Background

In eukaryotes, protein degradation is carried out by the proteasome. The integrative 26S proteasome is comprised of two sub particles: one or two terminal 19S regulatory particle(s) (RP), which serve as a proteasome activator; and the 20S core proteasome (CP), which degrades proteins. Most eukaryotic proteins are polyubiquitinated and channeled into 26S proteasome for degradation. In contrast, proteins that contain intrinsically disordered regions have been found to be directly destroyed by the ubiquitin-independent 20S proteasome (Ben-Nissan et al., 2014). Methods to purify and assembly the 20S proteasome in vitro are well established for mammalian cells and yeast. This has led to an understanding and appreciation of the numerous ways by which IDPs interact with the 20S proteasome (Leggett et al., 2005). However, to date a detailed and efficient protocol has not been reported for the purification of the 20S proteasome from plants. Book et al. (2010) developed an affinity-based strategy to effectively isolate the 26S proteasome from Arabidopsis. In their approach, PAG1, one of the 14 core proteasome polypeptides, is epitope-tagged and immunoprecipitated with epitope-specific antibodies such that the 26S proteasome is recovered. From purification studies of the proteasome complex, there are two important components to monitor during the purification scheme: ATP amount and salt concentration (Verma et al., 2000; Leggett et al., 2002). It is known that the integrity of the RP-CP complex relies on ATP, and the abundance of RP subunits is substantially reduced if all purification steps do not include ATP. Similarly, RP subunit abundance is reduced when immunoprecipitates (IPs) are washed with a high salt buffer (800 mM NaCl) before elution (Book et al., 2010). Based on previous methods, we designed a simple approach to specifically isolate the 20S proteasome by immunoprecipitating PAG1 complexes from total protein extracts of stable transgenic lines expressing gPAG1-Flag-4Myc (gPAG1-FM) under its native promoter. In our protocol, the protein extracts are not supplied with ATP, and IPs are washed with a buffer containing 800 mM NaCl, as this stringent condition has been reported to strip the 19S regulatory subunits away from the 20S core proteasome. For in vitro protein degradation assays, we modified a protocol from yeast work with the 20S proteasome (Hsieh et al., 2015). Taken together, this protocol is easy to follow and can provide an effective strategy to study degradation of IDPs in plants. We hope that this protocol will advance research in protein metabolism and regulation in plants.