Abstract
At present, several assays that use radioisotope labeling to quantify the degradation of long-lived proteins have been developed to measure autophagic flux. Here, we describe a nonradioactive pulse–chase protocol using L-azidohomoalanine (AHA) labeling to quantify long-lived protein degradation during autophagy. AHA is used as a surrogate for L-methionine, and, when added to cultured cells grown in methionine-free medium, AHA is incorporated into proteins during de novo protein synthesis. After a chase period to remove short-lived proteins, autophagy is induced by starvation or other stimuli. Cells then undergo a 'click' reaction between the azide group of AHA and a fluorescently tagged alkyne probe. The AHA-containing proteins can then be detected by flow cytometry. This protocol is nonradioactive, sensitive and quantitative, and it is easy to perform. It is also applicable to various cell culture systems. The whole protocol is estimated to take 4–5 d to complete.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Change history
07 September 2017
In the version of this article initially published, there was an error in Figure 1b. The structure of triazole, a 5-membered ring structure generated after the click reaction, was mistakenly drawn as a 6-membered ring. The error has been corrected in the HTML and PDF versions of the article.
References
Mizushima, N. & Komatsu, M. Autophagy: renovation of cells and tissues. Cell 147, 728–741 (2011).
Levine, B. & Kroemer, G. Autophagy in the pathogenesis of disease. Cell 132, 27–42 (2008).
Mizushima, N. Autophagy: process and function. Genes Dev. 21, 2861–2873 (2007).
Nakatogawa, H., Suzuki, K., Kamada, Y. & Ohsumi, Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat. Rev. Mol. Cell Biol. 10, 458–467 (2009).
Feng, Y., He, D., Yao, Z. & Klionsky, D.J. The machinery of macroautophagy. Cell Res. 24, 24–41 (2014).
Chan, E.Y. & Tooze, S.A. Evolution of Atg1 function and regulation. Autophagy 5, 758–765 (2009).
Laplante, M. & Sabatini, D.M. mTOR signaling in growth control and disease. Cell 149, 274–293 (2012).
Jung, C.H., Ro, S.H., Cao, J., Otto, N.M. & Kim, D.H. mTOR regulation of autophagy. FEBS Lett. 584, 1287–1295 (2010).
Klionsky, D.J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1–222 (2016).
Liu, X.D. et al. Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis. Oncogene 34, 2450–2460 (2015).
Shen, H.M. & Codogno, P. Autophagic cell death: Loch Ness monster or endangered species? Autophagy 7, 457–465 (2011).
Klionsky, D.J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4, 151–175 (2008).
Klionsky, D.J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445–544 (2012).
Ogier-Denis, E., Houri, J.J., Bauvy, C. & Codogno, P. Guanine nucleotide exchange on heterotrimeric Gi3 protein controls autophagic sequestration in HT-29 cells. J. Biol. Chem. 271, 28593–28600 (1996).
Roberts, E.A. & Deretic, V. Autophagic proteolysis of long-lived proteins in nonliver cells. Methods Mol. Biol. 445, 111–117 (2008).
Bauvy, C., Meijer, A.J. & Codogno, P. Assaying of autophagic protein degradation. Methods Enzymol. 452, 47–61 (2009).
Yewdell, J.W., Lacsina, J.R., Rechsteiner, M.C. & Nicchitta, C.V. Out with the old, in with the new? Comparing methods for measuring protein degradation. Cell Biol. Int. 35, 457–462 (2011).
Han, Q. & Hoffman, R.M. Nonradioactive enzymatic assay for plasma and serum vitamin B(6). Nat. Protoc. 3, 1815–1819 (2008).
Hein, C.D., Liu, X.M. & Wang, D. Click chemistry, a powerful tool for pharmaceutical sciences. Pharm. Res. 25, 2216–2230 (2008).
Moses, J.E. & Moorhouse, A.D. The growing applications of click chemistry. Chem. Soc. Rev. 36, 1249–1262 (2007).
Ullrich, M. et al. Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans. Nat. Protoc. 9, 2237–2255 (2014).
Dieterich, D.C. et al. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nat. Protoc. 2, 532–540 (2007).
Dieterich, D.C., Link, A.J., Graumann, J., Tirrell, D.A. & Schuman, E.M. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc. Natl. Acad. Sci. USA 103, 9482–9487 (2006).
Roth, S., Drewe, W.C. & Thomas, N.R. A concise and scalable route to L-azidohomoalanine. Nat. Protoc. 5, 1967–1973 (2010).
Link, A.J., Vink, M.K. & Tirrell, D.A. Preparation of the functionalizable methionine surrogate azidohomoalanine via copper-catalyzed diazo transfer. Nat. Protoc. 2, 1879–1883 (2007).
Millecamps, S. & Julien, J.P. [35S]Methionine metabolic labeling to study axonal transport of neuronal intermediate filament proteins in vivo. Methods Cell Biol. 78, 555–571 (2004).
Laughlin, S.T. & Bertozzi, C.R. Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation. Nat. Protoc. 2, 2930–2944 (2007).
Kachidian, P., Poulat, P., Marlier, L. & Privat, A. Immunohistochemical evidence for the coexistence of substance P, thyrotropin-releasing hormone, GABA, methionine-enkephalin, and leucin-enkephalin in the serotonergic neurons of the caudal raphe nuclei: a dual labeling in the rat. J. Neurosci. Res. 30, 521–530 (1991).
Dieterich, D.C. et al. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat. Neurosci. 13, 897–905 (2010).
Rosner, D., Schneider, T., Schneider, D., Scheffner, M. & Marx, A. Click chemistry for targeted protein ubiquitylation and ubiquitin chain formation. Nat. Protoc. 10, 1594–1611 (2015).
Gao, X. & Hannoush, R.N. Single-cell in situ imaging of palmitoylation in fatty-acylated proteins. Nat. Protoc. 9, 2607–2623 (2014).
Yang, J. et al. Global, in situ, site-specific analysis of protein S-sulfenylation. Nat. Protoc. 10, 1022–1037 (2015).
Heal, W.P., Wright, M.H., Thinon, E. & Tate, E.W. Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry. Nat. Protoc. 7, 105–117 (2012).
Zhang, J., Wang, J., Ng, S., Lin, Q. & Shen, H.M. Development of a novel method for quantification of autophagic protein degradation by AHA labeling. Autophagy 10, 901–912 (2014).
Kharaziha, P. et al. Sorafenib-induced defective autophagy promotes cell death by necroptosis. Oncotarget 6, 37066–37082 (2015).
Polletta, L. et al. SIRT5 regulation of ammonia-induced autophagy and mitophagy. Autophagy 11, 253–270 (2015).
Rashid, M.M., Runci, A., Russo, M.A. & Tafani, M. Muscle Lim protein (MLP)/CSRP3 at the crossroad between mechanotransduction and autophagy. Cell Death Dis. 6, e1940 (2015).
Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010).
Kimura, S., Fujita, N., Noda, T. & Yoshimori, T. Monitoring autophagy in mammalian cultured cells through the dynamics of LC3. Methods Enzymol. 452, 1–12 (2009).
Lelouard, H. et al. Transient aggregation of ubiquitinated proteins during dendritic cell maturation. Nature 417, 177–182 (2002).
Kawai, A., Takano, S., Nakamura, N. & Ohkuma, S. Quantitative monitoring of autophagic degradation. Biochem. Biophys. Res. Commun. 351, 71–77 (2006).
Shvets, E., Fass, E. & Elazar, Z. Utilizing flow cytometry to monitor autophagy in living mammalian cells. Autophagy 4, 621–628 (2008).
Kimura, S., Noda, T. & Yoshimori, T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3, 452–460 (2007).
Kiick, K.L., Saxon, E., Tirrell, D.A. & Bertozzi, C.R. Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc. Natl. Acad. Sci. USA 99, 19–24 (2002).
Nikic, I., Kang, J.H., Girona, G.E., Aramburu, I.V. & Lemke, E.A. Labeling proteins on live mammalian cells using click chemistry. Nat. Protoc. 10, 780–791 (2015).
Jiang, L. et al. Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry. Nat. Protoc. 11, 655–663 (2016).
Breton, G., Lee, J., Liu, K. & Nussenzweig, M.C. Defining human dendritic cell progenitors by multiparametric flow cytometry. Nat. Protoc. 10, 1407–1422 (2015).
Forment, J.V. & Jackson, S.P. A flow cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells. Nat. Protoc. 10, 1297–1307 (2015).
Wu, Y.T. et al. Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J. Biol. Chem. 285, 10850–10861 (2010).
Petiot, A., Ogier-Denis, E., Blommaart, E.F., Meijer, A.J. & Codogno, P. Distinct classes of phosphatidylinositol 3′-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J. Biol. Chem. 275, 992–998 (2000).
Yamamoto, A. et al. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct. Funct. 23, 33–42 (1998).
Hosokawa, N., Hara, Y. & Mizushima, N. Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size. FEBS Lett. 580, 2623–2629 (2006).
Kuma, A. et al. The role of autophagy during the early neonatal starvation period. Nature 432, 1032–1036 (2004).
Komatsu, M. et al. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J. Cell Biol. 169, 425–434 (2005).
Acknowledgements
This work was supported in part by the National Research Foundation-supported Interdisciplinary Research Group in Infectious Diseases of SMART (Singapore-MIT Alliance for Research and Technology) and by research grants from the National Medical Research Council Singapore (nos. NMRC-CIRG/1346/2012 and NMRC/CIRG/1373/2013) to H.-M.S. Y.M.L. was supported by NUS Research Scholarships. We also acknowledge support from the Chinese National Natural Sciences Foundation (grant nos. 81630092 and 81421091) and the Doctoral Station Science Foundation from the Chinese Ministry of Education of China (grant no. 20130091130003 to Z.-C.H.).
Author information
Authors and Affiliations
Contributions
J.W., J.Z., Q.L. and H.-M.S. designed the research. J.W. and J.Z. performed the experiments. Z.-C.H., S.N. and Y.S. assisted in the data analysis. J.W., J.Z., Y.M.L., Q.L. and H.-M.S. wrote the manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Wang, J., Zhang, J., Lee, Y. et al. Nonradioactive quantification of autophagic protein degradation with L-azidohomoalanine labeling. Nat Protoc 12, 279–288 (2017). https://doi.org/10.1038/nprot.2016.160
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2016.160
This article is cited by
-
Discovery and mechanism studies of a novel ATG4B inhibitor Ebselen by drug repurposing and its anti-colorectal cancer effects in mice
Cell & Bioscience (2022)
-
Quantitative analysis of global protein stability rates in tissues
Scientific Reports (2020)
-
Modification of messenger RNA by 2′-O-methylation regulates gene expression in vivo
Nature Communications (2019)
-
Translational attenuation and retinal degeneration in mice with an active integrated stress response
Cell Death & Disease (2018)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
