Abstract
Post-translational modifications play important roles in controlling protein function and can lead to altered protein stability. Protein stability can be determined after treatment with the protein synthesis inhibitor Cycloheximide. Cycloheximide is a translational inhibitor that inhibits protein synthesis via cytoplasmic ribosomes. Here we describe how to measure the stability of MYC2 in the context of regulation by FERONIA receptor kinase. First, we describe how to measure MYC2 stability in wild-type and feronia mutant; then we describe similar assays in transgenic plants expressing MYC2-FLAG and MYC2A12-FLAG (12 FERONIA phosphorylation sites are mutated to Alanine and the mutant protein is stabilized). MYC2 can be induced by mechanical touch, which can be a confounding factor in protein level measurement. In this protocol, we take that into consideration and try to achieve more accurate measurement.
Keywords: FERONIA, Receptor Kinase, MYC2, Transcription factor, Jasmonic acid signaling, Plant immunity, Protein phosphorylation, Protein degradation, Protein half-life
Background
We have shown that FERONIA receptor kinase can phosphorylate and destabilize MYC2, a major transcription factor in Jasmonic Acid (JA) signaling, which positively contributes to plant immunity (Guo et al., 2018). Under normal growth conditions, MYC2 is present at very low levels. Overnight treatment with MG132 allows MYC2 to accumulate to a higher level before Cycloheximide treatment (Jung et al., 2015; Jeong et al., 2017), which proves to be important for detection of such unstable proteins. Additionally, MYC2 can be induced by mechanical touch, which can lead to inconsistency in the protein level measurement. Here we provide a detailed protocol to help determine the half-life of proteins like MYC2. We illustrate how this approach can be used with both the endogenous protein and epitope-tagged proteins in transgenic plants. This protocol provides some general guidelines for determining the levels of proteins that exist at low levels and are sensitive to handling (e.g., mechanical touch).
Materials and Reagents
Note: For a complete list of reagents, please refer to Guo et al. (2018).
Equipment
Software
Procedure
Data analysis
After Western blotting, scan the film using a flat-bed scanner. Stain the membrane using Ponceau S and scan the stained membrane (Alternatively, this step can be done right after transfer. The membrane is then washed with distilled water before blotting). Use ImageJ to measure the intensity of the protein bands of interest and the intensity of Rubisco from the staining. Calculate the ratios of the protein of interest over the corresponding Rubisco. The protein half-life is the time when the ratio decreases by half. For an example, see Figures 3C and 3D in Guo et al. (2018). Alternatively, a housekeeping protein whose level doesn’t change dramatically due to Cycloheximide treatment can be monitored by western blotting and used as a reference instead of Rubisco.
Recipes
Acknowledgments
The authors thank Dr. Trevor Nolan for editing the manuscript. The research was supported by Signature Research Initiative of the College of Liberal Arts and Sciences and Plant Sciences Institute at Iowa State University.
Competing interests
Hongqing Guo and Yanhai Yin are co-inventors on the patent titled "Modulation of receptor-like kinases for promotion of plant growth", US9512440B2.
References
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