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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
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 12:1–222
Kirschke H, Langner J, Wiederanders B, Ansorge S, Bohley P (1977) Cathepsin L. A new proteinase from rat-liver lysosomes. Eur J Biochem 74:293–301
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 95:215–225
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 40:1531–1538
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 208:296–304
Furuno K, Ishikawa T, Kato K (1982) Appearance of autolysosomes in rat liver after leupeptin treatment. J Biochem 91:1485–1494
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 258:6093–6100
Ishikawa T, Furuno K, Kato K (1983) Ultrastructural studies on autolysosomes in rat hepatocytes after leupeptin treatment. Exp Cell Res 144:15–24
Kopitz J, Kisen GO, Gordon PB, Bohley P, Seglen PO (1990) Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes. J Cell Biol 111:941–953
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 274:15222–15229
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 266:18995–18999
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 1:84–91
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 44:279–289
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 37:223–234
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 51:618–631
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.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ueno, T., Komatsu, M. (2019). Measuring Nonselective and Selective Autophagy in the Liver. In: Ktistakis, N., Florey, O. (eds) Autophagy. Methods in Molecular Biology, vol 1880. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8873-0_34
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8873-0_34
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8872-3
Online ISBN: 978-1-4939-8873-0
eBook Packages: Springer Protocols