Abstract
In addition to generating N-degron-carrying substrates destined for proteolysis, N-terminal arginylation can globally upregulate selective macroautophagy via activation of the autophagic N-recognin and archetypal autophagy cargo receptor p62/SQSTM1/sequestosome-1. To evaluate the macroautophagic turnover of cellular substrates, including protein aggregates (aggrephagy) and subcellular organelles (organellophagy) mediated by N-terminal arginylation in vivo, we report here a protocol for assaying the activation of the autophagic Arg/N-degron pathway and degradation of cellular cargoes via N-terminal arginylation. These methods, reagents, and conditions are applicable across a wide spectrum of different cell lines, primary cultures, and/or animal tissues, thereby providing a general means for identification and validation of putative cellular cargoes degraded by Nt-arginylation-activated selective autophagy.
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
References
Ellis R, Minton A (2006) Protein aggregation in crowded environments. Biol Chem 387(5):485–497
Amm I, Sommer T, Wolf DH (2014) Protein quality control and elimination of protein waste: the role of the ubiquitin-proteasome system. Biochim Biophys Acta 1843(1):182–196
Ji CH, Kwon YT (2017) Crosstalk and interplay between the ubiquitin-proteasome system and autophagy. Mol Cells 40(7):441–449
Sriram SM, Kim BY, Kwon YT (2011) The N-end rule pathway: emerging functions and molecular principles of substrate recognition. Nat Rev Mol Cell Biol 12(11):735–747
Cha-Molstad H, Sung KS, Hwang J, Kim KA, Yu JE, Yoo YD, Jang JM, Han DH, Molstad M, Kim JG, Lee YJ, Zakrzewska A, Kim SH, Kim ST, Kim SY, Lee HG, Soung NK, Ahn JS, Ciechanover A, Kim BY, Kwon YT (2015) Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding. Nat Cell Biol 17(7):917–929
Cha-Molstad H, Yu JE, Lee SH, Kim JG, Sung KS, Hwang J, Yoo YD, Lee YJ, Kim ST, Lee DH, Ciechanover A, Kim BY, Kwon YT (2016) Modulation of SQSTM1/p62 activity by N-terminal arginylation of the endoplasmic reticulum chaperone HSPA5/ GRP78/BiP. Autophagy 12(2):426–428
Lee DH, Kim D, Kim ST, Jeong S, Kim JL, Shim SM, Heo AJ, Song X, Guo ZS, Bartlett DL, Oh SC, Lee J, Saito Y, Kim BY, Kwon YT, Lee YJ (2018) PARK7 modulates autophagic proteolysis through binding to the N-terminally arginylated form of the molecular chaperone HSPA5. Autophagy 14(11):1870–1885
Cha-Molstad H, Yu JE, Feng Z, Lee SH, Kim JG, Yang P, Han B, Sung KW, Yoo YD, Hwang J, McGuire T, Shim SM, Song HD, Ganipisetti S, Wang N, Jang JM, Lee MJ, Kim SJ, Lee KH, Hong JT, Ciechanover A, Mook-Jung I, Kim KP, Xie XQ, Kwon YT, Kim BY (2017) p62/SQSTM1/ Sequestosome-1 is an N-recognin of the N-end rule pathway which modulates autophagosome biogenesis. Nat Commun 8(1):102
Yoo YD, Mun SR, Ji CH, Sung KW, Kang KY, Heo AJ, Lee SH, An JY, Hwang J, Xie XQ, Ciechanover A, Kim BY, Kwon YT (2018) N-terminal arginylation generates a bimodal degron that modulates autophagic proteolysis. Proc Natl Acad Sci U S A 115(12):E2716–E2724
Ji CH, Kim HY, Lee MJ, Heo AJ, Park DY, Lim S, Shin S, Yang WS, Jung CA, Kim KY, Jeong EH, Park SH, Kim SB, Lee SJ, Na JE, Kang JI, Chi HM, Kim HT, Kim YK, Kim BY, Kwon YT (2022) The AUTOTAC chemical biology platform for targeted protein degradation via the autophagy-lysosome system. Nat Commun 13(1):904
Ji CH, Kim HY, Heo AJ, Lee SH, Lee MJ, Kim SB, Srinivasrao G, Mun SR, Cha-Molstad H, Ciechanover A, Choi CY, Lee HG, Kim BY, Kwon YT (2019) The N-Degron pathway mediates ER-phagy. Mol Cell 75(5):1058–1072.e9
Ji CH, Kim HY, Heo AJ, Lee MJ, Park DY, Kim DH, Kim BY, Kwon YT (2020) Regulation of reticulophagy by the N- degron pathway. Autophagy 16(2):373–375
Heo AJ, Kim SB, Ji CH, Han D, Lee SJ, Lee SH, Lee MJ, Lee JS, Ciechanover A, Kim BY, Kwon YT (2021) The N-terminal cysteine is a dual sensor of oxygen and oxidative stress. Proc Natl Acad Sci U S A. 118(50):e2107993118
Acknowledgments
This work was supported by the Basic Science Research Programs of the NRF funded by the Ministry of Science, ICT, and Future Planning (MSIP) (NRF-2020R1A5A1019023 and NRF-2021R1A2B5B03002614 to Y.T.K) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute and Korea Dementia Research Center (KDRC) funded by the MSIP (HU21C0201 to C.H.J.), and the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2022R1A6A3A13069514 to S.B.K. and 2022R1A6A3A1307128711 to M.J.L).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ji, C.H., Kim, S.B., Lee, M.J., Kwon, Y.T. (2023). Monitoring the Activation of Selective Autophagy via N-Terminal Arginylation. In: Kashina, A.S. (eds) Protein Arginylation. Methods in Molecular Biology, vol 2620. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2942-0_26
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2942-0_26
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2941-3
Online ISBN: 978-1-0716-2942-0
eBook Packages: Springer Protocols