Abstract
Highly regulated and targeted protein degradation plays a fundamental role in almost all cellular processes. Determination of the protein half-life by the chase assay serves as a powerful and popular strategy to compare the protein stability and study proteolysis pathways in cells. Here, we describe a chase assay in Haloferax volcanii, a halophilic archaeon as the model organism.
Keywords: Archaea, Ubiquitin-proteasome system, Targeted proteolysis, SAMP, Chase assay
Background
In eukaryotes, the ubiquitin proteasome system plays a major role in highly selective and targeted proteolysis (Glickman and Ciechanover, 2002). Recent evidence shows that small archaeal ubiquitin-like modifier proteins or SAMPs also function in targeting proteins for destruction by proteasomes (Maupin-Furlow, 2014; Anjum et al., 2015; Fu et al., 2016). Measurement of the protein half-life in vivo provides a direct way to study the proteolysis pathway. Cycloheximide chase and pulse-chase assays are commonly used to monitor the degradation of the targeted protein in eukaryotes (Zhou, 2004). The former method is utilized to determine the half-life of all cellular proteins after inhibition of translation elongation by cycloheximide; whereas, the pulse-chase assay measures the turnover of newly synthesized (pulse-labeled) proteins without interfering the normal cell growth. Compared with the eukaryotic system, a rapid and simple method to determine the half-life of a given protein in archaea is not well established. Therefore, we developed a protocol to measure the protein stability in vivo for the salt-loving archaeon Haloferax volcanii. Inhibitors of translation (anisomycin) and transcription (actinomycin D) are utilized to minimize the synthesis of new protein in this archaeon. TBP2, a TATA-binding protein (TBP) modified by ubiquitin-like isopeptide bonds in Haloferax volcanii, serves as the model protein substrate in this study.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Scan the region of X-ray film containing protein bands to produce a TIF file. Quantification of protein bands on the image is achieved by ImageJ software according to the user guidelines. Generally, purified TBP2 is used as the standard and a range of 0.5 ng to 50 ng protein serves as a range for the standard curve. Each TBP2 protein band is included in the same rectangle and the signal intensity reflected by the size of a peak is plotted. Details about quantification of protein by using ImageJ software can refer to (http://www.openwetware.org/wiki/Protein_Quantification_Using_ImageJ). Results are expressed as the percent change from time zero, which is set at 1.00. For example, measurement of the stability of TBP-StrepII and Flag-SAMP2 in the wild-type Haloferax volcanii strain by this chase assay has been published in mBio (Fu et al., 2016). Figure 2 for the representative data is originally published as Figure S2 by Fu et al. (2016). Figure 2. TBP2 degradation in wild-type strain. Chase assays were performed in the wild-type strain expressing Flag-SAMP2 and TBP2-StrepII. Log-phase cells were treated with 20 µg ml-1 actinomycin D and 50 µg ml-1 anisomycin for the indicated times and collected. TBP2 and SAMP2 protein levels were determined by anti-StrepII antibody and anti-Flag antibody, respectively. Equal loading was confirmed by CB staining. Experiments were performed in at least biological duplicates, and representative images are shown. ¥, coexpressed in trans. The dataset used in this figure was originally published in Fu et al. (2016).
Notes
Please note that the measure of TBP2 stability in this study was conducted when cells were grown in the standard culture condition. Knowing the environmental factors that trigger the turnover of targeted proteins and conducting the experiment in conditions where the targeted proteins are degraded will be highly recommended when other protein substrates are tested by this chase assay.
Recipes
Acknowledgments
This work was funded by US Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DE-FG02-05ER15650), USDA National Institute of Food and Agriculture (Hatch 1005900), and NIH | National Institute of General Medical Sciences (NIGMS) (NIH R01 GM57498-15).
References
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