G, Wada K, Okuno M, Kurosawa M, Nukina N (2011) Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins. Mol Cell 44:279–289CrossRefGoogle Scholar
  14. 14.
    Pilli M, Arko-Mensah J, Ponpuak M, Roberts E, Master S, Mandell MA et al (2012) TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation. Immunity 37:223–234CrossRefGoogle Scholar
  15. 15.
    Ichimura Y, Waguri S, Sou YS, Kageyama S, Hasegawa J, Ishimura R et al (2013) Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy. Mol Cell 51:618–631CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratory of Proteomics and Biomolecular Science, Research Support CenterJuntendo University Graduate School of MedicineTokyoJapan
  2. 2.Department of PhysiologyJuntendo University Graduate School of MedicineTokyoJapan

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