Assaying Transcription Factor Stability
Similar to the activities of transcription factors (TFs) in other eukaryotes, activities of many plant TFs are determined via regulated proteolysis by the ubiquitin/26S proteasome system. Thus, to fully understand the function of a TF, it is important to determine the fate of the active TF protein and unravel the environmental and intrinsic signals that control its total cellular level. Here we describe how to determine whether a TF of interest is targeted to the 26S proteasome for degradation. The given method combines analyses of the effects of translational inhibition and the inhibition of proteasome activity. An important requirement for these experiments is to monitor in parallel the effects of translational and proteasomal inhibition on the abundance of the TF and (1) on ubiquitin, which becomes rapidly depleted upon translational inhibition (2), on polyubiquitinated proteins, which accumulate upon successful inhibition of the 26S proteasome, and (3) on glutamine synthase, a very stable protein that is used as a general metabolic control. The method described here can be used to test TF stability under a variety of conditions and in different genetic backgrounds.
Key wordsImmunoblot analyses cycloheximide proteasome inhibitor transcription factor stability glutamine synthase ubiquitin
This work was supported by the KTRD Center in Lexington, KY, and by grants from NSF (# 0919991) and from the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service (#2005-35304-16043).
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