Abstract
Deciphering the molecular mechanisms underlying the regulation of the ATG4 protease is essential to understand the regulation of ATG8 lipidation, a key step in the biogenesis of the autophagosome and hence in autophagy progression. Here, we describe two complementary approaches to monitor ATG4 proteolytic activity in the model green alga Chlamydomonas reinhardtii: an in vitro assay using recombinant ATG4 and recombinant ATG8 as substrate, and a cell-free assay using soluble total protein extract from Chlamydomonas and recombinant Chlamydomonas ATG8 as substrate. Both assays are followed by non-reducing SDS-PAGE and immuno-blot analysis. Given the high evolutionary conservation of the ATG8 maturation process, these assays have also been validated to monitor ATG4 activity in yeast using Chlamydomonas ATG8 as substrate.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
He C, Klionsky DJ (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43:67–93. https://doi.org/10.1146/annurev-genet-102808-114910
Xie Z, Klionsky DJ (2007) Autophagosome formation: core machinery and adaptations. Nat Cell Biol 9:1102–1109. https://doi.org/10.1038/ncb1007-1102
Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y (2009) Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat Rev Mol Cell Biol 10:458–467. https://doi.org/10.1038/nrm2708
Mizushima N, Yoshimori T, Ohsumi Y (2011) The role of ATG proteins in autophagosome formation. Annu Rev Cell Dev Biol 27:107–132. https://doi.org/10.1146/annurev-cellbio-092910-154005
Díaz-Troya S, Pérez-Pérez ME, Florencio FJ, Crespo JL (2008) The role of TOR in autophagy regulation from yeast to plants and mammals. Autophagy 4:851–865. https://doi.org/10.4161/auto.6555
Liu Y, Bassham DC (2012) Autophagy: pathways for self-eating in plant cells. Annu Rev Plant Biol 63:215–237. https://doi.org/10.1146/annurev-arplant-042811-105441
Delorme-Axford E, Klionsky DJ (2018) Transcriptional and post-transcriptional regulation of autophagy in the yeast Saccharomyces cerevisiae. J Biol Chem 293:5396–5403. https://doi.org/10.1074/jbc.R117.804641
Pérez-Pérez ME, Florencio FJ, Crespo JL (2010) Inhibition of target of rapamycin signaling and stress activate autophagy in Chlamydomonas reinhardtii. Plant Physiol 152:1874–1888. https://doi.org/10.1104/pp.109.152520
Pérez-Pérez ME, Lemaire SD, Crespo JL (2016) Control of autophagy in chlamydomonas is mediated through redox-dependent inactivation of the ATG4 protease. Plant Physiol 172:2219–2234. https://doi.org/10.1104/pp.16.01582
Pérez-Pérez ME, Zaffagnini M, Marchand CH et al (2014) The yeast autophagy protease ATG4 is regulated by thioredoxin. Autophagy 10:1953–1964. https://doi.org/10.4161/auto.34396
Hutner SH, Provasoli L, Schatz A, Haskins CP (1950) Some approaches to the study of the role of metals in the metabolism of microorganisms. Proc Am Philos Soc 94:152–170
Acknowledgments
MEPP is supported by the Spanish Ministry of Science (grant PID2019-110080GB-I00), the Spanish Ministry of Economy and Competitiveness (grant BIO-2015-74432-JIN) and CSIC (grants 202040I006 and 2019AEP126 and Intramural SOLAUT_00033151). JLC is supported by the Spanish Ministry of Science, Innovation and Universities (grant PGC2018-099048B-100).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Crespo, J.L., Pérez-Pérez, M.E. (2022). Monitoring of ATG4 Protease Activity During Autophagy in the Model Microalga Chlamydomonas reinhardtii . In: Klemenčič, M., Stael, S., Huesgen, P.F. (eds) Plant Proteases and Plant Cell Death. Methods in Molecular Biology, vol 2447. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2079-3_17
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2079-3_17
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2078-6
Online ISBN: 978-1-0716-2079-3
eBook Packages: Springer Protocols