Exploring REGγ Target Protein

The eukaryotic20s proteasome is composed of four stacked rings with seven α unique subunits in the two outer rings and seven unique β subunits in the two inner rings forming a symmetrical barrel-shaped structure (α1-7β1-7β1-7a1-7). REGγ(PA28γ,PSME3), a member of11S proteasome activator family, can bind and activate the20s proteasome. To date, only a few REGγ target proteins have been found, such as SRC-3, p21, and Smurfl, Smurf2. REGγ promote degradation of these proteins in a ATP and ubiquitin independent manner, yet the degradation mechanism is still not clear and more target proteins need to be indentified. In this study, we purified20s proteasome from cattle red blood cells by multi-step liquid chromatographies, and purified REGγ by single affinity chromatography. We used purified REGγ and20s to select REGγ potential target protein in vitro. REGγ cannot stimulate PKA Ca degradation in vitro, but REGγ bind PKA Cα and regulate PKA Cα protein level in vivo. We also found GST fusion protein GST-REGγ only interact with in vitro translated α2and α6not other α subunits and GST-REGγ (N151Y) has this same trend, however, none of GST recombined α subunits can interact with in vitro translated REGy. These results suggest that some kind of α subunits modification is essential for the interaction between20s and REGγ. Taken together, our study provides a basis of REGγ target protein investigation and REGγ degradation mechanism exploration.